TW201144438A

TW201144438A - Recombinant soybean storage protein for lowering triglyceride level in blood and method for preparing the same and use thereof

Info

Publication number: TW201144438A
Application number: TW99118522A
Authority: TW
Inventors: Wen-Hwei Hsu; Ming-Te Yang; Liang-Jwu Chen; Menq-Jiau Tseng; Chang-Sheng Wang; Chin-Ho Lin; Wen-Ching Wang
Original assignee: Nat Univ Chung Hsing
Priority date: 2010-06-08
Filing date: 2010-06-08
Publication date: 2011-12-16
Also published as: TWI392734B

Abstract

The present invention provides a recombinant soybean storage protein having at least one VVYP active polypeptide capable of lowering triglyceride level in blood, wherein the at least one VVYP active polypeptide is inserted in a corresponding site in a non-core structural domain of a soybean storage protein; the soybean storage protein is selected from the group consisting of β -conglycinin β -subunit, glycinin Gy5 and glycinin Gy1; and said VVYP active polypeptide has a valine-valine-tyrosin-proline (VVYP) sequence and at least one enzyme cutting site. Since VVYP tetrapeptides can be expressed in combination with the storage protein, resulting in common expressed in plants, applications of the recombinant soybean storage protein according to the present invention can overcome obstacles of large demand for VVYP tetrapeptides in the art, resulting in increased values of agricultural products and preparations thereof.

Description

201144438 > 六、發明說明：【發明所屬之技術領域】本發明係有關於一種大豆重組蛋白，其包含有降血脂活性胜肽。本發明亦有關於一種可表現前述大豆重組蛋白基因的載體及其轉型微生物或植物細胞。本發明亦有關於含有前述大豆重組蛋白之大豆加工食品。【先前技術】利用具生物活性的機能性胜肽改善高血壓、心臟血管 • 疾病、高血脂、肥胖'疼痛和精神障礙等疾病’是現代醫學與保健專家積極研發的課題。目前已知一種活性胜肽，其胺基酸序列為纈胺酸-纈胺酸_酪胺酸_脯胺酸（vaiine_ valine-tyrosin-proline，ννγρ)(以下簡稱 ννγρ 活性胜肽），能抑制腸道中腊肪的吸收’同時增強肝臟中的三酸甘油脂脂肪酶（hepatic triglyceride lipase)的活性，而達到使個體快速清理***體内脂肪之㈣’因此可制作為體重控制以及降血脂相關產品。然而，囿於取得ννγρ活性胜肽之 • 方法目前主要係利用化學合成方法或者由含有VVYP活性胜肽蛋白質之消化產物萃取出，而前者方法成本昂貴，後者則需大量繁項的純化步驟，十分不便利，因此此技術領域仍存在對於有效率且可大量生產ννγρ活性胜肽之技術的需求。基於利用基因轉殖作物作為生物反應器以生產食品、飼料或生物醫藥的概念之興起，基因轉殖作物的利用成為大量生產含有活性胜肽之重要技術。研究人員係將胺基酸序列為異亮胺酸-異亮胺酸_谷胺酸_丙胺酸_離胺酸（neiie_ 201144438201144438 > SUMMARY OF THE INVENTION: TECHNICAL FIELD The present invention relates to a soybean recombinant protein comprising a hypolipidemic activity peptide. The present invention also relates to a vector capable of expressing the aforementioned soybean recombinant protein gene and a transformed microorganism or plant cell thereof. The present invention also relates to a soybean processed food containing the aforementioned soybean recombinant protein. [Prior Art] The use of bioactive functional peptides to improve hypertension, cardiovascular disease, hyperlipidemia, obesity, pain and mental disorders is a topic actively developed by modern medical and health care professionals. An active peptide is currently known, and its amino acid sequence is valine-valine-tyrosin-proline (ννγρ) (hereinafter referred to as ννγρ-active peptide), which can inhibit The absorption of fat in the intestine enhances the activity of hepatic triglyceride lipase in the liver, and achieves the ability to quickly clear and excrete body fat (4), thus making it a weight control and blood fat lowering product. . However, the method for obtaining ννγρ-active peptides is currently mainly extracted by chemical synthesis or by digestion products containing VVYP-active peptide proteins, which are expensive and the latter requires a large number of complicated purification steps. Inconvenient, there is still a need in the art for techniques that are efficient and can mass produce ννγρ-active peptides. Based on the rise of the concept of using genetically modified crops as bioreactors to produce food, feed or biopharmaceuticals, the use of genetically modified crops has become an important technology for the mass production of active peptides. The researchers used the amino acid sequence as isoleucine-isoleucine glutamate _ alanine _ lysine (neiie_ 201144438)

Glu-Ala-Lys, IIEAK)的胜肽片段***大豆球蛋白原AlaBlb (proglycinin AlaBlb)内’藉以利用重組大豆球蛋白原之表現大量製造含有IIEAK胜肽片段的重組蛋白質。利用尚等植物作為生物反應器的優點包括：（丨）相較於基因轉殖動物，基因轉殖植物只需要較低之成本，且目前已發展出有關植物之種植、收穫或加工等基礎技術；（2)植物並無與人類相通的病源菌；以及（3)高等植物具有較完整的真核生物蛋白質表現糸統。此外’台灣土地面積以及天 _ 然資源有限’因此極適合朝向植物生物反應器的方向發展。現有技術雖已揭露VVYP活性胜肽或其相關產品，另一方面，雖有其他胜肽片段（如ΙΙΕΑΚ)的重組蛋白或大豆基因轉殖作物，惟現有技術仍然沒有合適的ννγρ活性胜肽表現系統，特別是儲藏性蛋白可供***WYP活性胜肽之合適位點，且亦完全沒有經轉殖有ννγρ的基因改造作物或大豆。基於上述可見，本技術領域中仍然對於含有籲 VVYP《大豆重組基因以及其基因改造作物具有一迫切的需求。【發明内容】有鑒於現有技術的前述缺失，本發明之目的即在於克服大豆ma)難以轉殖之技術障礙，以成功大量獲得含有降血脂機能性的VVYP活性胜肽的基因轉殖作物，藉以滿足所屬技術領域中長期存在之需求。基於上述目的，本發明係提供一種用於降血脂的重組大豆儲藏性蛋白，其係於大豆儲藏性蛋白的非核心構造區 201144438 域予以***或置換成至少— 儲藏性蛋白包括選自於由下大豆球蛋白β-次單元、大豆 Gyl。 VVYP活性胜肽，其中該大豆列蛋白質所構成的群組：β_伴球蛋白Gy5以及大豆球蛋白在方面，依據本發明的用於降血脂的重組大豆儲藏性蛋白係—重組β·伴大豆球蛋白β·次單元如。_㈤ηβ_ 灿她)/肽片段，其中該大豆儲藏性蛋㈣β_伴大豆球蛋白β〜人早7C ’其係具有—如SEQ m耻W所示序列且其’丨於第229至230位置之間的胺基酸序列置換成VVYP 活性胜肽。面依據本發明的用於降血脂的重組大豆儲藏 :生蛋白係-重組大豆球蛋白Gy5多肽片段，其中該大豆儲藏1±蛋白係大且球蛋白Gy5，其係具有一如SEQ 恥：6 所不序列’且於其至少一選自於由下列者所構成的群組之胺基酸序列位置，予以置換成—ννγρ活性胜肽··介於第 203至207位置之間、第208至212位置之間以及第213至 _ 218位置之間的胺基酸序列。再方面’依據本發明的用於降血脂的重組大豆儲藏蛋白係重組大豆球蛋白Gyl多肽片段，其中該大豆儲藏性蛋白係大豆球蛋白Gyl，其係具有一如SEQ m N〇: 2 所示序列’且於其至少一選自於由下列者所構成的群組之胺基酸序列位置，予以置換成VVYP活性胜肽：介於第u 9 至126位置之間、第286至290位置之間以及第292至300 位置之間的胺基酸序列。本發明提供於一大豆種子儲藏性蛋白之非核心構造區 201144438 域***/置換使其帶有VVYP胜肽序列的重組基因及其編碼出的蛋白質，並在VVYP胜肽序列兩端安排適合消化道酵素剪切的胺基酸序列，由於VVYP胜肽***之位點為原本儲藏性蛋白之非核心構造區域，所以其並不影響改造後蛋白之表現與立體結構，非核心構造區域可藉由χ晶體繞射、生化特性及熱動力學演算、電腦模擬等推衍出來通常是非為α-螺旋（a-helix)或β-摺板（β-sheet)的外圍1〇〇ρ區域’且較佳的不影響蛋白質三級結構與四級結構。 • 依據本發明，該VVYP活性胜肽較佳的包含纈胺酸纈胺酸-酪胺酸_脯胺酸（valine_valine_tyrosinproline，序列以及至少一酵素辨識切點。例如，於ννγρ活性胜肽與重組大豆性蛋白之***位置之間形成另一酵素辨識切點，則VVYP活性胜肽内的該至少一酵素辨識切點與該插入位置的另一酵素辨識切點經消化切割，即可釋放出該 VVYP活性胜肽。較佳的，前述酵素辨識切點係為消化道酵素辨識切點。鲁依據本發明，該至少一消化道酵素辨識切點係選自於下列所構成的群組：膜蛋白酶（trypsin)的辨識切點、羧肽酶 B (carboxypeptidase B)的辨識切點、胰凝乳蛋白酶 (chymotrpsin)的辨識切點或者它們的組合。例如：胰蛋白轉（trypsin)疋切割在離胺酸（iySine，κ)或是精胺酸 (arginine，R)的 c 端，而敌肽酶 b (carboxypeptidase B)則是切在離胺酸（lysine，K)或是精胺酸（R)的n端；因此 RVVYPKVVYPKVVYPK胜肽序列（含三套VVYP)，先使用膜蛋白酶進行切割，所得到的胜肽序列為vvypk，接著 7 201144438 才以叛肽酶B再行切割而獲得所要的ννΥΡ胜肽序列。依據本發明，該ννγρ活性胜肽較佳的包含如下列公式所示的胺基酸序列：The peptide fragment of Glu-Ala-Lys, IIEAK) was inserted into the glycinin AlaBlb (proglycinin AlaBlb) to generate a recombinant protein containing the IIEAK peptide fragment in large quantities using recombinant glycinin. Advantages of using a still-based plant as a bioreactor include: (丨) Gene transfer plants require only a lower cost than genetically-transformed animals, and basic techniques for planting, harvesting, or processing have been developed. (2) plants have no pathogenic bacteria that are in contact with humans; and (3) higher plants have more complete eukaryotic protein expression systems. In addition, 'Taiwan's land area and limited resources are so limited' that it is ideally suited to the direction of plant bioreactors. Although the prior art has disclosed VVYP active peptide or related products, on the other hand, although there are other peptide fragments (such as sputum) recombinant protein or soybean gene transgenic crop, the prior art still has no suitable ννγρ activity peptide expression. Systems, particularly storage proteins, are available for insertion into the appropriate site of the WYP-active peptide, and there are no genetically modified crops or soybeans that have been transduced with ννγρ. Based on the above, there is still an urgent need in the art for the inclusion of VVYP "soybean recombinant gene and its genetically modified crops. SUMMARY OF THE INVENTION In view of the aforementioned shortcomings of the prior art, the object of the present invention is to overcome the technical obstacle that soybean ma) is difficult to transfer, and to successfully obtain a large number of genetically transgenic crops containing a VVYP active peptide having hypolipidemic function. It satisfies the long-standing needs in the field of technology. Based on the above object, the present invention provides a recombinant soybean storage protein for lowering blood fat, which is inserted or substituted into at least a non-core structural region 201144438 domain of a soybean storage protein, and is selected from the group consisting of Glycinin β-subunit, soybean Gyl. VVYP active peptide, wherein the group consisting of soybean protein: β_conglycinin Gy5 and glycinin, in terms of the recombinant soybean storage protein system for reducing blood fat according to the present invention - recombinant β·with soybean Globulin β·subunits such as. _ (five) ηβ_ 灿 / / peptide fragment, wherein the soybean storage egg (four) β_conglycinin β ~ human early 7C 'the line has - as shown in the sequence of SEQ m shame W and its 'between the positions 229 to 230 The amino acid sequence is replaced by a VVYP active peptide. Recombinant soybean storage for hypolipidemic according to the present invention: a raw protein-recombinant glycinin Gy5 polypeptide fragment, wherein the soybean stores 1± protein large and globulin Gy5, which has the same as SEQ shame: 6 Non-sequence ' and at least one selected from the group consisting of the amino acid sequence positions of the group, substituted with -ννγρ activity peptide · between positions 203 to 207, 208 to 212 Amino acid sequence between positions and between positions 213 to 218. Further, the recombinant soybean storage protein-based recombinant glycinin Gyl polypeptide fragment for reducing blood fat according to the present invention, wherein the soybean storage protein glycinin Gyl has a SEQ m N〇: 2 The sequence 'and at least one selected from the group consisting of the amino acid sequence positions of the group consisting of a VVYP-active peptide: between positions u 9 to 126 and positions 286 to 290 The amino acid sequence between the 292th and 300th positions. The present invention provides a recombinant gene having a VVYP peptide sequence inserted and substituted in a non-core structural region of a soybean seed storage protein, and a protein encoded thereby, and arranged at the two ends of the VVYP peptide sequence to fit the digestive tract. The amino acid sequence of the enzyme cleavage, because the site of VVYP peptide insertion is the non-core structural region of the original storage protein, it does not affect the expression and stereostructure of the modified protein, and the non-core structural region can be Crystal diffraction, biochemical properties and thermodynamic calculations, computer simulations, etc. are usually derived from the periphery of the α-helix (a-helix) or β-sheet (β-sheet) and preferably Does not affect the tertiary structure and quaternary structure of the protein. • According to the present invention, the VVYP active peptide preferably comprises valinate_valine_tyrosinproline (valine_valine_tyrosinproline, sequence and at least one enzyme recognition cut point. For example, ννγρ active peptide and recombinant soy Another enzyme identification cut point is formed between the insertion positions of the protein, and the VVYP active peptide is released by digesting and cutting the at least one enzyme recognition cut point in the VVYP active peptide and the other enzyme identification cut point at the insertion position. Preferably, the enzyme identification cut point is a digestive enzyme identification cut point. According to the invention, the at least one digestive enzyme identification cut point is selected from the group consisting of: the identification cut point of the membrane protease (trypsin), carboxy Identification of peptidase B (carboxypeptidase B), identification point of chymopsin (chymotpsin) or a combination thereof. For example: trypsin 疋 cleavage in lysine (iySine, κ) or arginine The c-terminus of (arginine, R), and the carboxypeptidase B is cleaved to the n-terminus of lysine (K) or arginine (R); thus RVV YPKVVYPKVVYPK peptide sequence (containing three sets of VVYP), first cleavage using membrane protease, the obtained peptide sequence is vvypk, and then 7 201144438 to re-cut with typeptidase B to obtain the desired νν ΥΡ peptide sequence. In the invention, the ννγρ-active peptide preferably comprises an amino acid sequence as shown in the following formula:

Xaa「(Val-Val-Tyr-Pro-Xaa2)n，其中Xaai、Xaa2分別與相鄰的纈胺酸（Val)以及脯胺酸（Pro)形成酵素辨識切點；以及η至少為丨，其中較佳的是，η 為 1、2、3、4、5、6、7、8、9 或 10。較佳的’刖述的Xaai為精胺酸（Arg);以及Xaa2為離 ^ 胺酸（Lys)。較佳的’依據本發明的用於降血脂的重組大豆儲藏性蛋白，具有一選自於由下列者所構成之群組的胺基酸序列： SEQ ID NO: 12、SEQ ID NO: 8 以及 SEQ ID NO: 4。又再一方面，本發明亦提供一種重組載體，其包含一控制序列以及一與該控制序列可操作的相連接（〇perably linked)的重組儲藏性蛋白基因序列，諸如重組卜伴大豆球蛋白β-次單TL基因序列、重組大豆球蛋白Gy5基因序列或籲重組大豆球蛋白Gyl基因序列，較佳的為具有SEq ID N〇: 11、7或3所示序列，而該重組儲藏性蛋白基因序列編碼一如前所述之重組大豆儲藏性蛋白，諸如，SEQIDN〇: 12、 8或4所示序列的蛋白質。依據本發明，所述的重組载體的控制序列係為適用於植物細胞的任何調節序列，諸如，但不限於：花椰菜嵌紋病毒 35S 啟動子（Cauliflower m〇saic 35s pr〇m〇ter，Xaa"(Val-Val-Tyr-Pro-Xaa2)n, wherein Xaai and Xaa2 form an enzyme recognition cut point with adjacent proline (Val) and proline (Pro), respectively; and η is at least 丨, of which Preferably, η is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferred 'Xaai of the above description is arginine (Arg); and Xaa2 is from the amine acid ( Lys). A preferred recombinant soybean storage protein for lowering blood lipids according to the present invention, having an amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO And SEQ ID NO: 4. In still another aspect, the present invention also provides a recombinant vector comprising a control sequence and a recombinant storage protein gene sequence operably linked to the control sequence , such as a recombinant pod glycinin β-sub-mono TL gene sequence, a recombinant glycinin Gy5 gene sequence or a recombinant glycinin Gyl gene sequence, preferably having an SEq ID N〇: 11, 7 or 3 a sequence, and the recombinant storage protein gene sequence encodes a recombinant soybean storage protein as described above, such as SEQ IDN〇: a protein of the sequence shown in 12, 8 or 4. According to the invention, the control sequence of the recombinant vector is any regulatory sequence suitable for use in plant cells, such as, but not limited to, broccoli mosaic virus 35S Promoter (Cauliflower m〇saic 35s pr〇m〇ter,

CaMV promoter)或大豆16_kD種子成熟蛋白GmpM9基因之啟動子（promoter of 16_kD soybean seed maturati〇n 201144438 protein gene, GmPM9 promoter) ° 本發明亦提供一種基因轉殖植物細胞，其包含一如前所述的重組储藏性蛋白基因序列，亦即如前所述重組卜伴大豆球蛋白β_次單元基因序列重組大豆球蛋白基因序列或重組大丑球蛋白Gyl基因序列，而該重組儲藏性蛋白基因=列編碼一如前所述之重組大豆儲藏性蛋白，其係可利用則述載體藉由已知的任何植物基因轉殖技術予以導入一植物細胞而得e Φ 又再一方面，本發明亦提供一種製備用於降血脂的重組大豆儲藏性蛋白之方法，其包括：將一種如前所述之重組載體導入一植物細胞内；以及令該植物細胞生長，藉以表現出用於降血脂的重組大豆儲藏性蛋白。依據本發明，所述的將一種如前所述之重組載體導入一植物細胞内的步驟，如此技術領域所知者，可藉由例如，仁不限於·農桿鹵Sp.)、粒子搶 (microprojectile bombardment)、電穿孔法（eiectrop〇mi〇n)、 • 微注射法（micr〇injecti〇n)以及超音波法。依據本發明，所述的植物細胞係衍生自各種可表現儲藏性蛋白（storage protein)的作物，例如，但不限於：大豆 (G/ycz’we max)、稻米（Oryza sp.)或玉米（Zea map)，只要將本發明的重組大豆儲藏性蛋白基因與適當的啟動子可操作的連接。综上所述，本發明特點在於藉由基因轉殖方式，改造植物種子之儲藏性蛋白之基因，以提高該蛋白之具有降血脂功效的VVYP活性胜肽的含量，其中該重組植物儲藏性 201144438 蛋白本身含有兩端接有消化道酵素辨識切點之一套咬多套 VVYP活性胜肽片段，因此可直接食用或經加工處理而獲得一植物機能性產品後食用，使其内含之ννγρ活性胜狀即可在腸道中被釋放出來，達到抑制脂肪吸收的效果，進 -步達到降低血脂之目的。另一方面，也達到增加作物或其產品之附加價值之目的。本發明的含降血脂胜肽VVYP的重組儲藏性蛋白，除了可應用於基因轉殖大豆外，其可應用於所有植物，較佳的是具有豐富儲藏性蛋白的植物，更佳的是應用於具有經濟價值的糧食性作物，例如大豆、稻米、玉米等，藉以進一步提升農作物的經濟價值。依據本發明的含降血脂胜肽 vvyp的重組儲藏性蛋白應用於基因轉殖大豆時，由於大豆種子富+ 40 i 50%的蛋白質’其中包括佔有及高比例的儲藏性蛋白，含降血脂胜& ννγρ㈣且儲藏性蛋白具有極大的優勢藉由大豆種子長成予以大量表現；又基因轉殖至大豆的成功案例不多，本發明的含降血脂胜肽ννγρ 的重組儲藏性蛋白可以突破大豆轉殖的技術障帛，可建立台灣農業生技的優勢。此外，大豆為國人傳統食物，於食品加工的利用性與所製得的商品價值極高。【實施方式】本發明將進一步藉由下面的實施例來作說明，但應明瞭的是，該等實施例僅為說明之用，而不應被視為本發明的實施上的限制。實施例 S2 於下述實把例中，分別針對大豆球蛋白（抑cinin)的 10 201144438CaMV promoter) or soybean 16_kD seed mature protein GmpM9 gene promoter (promoter of 16_kD soybean seed maturati〇n 201144438 protein gene, GmPM9 promoter) ° The present invention also provides a gene transfer plant cell, which comprises a Recombinant storage protein gene sequence, that is, the recombinant soy globulin β-subunit gene sequence recombinant glycinin gene sequence or recombinant large ugly globulin Gyl gene sequence, as described above, and the recombinant storage protein gene=column A recombinant soybean storage protein encoding the same as described above, which can be introduced into a plant cell by any known plant gene transfer technique to obtain e Φ. In still another aspect, the present invention also provides a A method for preparing a recombinant soybean storage protein for lowering blood fat, comprising: introducing a recombinant vector as described above into a plant cell; and growing the plant cell to thereby exhibit a recombinant soybean storage for lowering blood fat Sex protein. According to the present invention, the step of introducing a recombinant vector as described above into a plant cell, as known in the art, can be carried out, for example, by itself, not limited to Microprojectile bombardment), electroporation (eiectrop〇mi〇n), microinjection (micr〇injecti〇n), and ultrasonic method. According to the invention, the plant cell line is derived from various crops which can represent a storage protein, such as, but not limited to, soybean (G/ycz'we max), rice (Oryza sp.) or corn ( Zea map), as long as the recombinant soybean storage protein gene of the invention is operably linked to a suitable promoter. In summary, the invention is characterized in that the gene of the storage protein of the plant seed is modified by gene transfer method to increase the content of the VVYP active peptide having the blood lipid lowering effect of the protein, wherein the recombinant plant storage property 201144438 The protein itself contains a set of bite VVYP active peptide fragments with both ends of the digestive tract enzyme identification cut point, so it can be eaten directly or processed to obtain a plant functional product and eaten, so that its contained ννγρ activity wins. The shape can be released in the intestine to achieve the effect of inhibiting fat absorption, and further achieve the purpose of lowering blood fat. On the other hand, it also aims to increase the added value of crops or their products. The recombinant storage protein containing the hypolipidemic peptide VVYP of the present invention can be applied to all plants except for application to genetically transformed soybeans, preferably plants having abundant storage proteins, and more preferably applied to plants. Economical value of food crops, such as soybeans, rice, corn, etc., to further enhance the economic value of crops. The recombinant storage protein containing the hypolipidemic peptide vvyp according to the present invention is applied to genetically transgenic soybeans, because the soybean seeds are rich in +40 i 50% of the protein 'including possession and a high proportion of storage proteins, including blood fat reduction & ννγρ(4) and the storage protein has great advantages. It can be expressed in large quantities by the growth of soybean seeds. There are not many successful cases of gene transfer to soybean. The recombinant storage protein containing the hypolipidemic peptide ννγρ of the present invention can break through soybeans. The technical barriers to transfer can establish the advantages of Taiwan's agricultural technology. In addition, soybeans are traditional foods for Chinese people, and their use in food processing and the value of the products they produce are extremely high. The present invention will be further illustrated by the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be considered as limiting. EXAMPLES S2 In the following examples, respectively, for glycinin (cincin) 10 201144438

Gyl、Gy5基因以及β-伴大豆球蛋白（β-conglycinin)的β -次單元基因（β-subunit gene)進行基因改造。本發明係利用各蛋白質的立體結構預測結果’分別針對其非核心構造區域進行改造，其係於該區域内的適當胺基酸位點***或置換為含VVYP的序列，以獲得含有VVYP序列的重組蛋白。下述實施例係根據上述設計以基因工程方式，對前述基因進行改造，進一步將所得的重組基因透過蛋白質表現系統確認重組蛋白的表現，以及藉由轉殖構築體和農桿菌轉瘦 φ 法，將重組基因轉植入大豆殖株中進行大豆儲藏性蛋白的活體内（纟《 V!_VO)表現。實施例1.大豆球蛋白Gyl基因之改造一 '含有VVYP的Gyl重組蛋白及其重組基因之製備 (l)Gyl基因的選殖取21天未成熟大豆種子的總RNA為模板（tempiate)，依據NCBI基因庫已發表的大豆球蛋白Gy 1基因的cdna 序列（Genebank Accession NO: AB1 13349)為引子設計的依 Φ 據’引子含與SacI切位，分別為Gyl-23A (AGTCATCACCATGGCCAAGCTAG) (SEQ ID NO·· 13)以及 Gyl-30B (CCCGAGCTCCTAAGCCACAGCTCTCTTCT) (SEQ ID NO: 14)，以反轉錄-聚合酶鏈鎖反應（RT-PCR)的方式複製出1488 bp之大豆Gyl的cDNA基因序列片段（SEQ ID NO: 1)，其係編碼出一 Gyl蛋白（SEQ ID NO: 2)，再將此片段回收並構築於 pGEM-T easy Vector System (Promega Inc·) 中，以限制酵素作用，確定片段大小正確無誤，進行定序。將所得的大豆Gyl的cDNA基因序列片段構築於pET21b [S1 11 201144438 表現載體中，令大腸桿菌表現出上述蛋白質，並進行純化及以抗-Gyl抗血清經過西方墨點法（Western blot)偵測，結果顯示皆有56 kDa之Gyl蛋白的信號出現。 (2)重組Gyl基因的構築針對前述所得的Gyl蛋白的非核心構造區域進行改造，其藉由將 VVYP所組成的基因卡匣（gene cassette) [(RVVYP)n]置換與***非核心構造區域來達成，其中該基因卡匣係於VVYP序列兩端分別設計適合消化道酵素（胰蛋白酶和羧肽酶B)之剪切點，如第一 A圖所示，其中將Gy 1 cDNA基因所編碼出之蛋白的胺基酸序列第119至126的胺基酸置換為4套VVYP，命名為1QQ ;第286至290的胺基酸置換為1套VVYP，命名為5WF ;第292至300的胺基酸置換為2套VVYP，命名為2WW，因此最終得到置換與***有7套VVYP的Gyl重組基因，經定序確認，其序列如SEQ ID NO: 3所示，其編碼出重組Gyl多肽（SEQ ID NO: 4)，如第一 B圖所示，其中粗體字表示***VVYP的位置所在，底線表示VVYP卡S。二、含有7套VVYP的重組Gyl多肽之表現載體的構築與表現將上述經改造的Gyl重組基因接合到商業可取得的表現載體pET30a内，得到一重組載體命名為pET3〇a-7VVYP，藉由SDS-PAGE確認，前述重組載體成功表現帶有7套VVYP的重組Gyl多肽。進一步將改造後的重組Gyl 多肽以姨蛋白酶（trypsin)和叛肽酶B (carboxypeptidase B) 進行水解，經由MALDI-TOF進行質譜分析。結果顯示’ 12 201144438 符合VVYP標準品斷裂後的m/z，證實VVYP的生成。三、含有7套VVYP之Gyl重組基因的農桿菌轉殖載體之構築將具大豆種子專一性之GmPM9啟動子取代pBI121 上之35S啟動子，同時將改造過之Gyl重組基因片段，分別以及Acl剪接於GmPM9啟動子下游，再將構築過之pBI121上之GmPM9啟動子-Gyl-7基因-NOS終結子區域，分別以限制酵素切下接合到pCAMBIA1301和 φ PCAMBIA2301 上，完成 pCl-Gyl-7 和 pC2-Gy 1-7 轉殖載體之構築（參見第二及三圖所示）。將構築完成之前述轉殖載體送入農桿菌中，作為後續轉殖大豆使用。實施例2.大豆球蛋白Gy5基因之改造一、含有VVYP的Gy5重組蛋白及其重組基因之製備 (1 )Gy5基因的選殖The Gyl, Gy5 gene and the β-subunit gene of β-conglycinin were genetically engineered. The present invention utilizes the steric structure prediction results of each protein to individually modify its non-core structural region, and inserts or replaces the appropriate amino acid site in the region into a VVYP-containing sequence to obtain a VVYP-containing sequence. Recombinant protein. In the following examples, the above genes were genetically engineered according to the above design, and the resulting recombinant gene was further confirmed by the protein expression system to confirm the expression of the recombinant protein, and by the transgenic construct and the Agrobacterium-transduced φ method. The recombinant gene was transfected into the soybean plant for in vivo (纟"V!_VO) expression of the soybean storage protein. Example 1. Modification of Glycin Gyl Gene - Preparation of Gyl Recombinant Protein Containing VVYP and Its Recombinant Gene (1) Selection of Gyl Gene Total RNA of 21 days of immature soybean seeds was used as a template (tempiate), The NCBI gene library has published the cdna sequence of the glycinin Gy 1 gene (Genebank Accession NO: AB1 13349). The primer is designed to contain the SacI cleavage, which is Gyl-23A (AGTCATCACCATGGCCAAGCTAG) (SEQ ID NO ·· 13) and Gyl-30B (CCCGAGCTCCTAAGCCACAGCTCTCTTCT) (SEQ ID NO: 14), a 1488 bp cDNA sequence fragment of soybean Gyl was reproduced by reverse transcription-polymerase chain reaction (RT-PCR) (SEQ ID NO: 1), which encodes a Gyl protein (SEQ ID NO: 2), which is then recovered and constructed in pGEM-T easy Vector System (Promega Inc.) to limit the enzyme action and determine the correct fragment size. Incorrect, sequence. The obtained cDNA sequence fragment of soybean Gyl was constructed in pET21b [S1 11 201144438 expression vector, and Escherichia coli showed the above protein, and purified and detected by Western blot using anti-Gyl antiserum. The results showed that all of the 56 kDa Gyl protein signals appeared. (2) Construction of recombinant Gyl gene The non-core structural region of the Gyl protein obtained above was modified by replacing the gene cassette [(RVVYP)n] composed of VVYP into a non-core structural region. To achieve this, the gene cassette is designed at both ends of the VVYP sequence to design a cleavage point suitable for digestive enzymes (trypsin and carboxypeptidase B), as shown in Figure A, in which the Gy 1 cDNA gene is encoded. The amino acid of amino acid sequences 119 to 126 of the protein was replaced with 4 sets of VVYP, designated as 1QQ; the amino acid of 286 to 290 was replaced with 1 set of VVYP, designated 5WF; amines of 292 to 300 The base acid was replaced by two sets of VVYP, which was named 2WW, so that the Gyl recombinant gene with 7 sets of VVYP inserted and finally obtained was confirmed by sequencing, and its sequence is shown in SEQ ID NO: 3, which encodes a recombinant Gyl polypeptide ( SEQ ID NO: 4), as shown in the first B-picture, in which the boldface indicates where the VVYP is inserted and the bottom line indicates the VVYP card S. Construction and Expression of Expression Vector Containing 7 Sets of VVYP Recombinant Gyl Polypeptides The above-described engineered Gyl recombinant gene was ligated into the commercially available expression vector pET30a to obtain a recombinant vector named pET3〇a-7VVYP. SDS-PAGE confirmed that the aforementioned recombinant vector successfully expressed a recombinant Gyl polypeptide with 7 sets of VVYP. The engineered recombinant Gyl polypeptide was further hydrolyzed with trypsin and carboxypeptidase B, and subjected to mass spectrometry by MALDI-TOF. The results show that ' 12 201144438 meets the m/z after fracture of the VVYP standard, confirming the formation of VVYP. 3. Construction of Agrobacterium tumefaciens vector containing 7 sets of VVYP Gyl recombinant gene The GmPM9 promoter with soybean seed specificity was substituted for the 35S promoter on pBI121, and the modified Gyl recombinant gene fragment, respectively, and Acl splicing Downstream of the GmPM9 promoter, the GmPM9 promoter-Gyl-7 gene-NOS terminator region on pBI121 was constructed and ligated into pCAMBIA1301 and φ PCAMBIA2301 with restriction enzymes to complete pCl-Gyl-7 and pC2. -Gy 1-7 Construction of the transfer vector (see Figures 2 and 3). The previously constructed transfer vector which has been constructed is put into Agrobacterium and used as a subsequent transfer soybean. Example 2. Modification of glycinin Gy5 gene 1. Preparation of Gy5 recombinant protein containing VVYP and its recombinant gene (1) Selection of Gy5 gene

取2 1天未成熟大豆種子的總RNA為模板（template)，依據NCBI基因庫已發表的大豆球蛋白Gy5基因之cDNA 參序列（Genebank Accession NO: AB003680)為引子設計的依據引子含 iVcoI與 SacI切位，分別為 Gy5NcoI-26A (TCGTCCATGGGGAAGCCCTTCTTCAC) (SEQ ID NO: 15) 以及與 Gy5SacI-30B (GGCGGAGCTCTTATGGGTTGACCAAAGGGC) (SEQ ID NO: 16)，以反轉錄-聚合酶鏈鎖反應（RT-PCR)的方式複製出約1554 bp大小之大豆Gy5的cDNA基因序列片段（SEQ ID NO: 5)，其係編碼出一 Gy5蛋白（SEQ ID NO·· 6)，再將此片段回收並構築於 pGEM-T easy Vector System (Promega 13 201144438Take the total RNA of the immature soybean seeds for 21 days as a template, based on the published cDNA sequence of the glycinin Gy5 gene of the NCBI gene bank (Genebank Accession NO: AB003680). The primers are based on the introduction of iVcoI and SacI. The cleavage sites were Gy5NcoI-26A (TCGTCCATGGGGAAGCCCTTCTTCAC) (SEQ ID NO: 15) and Gy5SacI-30B (GGCGGAGCTCTTATGGGTTGACCAAAGGGC) (SEQ ID NO: 16) by reverse transcription-polymerase chain reaction (RT-PCR). A cDNA gene sequence fragment (SEQ ID NO: 5) of soybean Gy5 of about 1554 bp in size was cloned, which encodes a Gy5 protein (SEQ ID NO.. 6), which was then recovered and constructed on pGEM-T easy. Vector System (Promega 13 201144438

Inc，）中，以限制酵素作用，確定片段大小正確無誤，進行定序。將所得的大豆Gy5的cDNA基因序列片段構築於 PET21b表現載體中，令大腸桿菌表現出上述蛋白質，並進打純化及以抗-Gy5抗血清經過西方墨點法偵測，結果顯示皆有63 kDa之Gy5蛋白的信號出現。 (2)重組Gy5基因的構築針對前述所得的Gy5蛋白的非核心構造區域進行改造，其藉由將vvyp所組成的基因卡匣[(RVVYp)n]置換與 φ ***非核心構造區域來達成，參照第四A圖所示，其中係將前述Gy5 cDNA基因所編碼出的蛋白質之胺基酸序列第 203至207位置、第208至212位置以及第213至218位置 (如第四A圖中矩線標示）分別予以置換/***vvyp序列。依照不同***位點設計，分別獲得含有4套、5套、9套和10套VVYP之改造Gy5重組序列。以改造後含有1〇套 VVYP之Gy5重組蛋白為例，經定序確認後其序列如SEq① NO: 7所示，編碼出一重組Gy5多肽，序列如SEQ ID n〇:8 Φ 所示’如第四Β圖所示，其中粗體字表示***或置換VVYP 胜肽序列的位置，底線表示VVYP套組。二、含有10套VVYP之重組Gy5多肽表現載體之構築及表現以改造後含有10套VVYP之重組Gy5多肽為例，將上述經改造的Gy5重組基因序列接合到pET21b之表現載體，付到一重組載體命名為pET21b-Gy5nsp-v2535，並送入大腸菌Rosetta (DE3)(pLysS)中（寄存於財團法人食品工業研究發展所生物資源保存暨研究中心之pFT21b_Gy5nsp_ 201144438In Inc,), to limit the enzyme action, determine the correct size of the fragment, and sequence. The obtained cDNA sequence fragment of soybean Gy5 was constructed in a PET21b expression vector, and the above-mentioned protein was expressed by Escherichia coli, and purified and anti-Gy5 antiserum was detected by Western blotting method, and the results showed that all of them were 63 kDa. The signal of the Gy5 protein appears. (2) Construction of recombinant Gy5 gene The non-core structural region of the Gy5 protein obtained as described above was modified by inserting the gene cassette [[RVVYp)n] composed of vvyp into φ into a non-core structural region. Referring to Figure 4A, wherein the amino acid sequence of the protein encoded by the Gy5 cDNA gene is at positions 203 to 207, 208 to 212, and 213 to 218 (as in the fourth graph A). The line is marked) to replace/insert the vvyp sequence. According to different insertion site designs, the modified Gy5 recombination sequences containing 4 sets, 5 sets, 9 sets and 10 sets of VVYP were obtained respectively. Take the modified Gy5 recombinant protein containing 1 set of VVYP as an example. After sequence confirmation, the sequence is as shown in SEq1 NO: 7. A recombinant Gy5 polypeptide is encoded, and the sequence is as shown in SEQ ID n〇: 8 Φ. In the fourth diagram, the boldface indicates the position at which the VVYP peptide sequence is inserted or replaced, and the bottom line indicates the VVYP kit. 2. Construction and expression of recombinant Gy5 polypeptide expression vector containing 10 sets of VVYP. The recombinant Gy5 polypeptide containing 10 sets of VVYP was transformed into the expression vector of pET21b, and a recombinant was added to the expression vector of pET21b. The vector was named pET21b-Gy5nsp-v2535 and was sent to the coliform Rosetta (DE3) (pLysS) (stored in the Bioresource Conservation and Research Center of the Food Industry Research and Development Institute, pFT21b_Gy5nsp_ 201144438)

v2535 ’其寄存編號為BCRC 940584)表現。再以SDS-PAGE 分析，以抗-Gy5抗血清經過西方墨點法偵測，結果顯示前述重組載體成功表現帶有10套VVYP之重組Gy5多肽。三、含有10套VVYP之Gy5重組基因農桿菌轉殖載體之構築 pSK-Gy5-10藉由M/mI與酵素切位，將含有1〇套VVYP基因之序列置換入PUCBCAP-Gy5中，得到 pUCBCAP-Gy5-10 之載體構築。再利用 PUCBCAP-Gy5-10 φ 與載體PCAM1300分別藉由五I單一酵素切位，並利用兩種抗生素篩選’完成pCAMBCAP-Gy5-10之大豆轉殖載體構築（參見第五圖）。將構築完成之前述轉殖載體送入農桿菌中，作為後續轉殖大豆使用。四、含有10套VVYP之Gy5重組基因之轉殖株將上述帶有10套VVYP之Gy5重組基因之轉殖載體進行農桿菌轉殖大豆組織。取「高雄選10號」的大豆種子（來自亞洲蔬菜研究發展中心，台南縣善化鎮）於無菌培 φ 養基中發芽5至7天後，切取無菌苗之包括子葉節之子葉培植體進行農桿菌感染，感染後種植於B5W培養基（B5培養基含有 1.7 mg/1 BA，0.25 mg/1 GA3，3 % 蔗糖， carbenicillin 500〜100 mg/1，pH 5_8)(購自 PhytoTechnology, Laboratories);每二週繼代一次，在相同的B5W培養基可誘導出芽體；待抽出芽梢後，轉換到芽梢抽長培養基[MS 鹽類（MS salt)、維生素 B5、1 mg/1 BA 以及〇·5 mg/1 GA](購自Duchefa Biochemie)上誘導芽梢抽長，同時加入G418或潮黴素（hygromine)等抗生素以篩選轉殖植株；待芽梢抽長 m 15 201144438 約2至3公分即可移至MS發根培養基上誘導發根並予以健化，最後移至瓶外溫室馴化及定植。轉殖大豆經基因分析之結杲，顯示具有Gy5-10VP基因。實施例3· β-伴大豆球蛋白β-次單元基因之改造一、含有VVYP的β-伴大豆球蛋白β-次單元重組蛋白及其重組基因之製備 (1) β-伴大豆球蛋白β-次單元基因的選殖取2 1天未成熟大豆種子的總RNA為模板（template)，依據NCBI基因庫已發表的β-伴大豆球蛋白β-次單元基因序列（Genebank Accession NO: ΑΒ030841)為引子設計的依據，分別為 Beta-25A (ATGATGAGAGTGCGGTTTCCTTTGT) (SEQ ID NO: 17)以及 Gy5NcoI-26A 與 Beta-26B (TCAGTAGAGAGCACCTAAGATTGAAG) (SEQ ID NO: 18)，以反轉錄-聚合酶鏈鎖反應（RT-PCR)的方式複製出伴大豆球蛋白β-次單元的cDNA基因序列片段（SEQ ID NO: 9) ，其編碼出一伴大豆球蛋白β-次單元多肽（SEQ ID NO: 10) ，再將此片段回收並構築於pGEM-T easy Vector System (Pro meg a Inc.)中，以限制酵素作用，確定片段大小正確無誤，進行定序。將所得的β-伴大豆球蛋白β-次單元的cDNA 基因序列片段構築於pET2 lb表現載體中，令大腸桿菌表現出上述蛋白質，並進行純化，以抗-β-伴大豆球蛋白β-次單元血清經過西方墨點法偵測，結果顯示有β-伴大豆球蛋白β-次單元蛋白的信號出現。 (2) 重組β-伴大豆球蛋白β-次單元基因的構築針對前述所得的β-伴大豆球蛋白β-次單元的非核心構 16 201144438 造區域進行改造，其藉由使用 VVYP所組成的基因卡匣 [(RVVYP)n]置換與***非核心構造區域來達成，如第六A 圖所示，其中將β-伴大豆球蛋白β-次單元cDNA基因（SEQ ID NO: 9)所編碼出之蛋白的胺基酸序列229至230之位置 (如第六A圖中矩線標示）***7套VVYP序列置換為 VVYP。經定序確認，其序列如SEQ ID NO: 11所示，其編碼之重組β-伴大豆球蛋白β-次單元序列如SEQ ID NO: 12 所示，如第六B圖所示，其中粗體字表示***或置換VVYP 胜肽序列的位置，底線表示VVYP套組。二、含有7套VVYP之重組β-伴大豆球蛋白β-次單元多肽之表現載體的構築及表現將帶有7套VVYP之β-伴大豆球蛋白β-次單元重組基因接合於pET2lb之表現載體，得到一重組載以命名為 pET21-7Sbsnp-v57，並送入大腸菌 Rosetta (DE3)(pLysS)中 (寄存於財圑法人食品工業研究發展所生物資源保存暨研究中心之 pET21-7Sbsnp-V57，其寄存編號為 BCRC 940585) 表現。再以SDS-PAGE分析，以抗-β-伴大豆球蛋白β-次單元抗血清經過西方墨點法偵測，結果顯示前述重組載體成功表現帶有7套VVYP之重組β-次單元重組多肽（54.5 kDa)。三、含有7套VVYP之β-次單元重組基因的農桿菌轉殖載體之構築將上述帶有經改造之β-次單元重組基因質體，分別以具有Swal切位與SacI切位的引子增幅7S beta基因[所使用的引子序列為： 7S-*SmaI: 17 201144438 CCCGGGATGATGAGAGTGCGGTTTCCT (SEQ ID NO: 19)； 7S-5acI:GAGCTCTCAGTGGTGGTGGTGGTGGTG (SEQ ID NO: 20)]，再以 Smal/iSacI 限制酵素分別與 7S beta PCR 產物與PBI121作用後，回收預期之片段進行DNA的黏合作用後，得pCa-召-V57 (參見第七A圖）。將含有GmPM9 啟動子之 pCAMBIA 2301 與 pBI121 質體以 7//ηί/ΙΙΙ/ΖΖ?αΙ 限制酵素作用後，回收預期之片段進行DNA的黏合作用後，形成pGm9-gus質體，再以限制酵素分別與7S ^ beta PCR產物與pGm9-gus作用後，回收預期之片段，進行DNA的黏合作用，形成pGm- /3 -V57(參見第七B圖）。將構築完成之前述質體pCa-P-V57和pGm-p-V57以電穿孔方式送入農桿菌中，作為後續轉殖大豆使用。【圖式簡單說明】第一 A圖為本發明之一較佳的大豆球蛋白Gyl多肽片段之序列示意圖，其中標示處為胺基酸置換位置。第一 B圖為本發明的一較佳的重組大豆球蛋白Gyl多肽片段之序列示意圖，其中標示處為***的胺基酸序列，底線表示VVYP套組。第二圖係本發明的載體的較佳實施例pCl-Gyl-7的構築策略示意圖。第三圖係本發明的載體的另一較佳實施例pC2-Gyl-7 的構築策略示意圖。第四A圖為本發明之一較佳的大豆球蛋白Gy5多肽片段之序列示意圖，其中標示處為胺基酸置換位置。第四B圖為本發明的一較佳的重組大豆球蛋白Gy5多 18 201144438 肽片段之序列示意圖，其中標示處為***的胺基酸序列，底線表示VVYP套組。第五圖係係本發明的載體的較佳實施例pCAMBCAP_ Gy5-10的構築策略示意圖。第六A圖為本發明之一較佳的β·伴大豆球蛋白β次單元多肽片段之序列示意圖，其中標示處為胺基酸置換位置。第六Β圖為本發明的一較佳的重組ρ伴大豆球蛋白卜次單元多肽片段之序列示意圖，其中標示處為***的胺基 • 酸序列，底線表示VVYP套組。第七Α圖係係本發明的載體的較佳實施例pCa_点々π 的構築策略示意圖。第七B圖係係本發明的栽體的較佳實施例的構築策略示意圖。【主要元件符號說明】 (無）V2535 'its registration number is BCRC 940584) performance. Further, by SDS-PAGE analysis, the anti-Gy5 antiserum was detected by Western blotting, and the results showed that the recombinant vector successfully expressed 10 sets of VVYP recombinant Gy5 polypeptide. 3. Construction of a Gy5 recombinant gene Agrobacterium tumefaciens vector containing 10 sets of VVYP pSK-Gy5-10 was translocated with a set of VVYP gene into PUCBCAP-Gy5 by M/mI and enzyme cleavage to obtain pUCBCAP. -Gy5-10 carrier construction. The PUCBCAP-Gy5-10 φ and the vector PCAM1300 were separately ligated by a single enzyme and screened with two antibiotics to complete the construction of the soybean transfer vector of pCAMBCAP-Gy5-10 (see Figure 5). The previously constructed transfer vector which has been constructed is put into Agrobacterium and used as a subsequent transfer soybean. 4. Transplanting of Gy5 Recombinant Gene Containing 10 Sets of VVYP The above-mentioned transfer vector of Gy5 recombinant gene carrying 10 sets of VVYP was subjected to Agrobacterium-transplanted soybean tissue. Take the "Kaohsiung No. 10" soybean seeds (from the Asian Vegetable Research and Development Center, Shanhua County, Shanhua County) for 5 to 7 days after germination in the sterile culture medium, and then cut the cotyledon of the sterile seedlings including the cotyledonary nodes. Agrobacterium infection, planted in B5W medium after infection (B5 medium containing 1.7 mg/1 BA, 0.25 mg/1 GA3, 3% sucrose, carbenicillin 500~100 mg/1, pH 5_8) (purchased from PhytoTechnology, Laboratories); After two weeks of subculture, buds can be induced in the same B5W medium; after the shoots are removed, they are transferred to shoot growth medium [MS salt, vitamin B5, 1 mg/1 BA and 〇·5 Mg/1 GA] (purchased from Duchefa Biochemie) induces shoot growth, and add antibiotics such as G418 or hygromine to screen for transgenic plants; to grow shoots m 15 201144438 about 2 to 3 cm It can be moved to MS hair root medium to induce rooting and to be healthy, and finally moved to the greenhouse for domestication and colonization. The soybean was genetically analyzed and found to have the Gy5-10VP gene. Example 3·Modification of β-conglycinin β-subunit gene 1. Preparation of β-conglycinin β-subunit recombinant protein containing VVYP and preparation of recombinant gene thereof (1) β-conglycinin β - Subunit gene selection takes 21 days of total RNA of immature soybean seeds as a template, and the β-conglycinin β-subunit gene sequence published according to the NCBI gene bank (Genebank Accession NO: ΑΒ030841) The basis for the design of the primers was Beta-25A (ATGATGAGAGTGCGGTTTCCTTTGT) (SEQ ID NO: 17) and Gy5NcoI-26A and Beta-26B (TCAGTAGAGAGCACCTAAGATTGAAG) (SEQ ID NO: 18), with reverse transcription-polymerase chain reaction ( RT-PCR) a cDNA gene sequence fragment (SEQ ID NO: 9) encoding a conglycinin β-subunit, which encodes a conglycinin β-subunit polypeptide (SEQ ID NO: 10), This fragment was then collected and constructed in pGEM-T easy Vector System (Pro meg a Inc.) to limit the enzyme action, to determine the correct size of the fragment, and to sequence. The obtained cDNA gene sequence fragment of β-conglycinin β-subunit was constructed in pET2 lb expression vector, and Escherichia coli showed the above protein and purified to anti-β-conglycinin β-time The unit serum was detected by Western blotting and the results showed that β-conglycinin β-subunit protein signal appeared. (2) Construction of the recombinant β-conglycinin β-subunit gene The transformation of the non-core structure 16 201144438 region of the β-conglycinin β-subunit obtained as described above is carried out by using VVYP. The gene cassette [(RVVYP)n] is replaced by insertion into a non-core structural region, as shown in Figure 6A, in which the β-conglycinin β-subunit cDNA gene (SEQ ID NO: 9) is encoded. The position of the amino acid sequence 229 to 230 of the protein (as indicated by the rectangle in Figure 6A) was inserted into 7 sets of VVYP sequences and replaced with VVYP. Confirmed by sequencing, the sequence is as shown in SEQ ID NO: 11, and the encoded recombinant β-conglycinin β-subunit sequence is represented by SEQ ID NO: 12, as shown in Figure 6B, wherein The body line indicates the position at which the VVYP peptide sequence is inserted or replaced, and the bottom line indicates the VVYP set. 2. Construction and expression of a performance vector containing 7 sets of VVYP recombinant β-conglycinin β-subunit polypeptides. The performance of 7 sets of VVYP β-conglycinin β-subunit recombinant gene conjugated to pET2lb The vector was obtained as a recombinant plasmid named pET21-7Sbsnp-v57, and was sent to the coliform Rosetta (DE3) (pLysS) (pET21-7Sbsnp-V57 deposited in the Center for Biological Resources Preservation and Research, Food Research and Development Institute, Caisei) , its registration number is BCRC 940585) performance. Further, by SDS-PAGE analysis, the anti-β-conglycinin β-subunit antiserum was detected by western blotting method, and the recombinant vector successfully showed that the recombinant β-subunit recombinant polypeptide with 7 sets of VVYP was successfully expressed. (54.5 kDa). 3. Construction of Agrobacterium tumefaciens vector containing 7 sets of VVYP β-subunit recombinant genes The above-mentioned recombinant β-subunit recombinant gene plastids were amplified by primers with Swal cleavage and SacI cleavage. 7S beta gene [the primer sequence used is: 7S-*SmaI: 17 201144438 CCCGGGATGATGAGAGTGCGGTTTCCT (SEQ ID NO: 19); 7S-5acI: GAGCTCTCAGTGGTGGTGGTGGTGGTG (SEQ ID NO: 20)], and then Smal/iSacI restriction enzymes are respectively After the 7S beta PCR product was combined with PBI121, the expected fragment was recovered for DNA binding and pCa-call-V57 was obtained (see Figure 7A). After the pCAMBIA 2301 and pBI121 plastids containing the GmPM9 promoter were treated with 7//ηί/ΙΙΙ/ΖΖ?αΙ restriction enzymes, the expected fragments were recovered for DNA adhesion to form pGm9-gus plastids, and then the restriction enzymes were used. After reacting with the 7S ^ beta PCR product and pGm9-gus, respectively, the expected fragment was recovered and the DNA was viscous to form pGm- /3 -V57 (see Figure 7B). The previously constructed plastids pCa-P-V57 and pGm-p-V57 were electroporated into Agrobacterium and used as subsequent transgenic soybeans. BRIEF DESCRIPTION OF THE DRAWINGS The first A is a sequence diagram of a preferred glycinin Gyl polypeptide fragment of the present invention, wherein the label is an amino acid substitution site. The first B is a schematic representation of the sequence of a preferred recombinant glycinin Gyl polypeptide fragment of the present invention, wherein the label is the inserted amino acid sequence and the bottom line indicates the VVYP kit. The second drawing is a schematic representation of the construction strategy of the preferred embodiment of the vector of the present invention, pCl-Gyl-7. The third panel is a schematic diagram of the construction strategy of another preferred embodiment of the vector of the present invention, pC2-Gyl-7. Figure 4A is a schematic view showing the sequence of a preferred glycinin Gy5 polypeptide fragment of the present invention, wherein the label is an amino acid substitution site. Figure 4B is a schematic representation of the sequence of a preferred recombinant glycinin Gy5 poly 18 201144438 peptide fragment of the present invention, wherein the label is the inserted amino acid sequence and the bottom line indicates the VVYP kit. The fifth drawing is a schematic diagram of the construction strategy of the preferred embodiment pCAMBCAP_Gy5-10 of the vector of the present invention. Fig. 6A is a schematic view showing the sequence of a preferred β-conglycinin β-subunit polypeptide fragment of the present invention, wherein the label is an amino acid substitution position. The sixth schematic diagram is a sequence diagram of a preferred recombinant p-conglycinin subunit polypeptide fragment of the present invention, wherein the label is the inserted amino acid sequence and the bottom line indicates the VVYP kit. The seventh diagram is a schematic diagram of a construction strategy of a preferred embodiment of the vector of the present invention pCa_dot π. Fig. 7B is a schematic diagram showing the construction strategy of the preferred embodiment of the carrier of the present invention. [Main component symbol description] (none)

m 19 201144438 序列表 <110>國立中興大學 <120>用於降血脂之重組大豆儲藏性蛋白及其製備方法與應用 <130> P93684 <160> 6 <170> Patentln version 3.4 <210> 1 <211> 1488 <212> DNA <213〉 Glycine maxm 19 201144438 Sequence Listing <110> National Chung Hsing University <120> Recombinant soybean storage protein for lowering blood fat and preparation method and application <130> P93684 <160> 6 <170> Patentln version 3.4 <;210> 1 <211> 1488 <212> DNA <213> Glycine max

<220> <221> CDS <222〉（1)..(1485) <400〉 1 atg gcc aag eta gtt ttt tcc ett tgt ttt ctg ett ttc agt ggc tgc Met Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15 tgc ttc get ttc agt tcc aga gag cag cct cag caa aac gag tgc cag Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 48 96<220><221> CDS <222> (1)..(1485) <400> 1 atg gcc aag eta gtt ttt tcc ett tgt ttt ctg ett ttc agt ggc tgc Met Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15 tgc ttc get ttc agt tcc aga gag cag cct cag caa aac gag tgc cag Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 48 96

ate caa aaa etc aat gcc etc aaa ccg gat aac cgt ata gag tea gaa lie Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 45 144 gga ggg etc att gag aca tgg aac cct aac aac aag cca ttc cag tgt Gly Gly Leu lie Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60 192 gcc ggt gtt gcc etc tet ege tgc acc etc aac ege aac gcc ett cgt Ala Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70 75 80 aga cct tcc tac acc aac ggt ccc cag gaa ate tac ate caa caa ggt Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu He Tyr lie Gin Gin Gly 85 90 95 aag ggt att ttt ggc atg ata tac ccg ggt tgt cct age aca ttt gaa 240 288Ate caa aaa etc aat gcc etc aaa ccg gat aac cgt ata gag tea gaa lie Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 144 gga ggg etc att gag aca tgg aac cct aac aac aag cca ttc C g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g Asn Ala Leu Arg 65 70 75 80 aga cct tcc tac acc aac ggt ccc cag gaa ate tac ate caa caa ggt Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu He Tyr lie Gin Gin Gly 85 90 95 aag ggt att ttt ggc atg ata Tac ccg ggt tgt cct age aca ttt gaa 240 288

336 [ SI 384201144438 Lys Gly lie Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110 gag cct caa caa cct caa caa aga gga caa age age aga cca caa gac Glu Pro Gin Gin Pro Gin Gin Arg Gly Gin Ser Ser Arg Pro Gin Asp 115 120 125 cgt cac cag aag ate tat aac ttc aga gag ggt gat ttg ate gca gtg Arg His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val 130 135 140 cct act ggt gtt gca tgg tgg atg tac aac aat gaa gac act cct gtt Pro Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val 145 150 155 160 432 480336 [ SI 384201144438 Lys Gly lie Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110 gag cct caa caa cct caa caa aga gga caa age age aga cca caa gac Glu Pro Gin Gin Pro Gin Gin Arg Gly Gin Ser Ser Arg Pro Gin Asp 115 120 125 cgt cac cag aag ate tat aac ttc aga gag ggt gat ttg ate gca gtg Arg His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val 130 135 140 cct act ggt gtt gca tgg tgg Atg tac aac aat gaa gac act cct gtt Pro Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val 145 150 155 160 432 480

gtt gcc gtt tet att att gac acc aac age ttg gag aac cag etc gac Val Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp 165 170 175 528 cag atg cct agg aga ttc tat ett get ggg aac caa gag caa gag ttt Gin Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe 180 185 190 eta aaa tat cag caa gag caa gga ggt cat cag age cag aaa gga aag Leu Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys 195 200 205 576 624Gtt gcc gtt tet att att gac acc aac age ttg gag aac cag etc gac Val Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp 165 170 175 528 cag atg cct agg aga ttc tat ett get ggg aac caa gag caa Gag ttt Gin Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe 180 185 190 eta aaa tat cag caa gag caa gga ggt cat cag age cag aaa gga aag Leu Lys Tyr Gin Gin Gin Gin Gly Gly His Gin Ser Gin Lys Gly Lys 195 200 205 576 624

cat cag caa gaa gaa gaa aac gaa gga ggc age ata ttg agt ggc ttc His Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe 210 215 220 672 acc ctg gaa ttc ttg gaa cat gca ttc age gtg gac aag cag ata geg Thr Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala 225 230 235 240 720 aaa aac eta caa gga gag aac gaa ggg gaa gac aag gga gcc att gtg Lys Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala lie Val 245 250 255 aca gtg aaa gga ggt ctg age gtg ata aaa cca ccc aeg gac gag cag Thr Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin 260 ’ 265 270 caa caa aga ccc cag gaa gag gaa gaa gaa gaa gag gat gag aag cca 768 816 864 2 912201144438 Gin Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Glu Lys Pro 275 280 285 cag tgc aag ggt aaa gac aaa cac tgc caa cgc ccc cga gga age caa Gin Cys Lys Gly Lys Asp Lys His Cys Gin Arg Pro Arg Gly Ser Gin 290 295 300 age aaa age aga aga aat ggc att gac gag acc ata tgc acc atg aga Ser Lys Ser Arg Arg Asn Gly lie Asp Glu Thr lie Cys Thr Met Arg 305 310 315 320 960 ett cgc cac aac att ggc cag act tea tea cct gac ate tac aac cct Leu Arg His Asn lie Gly Gin Thr Ser Ser Pro Asp lie Tyr Asn Pro 325 330 335 1008Cat cag caa gaa gaa gaa aac gaa gga ggc age ata ttg agt ggc ttc His Gin Gin Glu Glu Glu Aslu Glu Gly Gly Ser lie Leu Ser Gly Phe 210 215 220 672 acc ctg gaa ttc ttg gaa cat gca ttc age gtg gac aag cag Ata geg Thr Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala 225 230 235 240 720 aaa aac eta caa gga gag aac gaa ggg gaa gac aag gga gcc att gtg Lys Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala lie Val 245 250 255 aca gtg aaa gga ggt ctg age gtg ata aaa cca ccc aeg gac gag cag Thr Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin 260 ' 265 270 caa caa aga ccc cag gaa Gag gaa gaa gaa gaa gag gat gag aag cca 768 816 864 2 912201144438 Gin Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Glu Aslu Glu Lys Pro 275 280 285 cag tgc aag ggt aaa gac aaa cac tgc caa cgc ccc cga gga age caa Gin Cys Lys Gly Lys Asp Lys His Cys Gin Arg Pro Arg Gly Ser Gin 290 295 300 age aaa age aga aga aat ggc att gac gag acc ata tgc acc atg aga Ser Lys Ser Arg Arg Asn Gly lie Asp Glu Thr lie Cys T Hr Met Arg 305 310 315 320 960 ett cgc cac aac att ggc cag act tea tea cct gac ate tac aac cct Leu Arg His Asn lie Gly Gin Thr Ser Ser Pro Asp lie Tyr Asn Pro 325 330 335 1008

caa gee ggt age gtc aca acc gee acc age ett gac ttc cca gee etc Gin Ala Gly Ser Val Thr Thr Ala Thr Ser Leu Asp Phe Pro Ala Leu 340 345 350 1056 teg tgg etc aga etc agt get gag ttt gga tet etc cgc aag aat gca Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly Ser Leu Arg Lys Asn Ala 355 360 365 1104 atg ttc gtg cca cac tac aac ctg aac geg aac age ata ata tac gca Met Phe Val Pro His Tyr Asn Leu Asn Ala Asn Ser lie lie Tyr Ala 370 375 380 1152Caa gee ggt age gtc aca acc gee acc age ett gac ttc cca gee etc Gin Ala Gly Ser Val Thr Thr Ala Thr Ser Leu Asp Phe Pro Ala Leu 340 345 350 1056 teg tgg etc aga etc agt get gag ttt gga tet etc cgc aag Aat gca Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly Ser Leu Arg Lys Asn Ala 355 360 365 1104 atg ttc gtg cca cac tac aac ctg aac geg aac age ata ata tac gca Met Phe Val Pro His Tyr Asn Leu Asn Ala Asn Ser Lie lie Tyr Ala 370 375 380 1152

ttg aat gga egg gca ttg ata caa gtg gtg aat tgc aac ggt gag aga Leu Asn Gly Arg Ala Leu lie Gin Val Val Asn Cys Asn Gly Glu Arg 385 + 390 395 400 1200 gtg ttt gat gga gag ctg caa gag gga egg gtg ctg ate gtg cca caa Val Phe Asp Gly Glu Leu Gin Glu Gly Arg Val Leu lie Val Pro Gin 405 410 415 1248 aac ttt gtg gtg get gca aga tea cag agt gac aac ttc gag tat gtg Asn Phe Val Val Ala Ala Arg Ser Gin Ser Asp Asn Phe Glu Tyr Val 420 425 430 1296 tea ttc aag acc aat gat aca ccc atg ate ggc act ett gca ggg gca Ser Phe Lys Thr Asn Asp Thr Pro Met lie Gly Thr Leu Ala Gly Ala 435 440 445 aac tea ttg ttg aac gca tta cca gag gaa gtg att cag cac act ttc 1344 1392Ttg aat gga egg gca ttg ata caa gtg gtg aat tgc aac ggt gag aga Leu Asn Gly Arg Ala Leu lie Gin Val Val Asn Cys Asn Gly Glu Arg 385 + 390 395 400 1200 gtg ttt gat gga gag ctg caa gag gga egg gtg ctg Ate gtg cca caa Val Phe Asp Gly Glu Leu Gin Glu Gly Arg Val Leu lie Val Pro Gin 405 410 415 1248 aac ttt gtg gtg get gca aga tea cag agt gac aac ttc gag tat gtg Asn Phe Val Val Ala Ala Arg Ser Gin Ser Asp Asn Phe Glu Tyr Val 420 425 430 1296 tea ttc aag acc aat gat aca ccc atg ate ggc act ett gca ggg gca Ser Phe Lys Thr Asn Asp Thr Pro Met lie Gly Thr Leu Ala Gly Ala 435 440 445 aac tea ttg ttg aac Gca tta cca gag gaa gtg att cag cac act ttc 1344 1392

[SI 3 201144438[SI 3 201144438

Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu Val lie Gin His Thr Phe 450 455 460 aac eta aaa age cag cag gcc agg cag ata aag aac aac aac cct ttc 1440Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu Val lie Gin His Thr Phe 450 455 460 aac eta aaa age cag cag gcc agg cag ata aag aac aac aac cct ttc 1440

Asn Leu Lys Ser Gin Gin Ala Arg Gin lie Lys Asn Asn Asn Pro Phe 465 470 475 480 aag ttc ctg gtt cca cct cag gag tet cag aag aga get gtg get tag 1488Asn Leu Lys Ser Gin Gin Ala Arg Gin lie Lys Asn Asn Asn Pro Phe 465 470 475 480 aag ttc ctg gtt cca cct cag gag tet cag aag aga get gtg get tag 1488

Lys Phe Leu Val Pro Pro Gin Glu Ser Gin Lys Arg Ala Val Ala 485 490 495Lys Phe Leu Val Pro Pro Gin Glu Ser Gin Lys Arg Ala Val Ala 485 490 495

<210> 2 <211〉 495 <212〉 PRT <213〉 Glycine max <400> 2<210> 2 <211> 495 <212> PRT <213> Glycine max <400> 2

Met Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15Met Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15

Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30

He Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 45He Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 45

Gly Gly Leu lie Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60Gly Gly Leu lie Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60

Ala Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70 75 80Ala Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70 75 80

Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr He Gin Gin Gly 85 90 95Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr He Gin Gin Gly 85 90 95

Lys Gly lie Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110Lys Gly lie Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110

Glu Pro Gin Gin Pro Gin Gin Arg Gly Gin Ser Ser Arg Pro Gin Asp 115 120 125Glu Pro Gin Gin Gin Gin Arg Gly Gin Ser Ser Arg Pro Gin Asp 115 120 125

Arg His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu He Ala Val 130 135 140Arg His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu He Ala Val 130 135 140

Pro Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val 145 150 155 160Pro Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val 145 150 155 160

Val Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp 165 170 175Val Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp 165 170 175

Gin Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe 180 185 190Gin Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe 180 185 190

[SI[SI

Leu Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys 4 201144438 195 200 205Leu Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys 4 201144438 195 200 205

His Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe 210 215 220His Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe 210 215 220

Thr Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala 225 230 235 240Thr Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala 225 230 235 240

Lys Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala lie Val 245 250 255Lys Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala lie Val 245 250 255

Thr Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin 260 265 270Thr Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin 260 265 270

Gin Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Glu Lys Pro 275 280 285Gin Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Glu Lys Pro 275 280 285

Gin Cys Lys Gly Lys Asp Lys His Cys Gin Arg Pro Arg Gly Ser Gin 290 295 300Gin Cys Lys Gly Lys Asp Lys His Cys Gin Arg Pro Arg Gly Ser Gin 290 295 300

Ser Lys Ser Arg Arg Asn Gly lie Asp Glu Thr lie Cys Thr Met Arg 305 310 315 320Ser Lys Ser Arg Arg Asn Gly lie Asp Glu Thr lie Cys Thr Met Arg 305 310 315 320

Leu Arg His Asn lie Gly Gin Thr Ser Ser Pro Asp lie Tyr Asn Pro 325 330 335Leu Arg His Asn lie Gly Gin Thr Ser Ser Pro Asp lie Tyr Asn Pro 325 330 335

Gin Ala Gly Ser Val Thr Thr Ala Thr Ser Leu Asp Phe Pro Ala Leu 340 345 350Gin Ala Gly Ser Val Thr Thr Ala Thr Ser Leu Asp Phe Pro Ala Leu 340 345 350

Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly Ser Leu Arg Lys Asn Ala 355 360 365Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly Ser Leu Arg Lys Asn Ala 355 360 365

Met Phe Val Pro His Tyr Asn Leu Asn Ala Asn Ser lie lie Tyr Ala 370 375 380Met Phe Val Pro His Tyr Asn Leu Asn Ala Asn Ser lie lie Tyr Ala 370 375 380

Leu Asn Gly Arg Ala Leu He Gin Val Val Asn Cys Asn Gly Glu Arg 385 390 395 400Leu Asn Gly Arg Ala Leu He Gin Val Val Asn Cys Asn Gly Glu Arg 385 390 395 400

Val Phe Asp Gly Glu Leu Gin Glu Gly Arg Val Leu lie Val Pro Gin 405 410 415Val Phe Asp Gly Glu Leu Gin Glu Gly Arg Val Leu lie Val Pro Gin 405 410 415

Asn Phe Val Val Ala Ala Arg Ser Gin Ser Asp Asn Phe Glu Tyr Val 420 425 430Asn Phe Val Val Ala Ala Arg Ser Gin Ser Asp Asn Phe Glu Tyr Val 420 425 430

Ser Phe Lys Thr Asn Asp Thr Pro Met lie Gly Thr Leu Ala Gly Ala 435 440 445Ser Phe Lys Thr Asn Asp Thr Pro Met lie Gly Thr Leu Ala Gly Ala 435 440 445

Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu Val lie Gin His Thr Phe 450 455 460Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu Val lie Gin His Thr Phe 450 455 460

Asn Leu Lys Ser Gin Gin Ala Arg Gin lie Lys Asn Asn Asn Pro Phe 465 470 475 480Asn Leu Lys Ser Gin Gin Ala Arg Gin lie Lys Asn Asn Asn As Pn 465 470 475 480

Lys Phe Leu Val Pro Pro Gin Glu Ser Gin Lys Arg Ala Val Ala 485 490 495 <210> 3 <211> 1554 <212> DNA <213> Glycine max 48 201144438 <220> <221〉 CDS <222〉（1)..(1551) <400〉 3 atg gcc aag eta gtt ttt tee ett tgt ttt ctg ett ttc agt ggc tgcLys Phe Leu Val Pro Pro Gin Glu Ser Gin Lys Arg Ala Val Ala 485 490 495 <210> 3 <211> 1554 <212> DNA <213> Glycine max 48 201144438 <220><221><222> (1)..(1551) <400> 3 atg gcc aag eta gtt ttt tee ett tgt ttt ctg ett ttc agt ggc tgc

Met Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15 96 tgc ttc get ttc agt tee aga gag cag cct cag caa aac gag tgc cagMet Ala Lys Leu Val Phe Ser Leu Cys Phe Leu Leu Phe Ser Gly Cys 15 10 15 96 tgc ttc get ttc agt tee aga gag cag cct cag caa aac gag tgc cag

Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 144Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 144

ate caa aaa etc aat gcc etc aaa ccg gat aac cgt ata gag tea gaa He Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg He Glu Ser Glu 35 40 45Ate caa aaa etc aat gcc etc aaa ccg gat aac cgt ata gag tea gaa He Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg He Glu Ser Glu 35 40 45

gga ggg etc att gag aca tgg aac cct aac aac aag cca ttc cag tgt Gly Gly Leu He Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60 gcc ggt gtt gcc etc tet ege tgc acc etc aac ege aac gcc ett cgt Ala Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70 75 80 aga cct tee tac acc aac ggt ccc cag gaa ate tac ate caa caa ggt Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr lie Gin Gin Gly 85 90 95 192 240 288 336 aag ggt att ttt ggc atg ata tac ccg ggt tgt cct age aca ttt gaaGg ggg etc att gag aca tgg aac cct aac aac aag cca ttc cag tgt Gly Gly Leu He Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60 gcc ggt gtt gcc etc tet ege tgc acc etc aac ege aac gcc ett Cgt Ala Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70 75 80 aga cct tee tac acc aac ggt ccc cag gaa ate tac ate caa caa ggt Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr lie Gin Gin Gly 85 90 95 192 240 288 336 aag ggt att ttt ggc atg ata tac ccg ggt tgt cct age aca ttt gaa

Lys Gly He Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110 384 gag cct caa caa cct caa caa cgt gtg gtg tat cca cgt gtt gtt tacLys Gly He Phe Gly Met lie Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110 384 gag cct caa caa cct caa caa cgt gtg gtg tat cca cgt gtt gtt tac

Glu Pro Gin Gin Pro Gin Gin Arg Val Val Tyr Pro Arg Val Val Tyr 115 120 125 432 cct agg gtt gtt tat cct cgt gtt gtt tat cct cgt cag caa gac cgtGlu Pro Gin Gin Pro Gin Gin Arg Val Val Tyr Pro Arg Val Val Tyr 115 120 125 432 cct agg gtt gtt tat cct cgt gtt gtt tat cct cgt cag caa gac cgt

Pro Arg Val Val Tyr Pro Arg Val Val Tyr Pro Arg Gin Gin Asp Arg 130 135 140 cac cag aag ate tat aac ttc aga gag ggt gat ttg ate gca gtg cctPro Arg Val Val Tyr Pro Arg Val Val Tyr Pro Arg Gin Gin Asp Arg 130 135 140 cac cag aag ate tat aac ttc aga gag ggt gat ttg ate gca gtg cct

[SI[SI

His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val Pro 6 480 528 201144438 145 150 155 160 act ggt gtt gca tgg tgg atg tac aac aat gaa gac act cct gtt gttHis Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val Pro 6 480 528 201144438 145 150 155 160 act ggt gtt gca tgg tgg atg tac aac aat gaa gac act cct gtt gtt

Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val Val 165 170 175 gcc gtt tct att att gac acc aac age ttg gag aac cag etc gac cagThr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val Val 165 170 175 gcc gtt tct att att gac acc aac age ttg gag aac cag etc gac cag

Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp Gin 180 185 190 atg cct agg aga ttc tat ett get ggg aac caa gag caa gag ttt etaAla Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp Gin 180 185 190 atg cct agg aga ttc tat ett get ggg aac caa gag caa gag ttt eta

Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe Leu 195 200 205 aaa tat cag caa gag caa gga ggt cat cag age cag aaa gga aag cat Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys His 210 215 220 cag caa gaa gaa gaa aac gaa gga ggc age ata ttg agt ggc ttc acc Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe Thr 225 230 235 240 ctg gaa ttc ttg gaa cat gca ttc age gtg gac aag cag ata geg aaa Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala Lys 245 250 255 aac eta caa gga gag aac gaa ggg gaa gac aag gga gcc att gtg aca Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala He Val Thr 260 265 270 gtg aaa gga ggt ctg age gtg ata aaa cca ccc aeg gac gag cag caaMet Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe Leu 195 200 205 aaa tat cag caa gag caa gga ggt cat cag age cag aaa gga aag cat Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys His 210 215 220 cag caa gaa gaa gaa aac gaa gga ggc age ata ttg agt ggc ttc acc Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe Thr 225 230 235 240 ctg gaa ttc ttg gaa cat gca ttc age gtg Gac aag cag ata geg aaa Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala Lys 245 250 255 aac eta caa gga gag aac gaa ggg gaa gac aag gga gcc att gtg aca Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala He Val Thr 260 265 270 gtg aaa gga ggt ctg age gtg ata aaa cca ccc aeg gac gag cag caa

Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin Gin 275 280 285 caa aga ccc cag gaa gag gaa gaa gaa gaa gag gat tgg aag gtt gtgVal Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin Gin 275 280 285 caa aga ccc cag gaa gag gaa gaa gaa gaa gag gat tgg aag gtt gtg

Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Trp Lys Val Val 290 295 300 tac cca aag ttt aaa tgg aaa gtt gtt tac cct ege gtt gtt tat ccaGin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Trp Lys Val Val 290 295 300 tac cca aag ttt aaa tgg aaa gtt gtt tac cct ege gtt gtt tat cca

Tyr Pro Lys Phe Lys Trp Lys Val Val Tyr Pro Arg Val Val Tyr Pro 305 310 315 320 cgt tgg ega gga age caa age aaa age aga aga aat ggc att gac gagTyr Pro Lys Phe Lys Trp Lys Val Val Tyr Pro Arg Val Val Tyr Pro 305 310 315 320 cgt tgg ega gga age caa age aaa age aga aga aat ggc att gac gag

Arg Trp Arg Gly Ser Gin Ser Lys Ser Arg Arg Asn Gly He Asp Glu 576 624 672Arg Trp Arg Gly Ser Gin Ser Lys Ser Arg Arg Asn Gly He Asp Glu 576 624 672

720 768 816 864 912 960 [S] 7 1008 1056 201144438 325 330 335 acc ata tgc acc atg aga ctt cgc cac aac att ggc cag act tea tea Thr lie Cys Thr Met Arg Leu Arg His Asn lie Gly Gin Thr Ser Ser 340 345 350 cct gac ate tac aac cct caa gcc ggt age gtc aca acc gee acc age Pro Asp lie Tyr Asn Pro Gin Ala Gly Ser Val Thr Thr Ala Thr Ser 355 360 365 1104 ctt gac ttc cca gcc etc teg tgg etc aga etc agt get gag ttt gga Leu Asp Phe Pro Ala Leu Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly 370 375 380 1152720 768 816 864 912 960 [S] 7 1008 1056 201144438 325 330 335 acc ata tgc acc atg aga ctt cgc cac aac att ggc cag act tea tea Thr lie Cys Thr Met Arg Leu Arg His Asn lie Gly Gin Thr Ser Ser 340 345 350 cct gac ate tac aac cct caa gcc ggt age gtc aca acc gee acc age Pro Asp lie Tyr Asn Pro Gin Ala Gly Ser Val Thr Thr Ala Thr Ser 355 360 365 1104 ctt gac ttc cca gcc etc teg tgg etc aga etc agt get Gag ttt gga Leu Asp Phe Pro Ala Leu Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly 370 375 380 1152

tet etc cgc aag aat gca atg ttc gtg cca cac tac aac ctg aac geg Ser Leu Arg Lys Asn Ala Met Phe Val Pro His Tyr Asn Leu Asn Ala 385 390 395 400 1200 aac age ata ata tac gca ttg aat gga egg gca ttg ata caa gtg gtg Asn Ser lie lie Tyr Ala Leu Asn Gly Arg Ala Leu lie Gin Val Val 405 410 415 1248 aat tgc aac ggt gag aga gtg ttt gat gga gag ctg caa gag gga egg Asn Cys Asn Gly Glu Arg Val Phe Asp Gly Glu Leu Gin Glu Gly Arg 420 425 430 1296 gtg ctg ate gtg cca caa aac ttt gtg gtg get gca aga tea cag agt Val Leu lie Val Pro Gin Asn Phe Val Val Ala Ala Arg Ser Gin Ser 435 440 445 1344Tet etc cgc aag aat gca atg ttc gtg cca cac tac aac ctg aac geg Ser Leu Arg Lys Asn Ala Met Phe Val Pro His Tyr Asn Leu Asn Ala 385 390 395 400 1200 aac age ata ata tac gca ttg aat gga egg gca ttg ata Caa gtg gtg Asn Ser lie lie Tyr Ala Leu Asn Gly Arg Ala Leu lie Gin Val Val 405 410 415 1248 aat tgc aac ggt gag aga gtg ttt gat gga gag ctg caa gag gga egg Asn Cys Asn Gly Glu Arg Val Phe Asp Gly Glu Leu Gin Glu Gly Arg 420 425 430 1296 gtg ctg ate gtg cca caa aac ttt gtg gtg get gca aga tea cag agt Val Leu lie Val Pro Gin Asn Phe Val Val Ala Ala Arg Ser Gin Ser 435 440 445 1344

gac aac ttc gag tat gtg tea ttc aag acc aat gat aca ccc atg ate Asp Asn Phe Glu Tyr Val Ser Phe Lys Thr Asn Asp Thr Pro Met lie 450 455 460 1392 ggc act ctt gca ggg gca aac tea ttg ttg aac gca tta cca gag gaa Gly Thr Leu Ala Gly Ala Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu 465 470 475 480 1440 gtg att cag cac act ttc aac eta aaa age cag cag gcc agg cag ata Val lie Gin His Thr Phe Asn Leu Lys Ser Gin Gin Ala Arg Gin lie 485 490 495 aag aac aac aac cct ttc aag ttc ctg gtt cca cct cag gag tet cag Lys Asn Asn Asn Pro Phe Lys Phe Leu Val Pro Pro Gin Glu Ser Gin 1488Gac aac ttc gag tat gtg tea ttc aag acc aat gat aca ccc atg ate Asp Asn Phe Glu Tyr Val Ser Phe Lys Thr Asn Asp Thr Pro Met lie 450 455 460 1392 ggc act ctt gca ggg gca aac tea ttg ttg aac gca tta cca Gag gaa Gly Thr Leu Ala Gly Ala Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu 465 470 475 480 1440 gtg att cag cac act ttc aac eta aaa age cag cag gcc agg cag ata Val lie Gin His Thr Phe Asn Leu Lys Ser Gin Gin Ala Arg Gin lie 485 490 495 aag aac aac aac cct ttc aag ttc ctg gtt cca cct cag gag tet cag Lys Asn Asn Asn Pro Phe Lys Phe Leu Val Pro Pro Gin Glu Ser Gin 1488

1536 [SI 8 201144438 500 505 510 1554 aag aga get gtg get tag Lys Arg Ala Val Ala 515 <210〉 4 <211> 517 <212> PRT <213> Glycine max <400> 41536 [SI 8 201144438 500 505 510 1554 aag aga get gtg get tag Lys Arg Ala Val Ala 515 <210> 4 <211> 517 <212> PRT <213> Glycine max <400> 4

Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 lie Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 45Cys Phe Ala Phe Ser Ser Arg Glu Gin Pro Gin Gin Asn Glu Cys Gin 20 25 30 lie Gin Lys Leu Asn Ala Leu Lys Pro Asp Asn Arg lie Glu Ser Glu 35 40 45

Gly Gly Leu He Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60Gly Gly Leu He Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gin Cys 50 55 60

Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr lie Gin Gin Gly 85 90 95Arg Pro Ser Tyr Thr Asn Gly Pro Gin Glu lie Tyr lie Gin Gin Gly 85 90 95

Lys Gly lie Phe Gly Met He Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110Lys Gly lie Phe Gly Met He Tyr Pro Gly Cys Pro Ser Thr Phe Glu 100 105 110

Glu Pro Gin Gin Pro Gin Gin Arg Val Val Tyr Pro Arg Val Val Tyr 115 120 125Glu Pro Gin Gin Pro Gin Gin Arg Val Val Tyr Pro Arg Val Val Tyr 115 120 125

Pro Arg Val Val Tyr Pro Arg Val Val Tyr Pro Arg Gin Gin Asp Arg 130 135 140Pro Arg Val Val Tyr Pro Arg Val Val Tyr Pro Arg Gin Gin Asp Arg 130 135 140

His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val Pro 145 150 155 160His Gin Lys lie Tyr Asn Phe Arg Glu Gly Asp Leu lie Ala Val Pro 145 150 155 160

Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val Val 165 170 175Thr Gly Val Ala Trp Trp Met Tyr Asn Asn Glu Asp Thr Pro Val Val 165 170 175

Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp Gin 180 185 190Ala Val Ser lie lie Asp Thr Asn Ser Leu Glu Asn Gin Leu Asp Gin 180 185 190

Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe Leu 195 200 205Met Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gin Glu Gin Glu Phe Leu 195 200 205

Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys His 210 215 220Lys Tyr Gin Gin Glu Gin Gly Gly His Gin Ser Gin Lys Gly Lys His 210 215 220

Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe Thr m 225 230 235 240 9 201144438Gin Gin Glu Glu Glu Asn Glu Gly Gly Ser lie Leu Ser Gly Phe Thr m 225 230 235 240 9 201144438

Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala Lys 245 250 255Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys Gin lie Ala Lys 245 250 255

Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala He Val Thr 260 265 270Asn Leu Gin Gly Glu Asn Glu Gly Glu Asp Lys Gly Ala He Val Thr 260 265 270

Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin Gin 275 280 285Val Lys Gly Gly Leu Ser Val lie Lys Pro Pro Thr Asp Glu Gin Gin 275 280 285

Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Trp Lys Val Val 290 295 300Gin Arg Pro Gin Glu Glu Glu Glu Glu Glu Glu Asp Trp Lys Val Val 290 295 300

Tyr Pro Lys Phe Lys Trp Lys Val Val Tyr Pro Arg Val Val Tyr Pro 305 310 315 320Tyr Pro Lys Phe Lys Trp Lys Val Val Tyr Pro Arg Val Val Tyr Pro 305 310 315 320

Arg Trp Arg Gly Ser Gin Ser Lys Ser Arg Arg Asn Gly lie Asp Glu 325 330 335Arg Trp Arg Gly Ser Gin Ser Lys Ser Arg Arg Asn Gly lie Asp Glu 325 330 335

Thr lie Cys Thr Met Arg Leu Arg His Asn lie Gly Gin Thr Ser Ser 340 345 350Thr lie Cys Thr Met Arg Leu Arg His Asn lie Gly Gin Thr Ser Ser 340 345 350

Pro Asp lie Tyr Asn Pro Gin Ala Gly Ser Val Thr Thr Ala Thr Ser 355 360 365Pro Asp lie Tyr Asn Pro Gin Ala Gly Ser Val Thr Thr Ala Thr Ser 355 360 365

Leu Asp Phe Pro Ala Leu Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly 370 375 380Leu Asp Phe Pro Ala Leu Ser Trp Leu Arg Leu Ser Ala Glu Phe Gly 370 375 380

Ser Leu Arg Lys Asn Ala Met Phe Val Pro His Tyr Asn Leu Asn Ala 385 390 395 400Ser Leu Arg Lys Asn Ala Met Phe Val Pro His Tyr Asn Leu Asn Ala 385 390 395 400

Asn Ser He He Tyr Ala Leu Asn Gly Arg Ala Leu He Gin Val Val 405 410 415Asn Ser He He Tyr Ala Leu Asn Gly Arg Ala Leu He Gin Val Val 405 410 415

Asn Cys Asn Gly Glu Arg Val Phe Asp Gly Glu Leu Gin Glu Gly Arg 420 425 430Asn Cys Asn Gly Glu Arg Val Phe Asp Gly Glu Leu Gin Glu Gly Arg 420 425 430

Val Leu lie Val Pro Gin Asn Phe Val Val Ala Ala Arg Ser Gin Ser 435 440 445Val Leu lie Val Pro Gin Asn Phe Val Val Ala Ala Arg Ser Gin Ser 435 440 445

Asp Asn Phe Glu Tyr Val Ser Phe Lys Thr Asn Asp Thr Pro Met lie 450 455 460Asp Asn Phe Glu Tyr Val Ser Phe Lys Thr Asn Asp Thr Pro Met lie 450 455 460

Gly Thr Leu Ala Gly Ala Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu 465 470 475 480Gly Thr Leu Ala Gly Ala Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu 465 470 475 480

Val lie Gin His Thr Phe Asn Leu Lys Ser Gin Gin Ala Arg Gin lie 485 490 495Val lie Gin His Thr Phe Asn Leu Lys Ser Gin Gin Ala Arg Gin lie 485 490 495

Lys Asn Asn Asn Pro Phe Lys Phe Leu Val Pro Pro Gin Glu Ser Gin 500 505 510Lys Asn Asn Asn Pro Phe Lys Phe Leu Val Pro Pro Gin Glu Ser Gin 500 505 510

Lys Arg Ala Val Ala 515 <210〉 5 <211> 1554 <212〉 DNA <213> Glycine max <220>Lys Arg Ala Val Ala 515 <210> 5 <211> 1554 <212> DNA <213> Glycine max <220>

<221> CDS 10 48201144438 <222〉（1)..(1551) <400〉 5 atg ggg aag ccc ttc ttc act etc tet ett tet tee ett tgc ttg eta Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 etc ttg teg agt gca tgc ttt get att acc tee age aag ttc aac gag Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30 tgc caa etc aac aac etc aac geg ttg gaa ccc gac cac ege gtt gag Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 96 144<221> CDS 10 48201144438 <222> (1)..(1551) <400> 5 atg ggg aag ccc ttc ttc act etc tet ett tet tee ett tgc ttg eta Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 etc ttg teg agt gca tgc ttt get att acc tee age aag ttc aac gag Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30 tgc caa etc aac aac etc Aac geg ttg gaa ccc gac cac ege gtt gag Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 96 144

tee gaa ggt ggt ett att gaa aca tgg aac tet caa cac cct gag ctg Ser Glu Gly Gly Leu He Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 192 caa tgc gee ggt gtc act gtt tee aaa ege acc etc aac ege aac ggc Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 etc cac ttg cca tet tac tea cct tat ccc caa atg ate att gtc gtt Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met lie lie Val Val 85 90 95 240 288Tee gaa ggt ggt ett att gaa aca tgg aac tet caa cac cct gag ctg Ser Glu Gly Gly Leu He Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 192 caa tgc gee ggt gtc act gtt tee aaa ege acc etc aac ege Aac ggc Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 etc cac ttg cca tet tac tea cct tat ccc caa atg ate att gtc gtt Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met Lie lie Val Val 85 90 95 240 288

caa ggg aag gga gca att gga ttt gca ttt ccg gga tgt cct gag aeg Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 HO 336 ttt gag aag cca caa caa caa tea acc aga aga ggc tea agg teg cag Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 384 cag caa eta caa gac agt cac cag aag att cgt cac ttc aat gaa gga Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 gac gta eta gtg att cct cct ggt gtt cct tac tgg acc tat aac act Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 432 480 ggc gat gaa cca gtt gtt gee ate agt cct ett gac acc tee aac ttcCaa ggg aag gga gca att gga ttt gca ttt ccg gga tgt cct gag aeg Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 HO 336 ttt gag aag cca caa caa caa caa tea acc aga aga ggc tea agg Teg cag Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 384 cag caa eta caa gac agt cac cag aag att cgt cac ttc aat gaa gga Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 gac gta eta gtg att cct cct ggt gtt cct tac tgg acc tat aac act Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 432 480 ggc gat gaa cca gtt Gtt gee ate agt cct ett gac acc tee aac ttc

528 i SI 11 576201144438 Gly Asp Glu Pro Val Val Ala lie Ser Pro Leu Asp Thr Ser Asn Phe 165 170 175 aac aat cag ctt gat caa aac ccc aga gta ttt tac ctt get ggg aac Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 cca gat ata gag cac cca gag acc atg caa cag cag cag cag cag aag Pro Asp He Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Lys 195 200 205 624 agt cat ggt gga ege aag cag ggg caa cac cag caa cag gag gaa gaa Ser His Gly Gly Arg Lys Gin Gly Gin His Gin Gin Gin Glu Glu Glu 210 215 220 672528 i SI 11 576201144438 Gly Asp Glu Pro Val Val Ala lie Ser Pro Leu Asp Thr Ser Asn Phe 165 170 175 aac aat cag ctt gat caa aac ccc aga gta ttt tac ctt get ggg aac Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 cca gat ata gag cac cca gag acc atg caa cag cag cag cag cag ag Pro Asp He Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Lys 195 200 205 624 agt cat ggt gga ege Aag cag ggg caa cac cag caa cag gag gaa gaa Ser His Gly Gly Arg Lys Gin Gin Gin His Gin Gin Gin Glu Glu Glu 210 215 220 672

ggt ggc agt gtg etc agt ggc ttc age aaa cat ttc tta gca caa tcc Gly Gly Ser Val Leu Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser 225 230 235 240 720 ttc aac acc aac gag gac aca get gag aaa ctt egg tet cca gat gac Phe Asn Thr Asn Glu Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp 245 250 255 gaa agg aag cag ate gtg aca gtg gag gga ggc etc age gtt ate age Glu Arg Lys Gin lie Val Thr Val Glu Gly Gly Leu Ser Val lie Ser 260 265 270 768 816Ggt ggc agt gtg etc agt ggc ttc age aaa cat ttc tta gca caa tcc Gly Gly Ser Val Leu Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser 225 230 235 240 720 ttc aac acc aac gag gac aca get gag aaa ctt egg tet Cca gat gac Phe Asn Thr Asn Glu Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp 245 250 255 gaa agg aag cag ate gtg aca gtg gag gga ggc etc age gtt ate age Glu Arg Lys Gin lie Val Thr Val Glu Gly Gly Leu Ser Val lie Ser 260 265 270 768 816

ccc aag egg caa gaa cag gaa gac gaa gac gaa gat gaa gac gaa gaa Pro Lys Arg Gin Glu Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu 275 280 285 864 tat gaa caa act ccc tet tac cct cca ega ega cca ate cat gga aag Tyr Glu Gin Thr Pro Ser Tyr Pro Pro Arg Arg Pro lie His Gly Lys 290 295 300 912 cat gaa gat gac gag gac gag gac gaa gaa gaa gat caa cct cgt cct His Glu Asp Asp Glu Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro 305 310 315 320 960 gat cac cct cca cag ega cca age agg ccc gaa caa caa gaa cca cgt Asp His Pro Pro Gin Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg 325 330 335 1008 gga aga gga tgt cag act aga aat ggg gtt gag gaa aat att tgc acc 1056 [s】 12 201144438Ccc aag egg caa gaa cag gaa gac gaa gac gaa gat gaa gac gaa gaa Pro Lys Arg Gin Glu Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu 275 280 285 864 tat gaa caa act ccc tet tac cct cca ega ega cca ate cat Gga aag Tyr Glu Gin Thr Pro Ser Tyr Pro Pro Arg Arg Pro lie His Gly Lys 290 295 300 912 cat gaa gat gac gag gac gag gac gaa gaa gaa gat caa cct cgt cct His Glu Asp Asp Glu Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro 305 310 315 320 960 gat cac cct cca cag ega cca age agg ccc gaa caa caa gaa cca cgt Asp His Pro Pro Gin Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg 325 330 335 1008 gga aga gga tgt cag Act aga aat ggg gtt gag gaa aat att tgc acc 1056 [s] 12 201144438

Gly Arg Gly Cys Gin Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr 340 345 350 atg aag ctt cac gag aac att get ege cct tea cgt get gac ttc tac 1104Gly Arg Gly Cys Gin Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr 340 345 350 atg aag ctt cac gag aac att get ege cct tea cgt get gac ttc tac 1104

Met Lys Leu His Glu Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr 355 360 365 aac cct aaa get ggt ege att age acc etc aac agt etc acc etc cca 1152Met Lys Leu His Glu Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr 355 360 365 aac cct aaa get ggt ege att age acc etc aac agt etc acc etc cca 1152

Asn Pro Lys Ala Gly Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro 370 375 380 gee etc ege caa ttc gga etc agt gee caa tat gtt gtc etc tac agg 1200Asn Pro Lys Ala Gly Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro 370 375 380 gee etc ege caa ttc gga etc agt gee caa tat gtt gtc etc tac agg 1200

Ala Leu Arg Gin Phe Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg 385 390 395 400 1248Ala Leu Arg Gin Phe Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg 385 390 395 400 1248

aat gga att tac tet cca cat tgg aac ttg aac geg aac agt gtg ate Asn Gly lie.Tyr Ser Pro His Trp Asn Leu Asn Ala Asn Ser Val lie 405 410 415 tat gtg act ega ggg aaa gga aga gtt aga gtg gtg aac tgc caa ggg 1296Aat gga att tac tet cca cat tgg aac ttg aac geg aac agt gtg ate Asn Gly lie.Tyr Ser Pro His Trp Asn Leu Asn Ala Asn Ser Val lie 405 410 415 tat gtg act ega ggg aaa gga aga gtt aga gtg gtg aac tgc Caa ggg 1296

Tyr Val Thr Arg Gly Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly 420 425 430 aat gca gtg ttc gac ggt gag eta agg agg gga caa ttg eta gtg gtg 1344Tyr Val Thr Arg Gly Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly 420 425 430 aat gca gtg ttc gac ggt gag eta agg agg gga caa ttg eta gtg gtg 1344

Asn Ala Val Phe Asp Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val 435 440 445 1392 ccg cag aac ttt gtg gtg get gag caa ggg gga gaa caa gga ttg gaa Pro Gin Asn Phe Val Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu 450 455 460 tac gta gtg ttc aag aca cac cac aac gee gtg age age tac att aag 1440Asn Ala Val Phe Asp Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val 435 440 445 1392 ccg cag aac ttt gtg gtg get gag caa ggg gga gaa caa gga ttg gaa Pro Gin Asn Phe Val Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu 450 455 460 tac gta gtg ttc aag aca cac cac aac gee gtg age age tac att aag 1440

Tyr Val Val Phe Lys Thr His His Asn Ala Val Ser Ser Tyr lie Lys 465 470 475 480 gat gtg ttt agg gca ate cct teg gag gtt ctt tee aat tet tac aac 1488Tyr Val Val Phe Lys Thr His His Asn Ala Val Ser Ser Tyr lie Lys 465 470 475 480 gat gtg ttt agg gca ate cct teg gag gtt ctt tee aat tet tac aac 1488

Asp Val Phe Arg Ala lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn 485 490 495 ctt ggc cag agt caa gtg cgt cag etc aag tat caa gga aac tee ggc 1536Asp Val Phe Arg Ala lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn 485 490 495 ctt ggc cag agt caa gtg cgt cag etc aag tat caa gga aac tee ggc 1536

Leu Gly Gin Ser Gin Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly 500 505 510 m cct ttg gtc aac cca taa 1554 13 201144438Leu Gly Gin Ser Gin Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly 500 505 510 m cct ttg gtc aac cca taa 1554 13 201144438

Pro Leu Val Asn Pro 515 <210> 6 <211> 517 <212> PRT <213〉 Glycine max <400〉 6Pro Leu Val Asn Pro 515 <210> 6 <211> 517 <212> PRT <213> Glycine max <400> 6

Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15

Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30

Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45

Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60

Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80

Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met lie lie Val Val 85 90 95Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met lie lie Val Val 85 90 95

Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110

Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125

Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140

Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160

Gly Asp Glu Pro Val Val Ala lie Ser Pro Leu Asp Thr Ser Asn Phe 165 170 175Gly Asp Glu Pro Val Val Ala lie Ser Pro Leu Asp Thr Ser Asn Phe 165 170 175

Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190

Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Lys 195 200 205Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Lys 195 200 205

Ser His Gly Gly Arg Lys Gin Gly Gin His Gin Gin Gin Glu Glu Glu 210 215 220Ser His Gly Gly Arg Lys Gin Gly Gin Gin Gin Gin Glu Glu Glu 210 215 220

Gly Gly Ser Val Leu Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser 225 230 235 240Gly Gly Ser Val Leu Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser 225 230 235 240

Phe Asn Thr Asn Glu Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp 245 250 255Phe Asn Thr Asn Glu Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp 245 250 255

Glu Arg Lys Gin lie Val Thr Val Glu Gly Gly Leu Ser Val lie Ser 260 265 270 14 201144438Glu Arg Lys Gin lie Val Thr Val Glu Gly Gly Leu Ser Val lie Ser 260 265 270 14 201144438

Pro Lys Arg Gin Glu Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu 275 280 285Pro Lys Arg Gin Glu Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu 275 280 285

Tyr Glu Gin Thr Pro Ser Tyr Pro Pro Arg Arg Pro lie His Gly Lys 290 295 300Tyr Glu Gin Thr Pro Ser Tyr Pro Pro Arg Arg Pro lie His Gly Lys 290 295 300

His Glu Asp Asp Glu Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro 305 310 315 320His Glu Asp Asp Glu Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro 305 310 315 320

Asp His Pro Pro Gin Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg 325 330 335Asp His Pro Pro Gin Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg 325 330 335

Gly Arg Gly Cys Gin Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr 340 345 350Gly Arg Gly Cys Gin Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr 340 345 350

Met Lys Leu His Glu Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr 355 360 365Met Lys Leu His Glu Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr 355 360 365

Asn Pro Lys Ala Gly Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro 370 375 380Asn Pro Lys Ala Gly Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro 370 375 380

Ala Leu Arg Gin Phe Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr ArgAla Leu Arg Gin Phe Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg

385 390 395 400385 390 395 400

Asn Gly lie Tyr Ser Pro His Trp Asn Leu Asn Ala Asn Ser Val lie 405 410 415Asn Gly lie Tyr Ser Pro His Trp Asn Leu Asn Ala Asn Ser Val lie 405 410 415

Tyr Val Thr Arg Gly Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly 420 425 430Tyr Val Thr Arg Gly Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly 420 425 430

Asn Ala Val Phe Asp Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val 435 440 445Asn Ala Val Phe Asp Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val 435 440 445

Pro Gin Asn Phe Val Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu 450 455 460Pro Gin Asn Phe Val Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu 450 455 460

Tyr Val Val Phe Lys Thr His His Asn Ala Val Ser Ser Tyr lie Lys 465 470 475 480Tyr Val Val Phe Lys Thr His His Asn Ala Val Ser Ser Tyr lie Lys 465 470 475 480

Asp Val Phe Arg Ala lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn 485 490 495Asp Val Phe Arg Ala lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn 485 490 495

Leu Gly Gin Ser Gin Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly 500 505 510Leu Gly Gin Ser Gin Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly 500 505 510

Pro Leu Val Asn Pro 515 <210〉 7 <211> 1701 <212〉 DNA <213〉 Glycine max <220〉 <221> CDS <222〉（1)..(1698) <400> 7 48 m atg ggg aag ccc ttc ttc act etc tet ett tet tcc ett tgc ttg eta 15 201144438Pro Leu Val Asn Pro 515 <210> 7 <211> 1701 <212> DNA <213> Glycine max <220> <221> CDS <222> (1).. (1698) <400> 7 48 m atg ggg aag ccc ttc ttc act etc tet ett tet tcc ett tgc ttg eta 15 201144438

Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 etc ttg teg agt gca tgc ttt get att acc tcc age aag ttc aac gag 96Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 etc ttg teg agt gca tgc ttt get att acc tcc age aag ttc aac gag 96

Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30 tgc caa etc aac aac etc aac geg ttg gaa ccc gac cac ege gtt gag 144Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ly Lys Phe Asn Glu 20 25 30 tgc caa etc aac aac etc aac geg ttg gaa ccc gac cac ege gtt gag 144

Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 tcc gaa ggt ggt ett att gaa aca tgg aac tet caa cac cct gag ctg 192Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 tcc gaa ggt ggt ett att gaa aca tgg aac tet caa cac cct gag ctg 192

Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 240Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 240

caa tgc gcc ggt gtc act gtt tcc aaa ege acc etc aac ege aac ggc Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 etc cac ttg cca tet tac tea cct tat ccc caa atg ate att gtc gtt 288Caa tgc gcc ggt gtc act gtt tcc aaa ege acc etc aac ege aac ggc Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 etc cac ttg cca tet tac tea cct tat ccc caa atg ate att Gtc gtt 288

Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met lie He Val Val 85 90 95 caa ggg aag gga gca att gga ttt gca ttt ccg gga tgt cct gag aeg 336Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met lie He Val Val 85 90 95 caa ggg aag gga gca att gga ttt gca ttt ccg gga tgt cct gag aeg 336

Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110 384 432 ttt gag aag cca caa caa caa tea acc aga aga ggc tea agg teg cag Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 cag caa eta caa gac agt cac cag aag att cgt cac ttc aat gaa gga Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 gac gta eta gtg att cct cct ggt gtt cct tac tgg acc tat aac act 480Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110 384 432 ttt gag aag cca caa caa caa tea acc aga aga ggc tea agg teg cag Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 cag caa eta caa gac agt cac cag aag att cgt cac ttc aat gaa gga Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 gac gta eta gtg att cct cct ggt gtt Cct tac tgg acc tat aac act 480

Asp Val Leu Val He Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 ggc gat gaa cca gtt gtt gcc ate agt ett ett gac acc tcc aac ttc 528Asp Val Leu Val He Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 ggc gat gaa cca gtt gtt gcc ate agt ett ett gac acc tcc aac ttc 528

Gly Asp Glu Pro Val Val Ala lie Ser Leu Leu Asp Thr Ser Asn Phe 165 170 175Gly Asp Glu Pro Val Val Ala lie Ser Leu Leu Asp Thr Ser Asn Phe 165 170 175

[SI aac aat cag ett gat caa aac ccc aga gta tit tac ett get ggg aac 576 16 624201144438[SI aac aat cag ett gat caa aac ccc aga gta tit tac ett get ggg aac 576 16 624201144438

Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 cca gat ata gag cac cca gag acc atg caa caa cag cag cag cag cag Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Gin 195 200 205 cgc gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag Arg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 210 215 220 672 gtg gtg tat ccg aag gtg gtg tat ccg aag cag cgc gtg gtg tat ccg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Gin Arg Val Val Tyr Pro 225 230 235 240 720Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 cca gat ata gag cac cca gag acc atg caa caa cag cag cag cag cag cag Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Gin 195 200 205 cgc gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag Arg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 210 215 220 672 gtg gtg tat ccg aag gtg gtg tat ccg aag Cag cgc gtg gtg tat ccg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Gin Arg Val Val Tyr Pro 225 230 235 240 720

aag gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 245 250 255 768 gtg gtg tat ccg aag cag caa cag gag gaa gaa ggt ggc agt gtg etc Val Val Tyr Pro Lys Gin Gin Gin Glu Glu Glu Gly Gly Ser Val Leu 260 265 270 816 agt ggc ttc age aaa cat ttc tta gca caa tcc ttc aac acc aac gag Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser Phe Asn Thr Asn Glu 275 280 285 864Aag gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 245 250 255 768 gtg gtg tat ccg aag cag caa cag gag gaa gaa ggt ggc agt Gtg et Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Asn Thr Asn Glu 275 280 285 864

gac aca get gag aaa ett egg tet cca gat gac gaa agg aag cag ate Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp Glu Arg Lys Gin lie 290 295 300 912 gtg aca gtg gag gga ggc etc age gtt ate age ccc aag tgg caa gaa Val Thr Val Glu Gly Gly Leu Ser Val lie Ser Pro Lys Trp Gin Glu 305 310 315 320 caa gaa gac gaa gac gaa gat gaa gac gaa gaa tat gaa caa act ccc Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu Tyr Glu Gin Thr Pro 325 330 335 tet tac cct cca ega ega cca age cat gga aag cat gaa gat eac gag Ser Tyr Pro Pro Arg Arg Pro Ser His Gly Lys His Glu Asp Asp Glu 340 345 350 960 1008 1056 gac gag gac gaa gaa gaa gat caa cct cgt cct gat cac cct cca cag 1104 m 17 201144438Gac aca get gag aaa ett egg tet cca gat gac gaa agg ag cag ate Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp Glu Arg Lys Gin lie 290 295 300 912 gtg aca gtg gag gga ggc etc age gtt ate age ccc aag tgg Caa gaa Val Thr Val Glu Gly Gly Leu Ser Val lie Ser Pro Lys Trp Gin Glu 305 310 315 320 caa gaa gac gaa gac gaa gat gaa gac gaa gaa tat gaa caa act ccc Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu Tyr Glu Gin Thr Pro 325 330 335 tet tac cct cca ega ega cca age cat gga aag cat gaa gat eac gag Ser Tyr Pro Pro Arg Arg Pro Ser His Gly Lys His Glu Asp Asp Glu 340 345 350 960 1008 1056 gac gag gac gaa gaa Gaa gat caa cct cgt cct gat cac cct cca cag 1104 m 17 201144438

Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro Asp His Pro Pro Gin 355 360 365 cga cca age agg ccc gaa caa caa gaa cca cgt gga aga gga tgt cag 1152Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro Asp His Pro Pro Gin 355 360 365 cga cca age agg ccc gaa caa caa gaa cca cgt gga aga gga tgt cag 1152

Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg Gly Arg Gly Cys Gin 370 375 380 act aga aat ggg gtt gag gaa aat att tgc acc atg aag ett cac gag 1200Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg Gly Arg Gly Cys Gin 370 375 380 act aga aat ggg gtt gag gaa aat att tgc acc atg aag ett cac gag 1200

Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr Met Lys Leu His Glu 385 390 395 400 aac att get ege cct tea cgt get gac ttc tac aac cct aaa get ggt 1248Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr Met Lys Leu His Glu 385 390 395 400 aac att get ege cct tea cgt get gac ttc tac aac cct aaa get ggt 1248

Asn He Ala Arg Pro Ser Arg Ala Asp Phe Tyr Asn Pro Lys Ala Gly 405 410 415 1296Asn He Ala Arg Pro Ser Arg Ala Asp Phe Tyr Asn Pro Lys Ala Gly 405 410 415 1296

ege att age acc etc aac agt etc acc etc cca gee etc ege caa ttc Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro Ala Leu Arg Gin Phe 420 425 430 gga etc agt gee caa tat gtt gtc etc tac agg aat gga att tac tet 1344Ege att age acc etc aac agt etc acc etc cca gee etc ege caa ttc Arg lie Ser Thr Leu Asn Ser Leu Thr Leu Pro Ala Leu Arg Gin Phe 420 425 430 gga etc agt gee caa tat gtt gtc etc tac agg aat gga att tac Tet 1344

Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg Asn Gly He Tyr Ser 435 440 445 cca cat tgg aac ttg aac geg aac agt gtg ate tat gtg act cga ggg 1392Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg Asn Gly He Tyr Ser 435 440 445 cca cat tgg aac ttg aac geg aac agt gtg ate tat gtg act cga ggg 1392

Pro His Trp Asn Leu Asn Ala Asn Ser Val lie Tyr Val Thr Arg Gly 450 455 460 1440 aaa gga aga gtt aga gtg gtg aac tgc caa ggg aat gca gtg ttc gac Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly Asn Ala Val Phe Asp 465 470 475 480 ggt gag eta agg agg gga caa ttg eta gtg gtg ccg cag aac ttt gtg 1488Pro His Trp Asn Leu Asn Ala Asn Ser Val lie Tyr Val Thr Arg Gly 450 455 460 1440 aaa gga aga gtt aga gtg gtg aac tgc caa ggg aat gca gtg ttc gac Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly Asn Ala Val Phe Asp 465 470 475 480 ggt gag eta agg agg gga caa ttg eta gtg gtg ccg cag aac ttt gtg 1488

Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val Pro Gin Asn Phe Val 485 490 495 gtg get gag caa ggg gga gaa caa gga ttg gaa tac gta gtg ttc aag 1536Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val Pro Gin Asn Phe Val 485 490 495 gtg get gag caa ggg gga gaa caa gga ttg gaa tac gta gtg ttc aag 1536

Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu Tyr Val Val Phe Lys 500 505 510 aca cac cac aac gee gtg age age tac att aag gat gtg ttt agg gca 1584Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu Tyr Val Val Phe Lys 500 505 510 aca cac cac aac gee gtg age age tac att aag gat gtg ttt agg gca 1584

Thr His His Asn Ala Val Ser Ser Tyr lie Lys Asp Val Phe Arg Ala 515 520 525 [S] ate cct teg gag gtt ett tee aat tet tac aac ett ggc cag agt caa 1632 18 201144438 lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn Leu Gly Gin Ser Gin 530 535 540 gtg cgt cag etc aag tat caa gga aac tcc ggc cct ttg gtc aac cca Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly Pro Leu Val Asn Pro 545 550 555 560 1680 cac cac cac cac cac cac taa His His His His His His 565 1701 <210〉 <211〉 <212〉 <213>Thr His His Asn Ala Val Ser Ser Tyr lie Lys Asp Val Phe Arg Ala 515 520 525 [S] ate cct teg gag gtt ett tee aat tet tac aac ett ggc cag agt caa 1632 18 201144438 lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn Leu Gly Gin Ser Gin 530 535 540 gtg cgt cag etc aag tat caa gga aac tcc ggc cct ttg gtc aac cca Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly Pro Leu Val Asn Pro 545 550 555 560 1680 cac cac cac Cac cac cac taa His His His His His His 565 1701 <210> <211> <212> <213>

<400〉 S 566 PRT Glycine max 8<400〉 S 566 PRT Glycine max 8

Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30 Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met He lie Val Val 85 90 95 Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110 Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 Gly Asp Glu Pro Val Val Ala lie Ser Leu Leu Asp Thr Ser Asn Phe 165 170 175 Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Gin 195 200 205 [S] 19 201144438Met Gly Lys Pro Phe Phe Thr Leu Ser Leu Ser Ser Leu Cys Leu Leu 15 10 15 Leu Leu Ser Ser Ala Cys Phe Ala lie Thr Ser Ser Lys Phe Asn Glu 20 25 30 Cys Gin Leu Asn Asn Leu Asn Ala Leu Glu Pro Asp His Arg Val Glu 35 40 45 Ser Glu Gly Gly Leu lie Glu Thr Trp Asn Ser Gin His Pro Glu Leu 50 55 60 Gin Cys Ala Gly Val Thr Val Ser Lys Arg Thr Leu Asn Arg Asn Gly 65 70 75 80 Leu His Leu Pro Ser Tyr Ser Pro Tyr Pro Gin Met He lie Val Val 85 90 95 Gin Gly Lys Gly Ala lie Gly Phe Ala Phe Pro Gly Cys Pro Glu Thr 100 105 110 Phe Glu Lys Pro Gin Gin Gin Ser Thr Arg Arg Gly Ser Arg Ser Gin 115 120 125 Gin Gin Leu Gin Asp Ser His Gin Lys lie Arg His Phe Asn Glu Gly 130 135 140 Asp Val Leu Val lie Pro Pro Gly Val Pro Tyr Trp Thr Tyr Asn Thr 145 150 155 160 Gly Asp Glu Pro Val Val Ala lie Ser Leu Leu Asp Thr Ser Asn Phe 165 170 175 Asn Asn Gin Leu Asp Gin Asn Pro Arg Val Phe Tyr Leu Ala Gly Asn 180 185 190 Pro Asp lie Glu His Pro Glu Thr Met Gin Gin Gin Gin Gin Gin Gin 195 200 205 [ S] 19 201144438

Arg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 210 215 220Arg Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 210 215 220

Val Val Tyr Pro Lys Val Val Tyr Pro Lys Gin Arg Val Val Tyr Pro 225 230 235 240Val Val Tyr Pro Lys Val Val Tyr Pro Lys Gin Arg Val Val Tyr Pro 225 230 235 240

Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 245 250 255Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys 245 250 255

Val Val Tyr Pro Lys Gin Gin Gin Glu Glu Glu Gly Gly Ser Val Leu 260 265 270Val Val Tyr Pro Lys Gin Gin Gin Glu Glu Glu Gly Gly Ser Val Leu 260 265 270

Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser Phe Asn Thr Asn Glu 275 ' 280 285Ser Gly Phe Ser Lys His Phe Leu Ala Gin Ser Phe Asn Thr Asn Glu 275 ' 280 285

Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp Glu Arg Lys Gin lie 290 295 300Asp Thr Ala Glu Lys Leu Arg Ser Pro Asp Asp Glu Arg Lys Gin lie 290 295 300

Val Thr Val Glu Gly Gly Leu Ser Val lie Ser Pro Lys Trp Gin Glu 305 310 315 320Val Thr Val Glu Gly Gly Leu Ser Val lie Ser Pro Lys Trp Gin Glu 305 310 315 320

Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu Tyr Glu Gin Thr ProGin Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu Tyr Glu Gin Thr Pro

325 330 335325 330 335

Ser Tyr Pro Pro Arg Arg Pro Ser His Gly Lys His Glu Asp Asp Glu 340 345 350Ser Tyr Pro Pro Arg Arg Pro Ser His Gly Lys His Glu Asp Asp Glu 340 345 350

Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro Asp His Pro Pro Gin 355 360 365Asp Glu Asp Glu Glu Glu Asp Gin Pro Arg Pro Asp His Pro Pro Gin 355 360 365

Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg Gly Arg Gly Cys Gin 370 375 380Arg Pro Ser Arg Pro Glu Gin Gin Glu Pro Arg Gly Arg Gly Cys Gin 370 375 380

Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr Met Lys Leu His Glu 385 390 395 400Thr Arg Asn Gly Val Glu Glu Asn lie Cys Thr Met Lys Leu His Glu 385 390 395 400

Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr Asn Pro Lys Ala Gly 405 410 415Asn lie Ala Arg Pro Ser Arg Ala Asp Phe Tyr Asn Pro Lys Ala Gly 405 410 415

Arg He Ser Thr Leu Asn Ser Leu Thr Leu Pro Ala Leu Arg Gin Phe 420 425 430Arg He Ser Thr Leu Asn Ser Leu Thr Leu Pro Ala Leu Arg Gin Phe 420 425 430

Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg Asn Gly lie Tyr Ser 435 440 445Gly Leu Ser Ala Gin Tyr Val Val Leu Tyr Arg Asn Gly lie Tyr Ser 435 440 445

Pro His Trp Asn Leu Asn Ala Asn Ser Val lie Tyr Val Thr Arg Gly 450 455 460Pro His Trp Asn Leu Asn Ala Asn Ser Val lie Tyr Val Thr Arg Gly 450 455 460

Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly Asn Ala Val Phe Asp 465 470 475 480Lys Gly Arg Val Arg Val Val Asn Cys Gin Gly Asn Ala Val Phe Asp 465 470 475 480

Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val Pro Gin Asn Phe Val 485 490 495Gly Glu Leu Arg Arg Gly Gin Leu Leu Val Val Pro Gin Asn Phe Val 485 490 495

Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu Tyr Val Val Phe Lys 500 505 510Val Ala Glu Gin Gly Gly Glu Gin Gly Leu Glu Tyr Val Val Phe Lys 500 505 510

Thr His His Asn Ala Val Ser Ser Tyr lie Lys Asp Val Phe Arg Ala 515 520 525 lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn Leu Gly Gin Ser Gin 530 535 540Thr His His Asn Ala Val Ser Ser Tyr lie Lys Asp Val Phe Arg Ala 515 520 525 lie Pro Ser Glu Val Leu Ser Asn Ser Tyr Asn Leu Gly Gin Ser Gin 530 535 540

Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly Pro Leu Val Asn Pro m 545 550 555 560 20 201144438 His His His His His His 565 <210> 9 <211> 1320 <212> DNA <213〉 Glycine max <220> <221> CDS <222〉（1)..(1317)Val Arg Gin Leu Lys Tyr Gin Gly Asn Ser Gly Pro Leu Val Asn Pro m 545 550 555 560 20 201144438 His His His His His His 565 <210> 9 <211> 1320 <212> DNA <213> Glycine Max <220><221> CDS <222〉 (1)..(1317)

<400〉 9 atg atg aga gtg egg ttt cct ttg ttg gtg ttg ctg gga act gtt ttc Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15 48 ctg gca tea gtt tgt gtc tea tta aag gtg aga gag gat gag aat aac Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30 96 cct ttc tac ttg aga age tet aac age ttc caa act etc ttt gag aac Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45 144 caa aac ggt ege att cgt etc etc cag aga ttc aac aaa ege tee cca Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60 192<400> 9 atg atg aga gtg egg ttt cct ttg ttg gtg ttg ctg gga act gtt ttc Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15 48 ctg gca tea gtt tgt gtc tea tta aag Gtg aga gag gat gag aat aac Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30 96 cct ttc tac ttg aga age tet aac age ttc caa act etc ttt gag aac Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45 144 caa aac ggt ege att cgt etc etc cag aga ttc aac aaa ege tee cca Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60 192

caa ett gag aac ett ega gac tac egg att gtc cag ttt cag tea aaa Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 65 70 75 80 ccc aac aca ate ett etc ccc cac cat get gac gee gat ttc ttc etc Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95 ttt gtc ett age ggg aga gee ata ett acc ttg gtg aac aac gac gac Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110 aga gac tee tac aac ett cac cct ggc gat gee cag aga ate cca get Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 240 288 336Caa ett gag aac ett ega gac tac egg att gtc cag ttt cag tea aaa Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 65 70 75 80 ccc aac aca ate ett etc ccc cac cat get gac gee gat ttc Ttc etc Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95 ttt gtc ett age ggg aga gee ata ett acc ttg gtg aac aac gac gac Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110 aga gac tee tac aac ett cac cct ggc gat gee cag aga ate cca get Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 240 288 336

l SI 21 384 432201144438 115 120 125 gga acc act tac tat ttg gtt aac cct cac gac cac cag aat etc aaa Gly Thr Thr Tyr Tyr Leu Val Asn Pro His Asp His Gin Asn Leu Lys 130 135 140 ata ate aaa ett gee ata ccc gtc aac aaa cct ggc aga tat gat gat lie lie Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160 ttc ttc tta tet age act caa gee caa cag tee tac ttg caa ggc ttc Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175 480 528l s. Gtc aac aaa cct ggc aga tat gat gat lie lie Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160 ttc ttc tta tet age act caa gee caa cag tee tac ttg caa ggc ttc Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175 480 528

age cat aat att eta gag acc tee ttc cat age gaa ttc gag gag ata Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190 576 aac agg gtt ttg ttt gga gag gaa gag gag cag agg cag caa gag gga Asn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Arg Gin Gin Glu Gly 195 200 205 624 gtg ate gtg gaa etc tea aag gaa caa att egg caa ctg age aga cgt Val lie Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220 672Age cat aat att eta gag acc tee ttc cat age gaa ttc gag gag ata Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190 576 aac agg gtt ttg ttt gga gag gaa gag gag cag agg cag caa Gag gga Asn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Arg Gin Gin Glu Gly 195 200 205 624 gtg ate gtg gaa etc tea aag gaa caa att egg caa ctg age aga cgt Val lie Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220 672

gee aaa tet agt tea agg aaa acc att tee tee gaa gat gaa cca ttc Ala Lys Ser Ser Ser Arg Lys Thr He Ser Ser Glu Asp Glu Pro Phe 225 230 235 240 720 aac ttg aga age ege aac ccc ate tat tee aac aac ttt gga aag ttc Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly Lys Phe 245 250 255 ttt gag ate acc cct gag aaa aac cca cag ett egg gac ttg gat ate Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu Asp lie 260 265 270 ttc etc agt tet gtg gat ate aac gaa gga get ett ett eta cca cac Phe Leu Ser Ser Val Asp lie Asn Glu Gly Ala Leu Leu Leu Pro His 275 280 285 ttc aat tea aag gee ata gtg ata eta gtg att aat gaa gga gat gca Phe Asn Ser Lys Ala He Val lie Leu Val He Asn Glu Gly Asp Ala 768 816 864 [S] 22 912 960201144438 290 295 300 aac att gaa ctt gtt ggc att aaa gaa caa caa cag aag cag aaa cag Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin Lys Gin 305 310 315 320 gaa gag gaa cct ttg gaa gtg caa agg tac aga get gaa ttg tet gaa Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu Ser Glu 325 330 335 gac gat gta ttt gta att cca gca get tat cca ttt gtc gtc aac get Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val Asn Ala 340 345 350 1008 1056Gee aaa tet agt tea agg aaa acc att tee tee gaa gat gaa cca ttc Ala Lys Ser Ser Ser Arg Lys Thr He Ser Ser Glu Asp Glu Pro Phe 225 230 235 240 720 aac ttg aga age ege aac ccc ate tat tee aac aac ttt Gga aag ttc Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly Lys Phe 245 250 255 ttt gag ate acc cct gag aaa aac cca cag ett egg gac ttg gat ate Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu Asp lie 260 265 270 ttc etc agt tet gtg gat ate aac gaa gga get ett ett eta cca cac Phe Leu Ser Ser Val Asp lie Asn Glu Gly Ala Leu Leu Leu Pro His 275 280 285 ttc aat tea aag gee ata gtg ata Eta gtg att aat gaa gga gat gca phe Asn Ser Lys Ala He Val lie Leu Val He Asn Glu Gly Asp Ala 768 816 864 [S] 22 912 960201144438 290 295 300 aac att gaa ctt gtt ggc att aaa gaa caa caa cag aag cag Aaa cag Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin Lys Gin 305 310 315 320 gaa gag gaa cct ttg gaa gtg caa agg tac aga get gaa ttg tet gaa Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu Ser Glu 325 330 335 gac gat gta ttt gta att cca gca get tat cca ttt gtc gtc aac get Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val Asn Ala 340 345 350 1008 1056

acc tea aac etc aat ttc ctt get ttt ggt ate aat get gag aac aac Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu Asn Asn 355 360 365 1104 cag agg aac ttc ctt gca ggc gag aaa gac aat gtg gta agg cag ata Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg Gin lie 370 375 380 1152 gaa aga caa gtg cag gag ctt geg ttc cct ggg tet gca caa gat gtt Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin Asp Val 385 390 395 400 1200 gag agg eta tta aag aag cag agg gaa tee tac ttt gtt gat get cag Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp Ala Gin 405 410 415 1248Acc tea aac etc aat ttc ctt get ttt ggt ate aat get gag aac aac Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu Asn Asn 355 360 365 1104 cag agg aac ttc ctt gca ggc gag aaa gac aat gtg gta agg Cag ata Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg Gin lie 370 375 380 1152 gaa aga caa gtg cag gag ctt geg ttc cct ggg tet gca caa gat gtt Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin Asp Val 385 390 395 400 1200 gag agg eta tta aag aag cag agg gaa tee tac ttt gtt gat get cag Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp Ala Gin 405 410 415 1248

cct cag cag aag gag gag ggg agt aag gga aga aag ggt cct ttt cct Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro Phe Pro 420 425 430 tea ate tta ggt get etc tac tga Ser lie Leu Gly Ala Leu Tyr 435 1296 1320 <210〉 10 <211> 439 <212〉 PRT <213〉 Glycine maxCct cag cag ag gag gag ggg agt aag gga aga aag ggt cct ttt cct Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro Phe Pro 420 425 430 tea ate tta ggt get etc tac tga Ser lie Leu Gly Ala Leu Tyr 435 1296 1320 <210> 10 <211> 439 <212> PRT <213> Glycine max

[SI 23 201144438 <400> 10[SI 23 201144438 <400> 10

Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15

Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30

Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45

Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60

Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 65 70 75 80Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 65 70 75 80

Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95

Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110

Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 115 120 125Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 115 120 125

Gly Thr Thr Tyr Tyr Leu Val Asn Pro His Asp His Gin Asn Leu Lys 130 135 140Gly Thr Thr Tyr Tyr Leu Val Asn Pro His Asp His Gin Asn Leu Lys 130 135 140

He lie Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160He lie Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160

Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175

Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu He 180 185 190Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu He 180 185 190

Asn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Arg Gin Gin Glu Gly 195 200 205Asn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Arg Gin Gin Glu Gly 195 200 205

Val lie Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220Val lie Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220

Ala Lys Ser Ser Ser Arg Lys Thr lie Ser Ser Glu Asp Glu Pro Phe 225 230 235 240Ala Lys Ser Ser Ser Arg Lys Thr lie Ser Ser Glu Asp Glu Pro Phe 225 230 235 240

Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly Lys Phe 245 250 255Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly Lys Phe 245 250 255

Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu Asp lie 260 265 270Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu Asp lie 260 265 270

Phe Leu Ser Ser Val Asp He Asn Glu Gly Ala Leu Leu Leu Pro His 275 280 285Phe Leu Ser Ser Val Asp He Asn Glu Gly Ala Leu Leu Leu Pro His 275 280 285

Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly Asp Ala 290 295 300Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly Asp Ala 290 295 300

Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin Lys Gin 305 310 315 320Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin Lys Gin 305 310 315 320

Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu Ser GluGlu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu Ser Glu

[SI 325 330 335 24 201144438[SI 325 330 335 24 201144438

Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val Asn Ala 340 345 350Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val Asn Ala 340 345 350

Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu Asn Asn 355 360 365Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu Asn Asn 355 360 365

Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg Gin lie 370 375 380Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg Gin lie 370 375 380

Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin Asp Val 385 390 395 400Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin Asp Val 385 390 395 400

Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp Ala Gin 405 410 415Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp Ala Gin 405 410 415

Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro Phe Pro 420 425 430Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro Phe Pro 420 425 430

Ser lie Leu Gly Ala Leu Tyr 435Ser lie Leu Gly Ala Leu Tyr 435

<210> 11 <211> 1446 <212> DNA <213> Glycine max <220〉 <221> CDS <222> (1)..(1443) <400> 11 atg atg aga gtg egg ttt cct ttg ttg gtg ttg ctg gga act gtt ttc 48<210> 11 <211> 1446 <212> DNA <213> Glycine max <220><221> CDS <222> (1)..(1443) <400> 11 atg atg aga Gtg egg ttt cct ttg ttg gtg ttg ctg gga act gtt ttc 48

Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15 ctg gca tea gtt tgt gtc tea tta aag gtg aga gag gat gag aat aac 96Met Met Arg Val Arg Phe Pro Leu Leu Val Leu Leu Gly Thr Val Phe 15 10 15 ctg gca tea gtt tgt gtc tea tta aag gtg aga gag gat gag aat aac 96

Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30 cct ttc tac ttg aga age tet aac age ttc caa act etc ttt gag aac 144Leu Ala Ser Val Cys Val Ser Leu Lys Val Arg Glu Asp Glu Asn Asn 20 25 30 cct ttc tac ttg aga age tet aac age ttc caa act etc ttt gag aac 144

Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45 caa aac ggt ege att cgt etc etc cag aga ttc aac aaa ege tcc cca 192Pro Phe Tyr Leu Arg Ser Ser Asn Ser Phe Gin Thr Leu Phe Glu Asn 35 40 45 caa aac ggt ege att cgt etc etc cag aga ttc aac aaa ege tcc cca 192

Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60 caa ett gag aac ett ega gac tac egg att gtc cag ttt cag tea aaa 240 [S]Gin Asn Gly Arg lie Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60 caa ett gag aac ett ega gac tac egg att gtc cag ttt cag tea aaa 240 [S]

Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 25 288201144438 65 70 75 80 ccc aac aca ate ett etc ccc cac cat get gac gee gat ttc ttc etc Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95 ttt gtc ett age ggg aga gee ata ett acc ttg gtg aac aac gac gac Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110 aga gac tee tac aac ett cac cct ggc gat gee cag aga ate cca get Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 115 120 125 336 384Gin Leu Glu Asn Leu Arg Asp Tyr Arg lie Val Gin Phe Gin Ser Lys 25 288201144438 65 70 75 80 ccc aac aca ate ett etc ccc cac cat get gac gee gat ttc ttc etc Pro Asn Thr lie Leu Leu Pro His His Ala Asp Ala Asp Phe Phe Leu 85 90 95 ttt gtc ett age ggg aga gee ata ett acc ttg gtg aac aac gac gac Phe Val Leu Ser Gly Arg Ala lie Leu Thr Leu Val Asn Asn Asp Asp 100 105 110 aga gac tee tac aac ett cac cct Ggc gat gee cag aga ate cca get Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg lie Pro Ala 115 120 125 336 384

gga acc act tac tat ttg gtt aac cct cac gac cac cag aat etc aaa Gly Thr Thr Tyr Tyr Leu Val Asn Pro His Asp His Gin Asn Leu Lys 130 135 140 432 ata ate aaa ett gee ata ccc gtc aac aaa cct ggc aga tat gat gat He lie Lys Leu Ala He Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160 ttc ttc tta tet age act caa gee caa cag tee tac ttg caa ggc ttc Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175 480 528 age cat aat att eta gag acc tee ttc cat age gaa ttc gag gag ata Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190 576Gga acc act tac tat ttg gtt aac cct cac gac cac cag aat etc aaa Gly Thr Thr Tyr Tyr Leu Val Asn Pro His Asp His Gin Asn Leu Lys 130 135 140 432 ata ate aaa ett gee ata ccc gtc aac aaa cct ggc aga tat Gat gat He lie Lys Leu Ala He Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 145 150 155 160 ttc ttc tta tet age act caa gee caa cag tee tac ttg caa ggc ttc Phe Phe Leu Ser Ser Thr Gin Ala Gin Gin Ser Tyr Leu Gin Gly Phe 165 170 175 480 528 age cat aat att eta gag acc tee ttc cat age gaa ttc gag gag ata Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190 576

624 aac agg gtt ttg ttt gga gag gaa gag gag cag agg cag caa gag gga624 aac agg gtt ttg ttt gga gag gaa gag gag cag agg cag caa gag gga

Asn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Arg Gin Gin Glu Gly 195 200 205 672 gtg ate gtg gaa etc tea aag gaa caa att egg caa ctg age aga cgtAsn Arg Val Leu Phe Gly Glu Glu Glu Glu Gin Gin Gin Glu Gly 195 200 205 672 gtg ate gtg gaa etc tea aag gaa caa att egg caa ctg age aga cgt

Val He Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220 720 gee aaa tet agt cag gtg gtg tat ccg aag gtg gtg tat ccg aag egeVal He Val Glu Leu Ser Lys Glu Gin lie Arg Gin Leu Ser Arg Arg 210 215 220 720 gee aaa tet agt cag gtg gtg tat ccg aag gtg gtg tat ccg aag ege

Ala Lys Ser Ser Gin Val Val Tyr Pro Lys Val Val Tyr Pro Lys Arg 225 230 235 240 gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag gtg [S]Ala Lys Ser Ser Gin Val Val Tyr Pro Lys Val Val Tyr Pro Lys Arg 225 230 235 240 gtg gtg tat ccg aag gtg gtg tat ccg aag gtg gtg tat ccg aag gtg [S]

Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val 26 768 816 201144438 245 250 255 gtg tat ccg aag gtg gtg tat ccg aaa acc att tcc tcc gaa gat gaa Val Tyr Pro Lys Val Val Tyr Pro Lys Thr lie Ser Ser Glu Asp Glu 260 265 270 cca ttc aac ttg aga age ege aac ccc ate tat tcc aac aac ttt gga Pro Phe Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly 275 280 285 aag ttc ttt gag ate acc cct gag aaa aac cca cag ett egg gac ttg Lys Phe Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu 290 295 300 864 912Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val 26 768 816 201144438 245 250 255 gtg tat ccg aag gtg gtg tat ccg aaa acc att tcc tcc gaa gat gaa Val Tyr Pro Lys Val Val Tyr Pro Lys Thr lie Ser Ser Glu Asp Glu 260 265 270 cca ttc aac ttg aga age ege aac ccc ate tat tcc aac aac ttt gga Pro Phe Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly 275 280 285 aag ttc ttt gag ate acc cct Gag aaa aac cca cag ett egg gac ttg Lys Phe Phe Glu lie Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu 290 295 300 864 912

gat ate ttc etc agt tet gig gat ate aac gaa gga get ett ett eta Asp lie Phe Leu Ser Ser Val Asp He Asn Glu Gly Ala Leu Leu Leu 305 310 315 320 960 cca cac ttc aat tea aag gee ata gtg ata eta gtg att aat gaa gga Pro His Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly 325 330 335 1008 gat gca aac att gaa ett gtt ggc att aaa gaa caa caa cag aag cag Asp Ala Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin 340 345 350 1056 aaa cag gaa gag gaa cct ttg gaa gtg caa aeg tac aga get gaa ttg Lys Gin Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu 355 360 365 1104Gat ate ttc etc agt tet gig gat ate aac gaa gga get ett ett eta Asp lie Phe Leu Ser Ser Val Asp He Asn Glu Gly Ala Leu Leu Leu 305 310 315 320 960 cca cac ttc aat tea aag gee ata gtg ata eta gtg att Aat gaa gga Pro His Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly 325 330 335 1008 gat gca aac att gaa ett gtt ggc att aaa gaa caa caa cag aag cag Asp Ala Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin 340 345 350 1056 aaa cag gaa gag gaa cct ttg gaa gtg caa aeg tac aga get gaa ttg Lys Gin Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu 355 360 365 1104

tet gaa gac gat gta ttt gta att cca gca get tat cca ttt gtc gtc Ser Glu Asp Asp Val Phe Val He Pro Ala Ala Tyr Pro Phe Val Val 370 375 380 aac get acc tea aac etc aat ttc ett get ttt ggt ate aat get gag Asn Ala Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu 385 390 395 400 aac aac cag agg aac ttc ett gca ggc gag aaa gac aat gtg gta agg Asn Asn Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg 405 410 415 cag ata gaa aga caa gtg cag gag ett geg ttc cct ggg tet gca caa Gin lie Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin 1152 1200 1248 [S] 27 1296 201144438 420 425 430 gat gtt gag agg eta tta aag aag cag agg gaa tee tac ttt gtt gat 1344Tet gaa gac gat gta ttt gta att cca gca get tat cca ttt gtc gtc Ser Glu Asp Asp Val Phe Val He Pro Ala Ala Tyr Pro Phe Val Val 370 375 380 aac get acc tea aac etc aat ttc ett get ttt ggt ate aat get Gag Asn Ala Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu 385 390 395 400 aac aac cag agg aac ttc ett gca ggc gag aaa gac aat gtg gta agg Asn Asn Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg 405 410 415 cag ata gaa aga caa gtg cag gag ett geg ttc cct ggg tet gca caa Gin lie Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin 1152 1200 1248 [S] 27 1296 201144438 420 425 430 Gat gtt gag agg eta tta aag aag cag agg gaa tee tac ttt gtt gat 1344

Asp Val Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp 435 440 445 get cag cct cag cag aag gag gag ggg agt aag gga aga aag ggt cct 1392Asp Val Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp 435 440 445 get cag cct cag cag aag gag gag ggg agt aag gga aga aag ggt cct 1392

Ala Gin Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro 450 455 460 ttt cct tea ate tta ggt get etc tac etc gag cac cac cac cac cac 1440Ala Gin Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro 450 455 460 ttt cct tea ate tta ggt get etc tac etc gag cac cac cac cac cac 1440

Phe Pro Ser lie Leu Gly Ala Leu Tyr Leu Glu His His His His His 465 470 475 480 cac tga 1446Phe Pro Ser lie Leu Gly Ala Leu Tyr Leu Glu His His His His His 465 470 475 480 cac tga 1446

<210〉 12 <211> 481 <212> PRT <213> Glycine max <400> 12<210> 12 <211> 481 <212> PRT <213> Glycine max <400> 12

Gin Asn Gly Arg He Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60Gin Asn Gly Arg He Arg Leu Leu Gin Arg Phe Asn Lys Arg Ser Pro 50 55 60

Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg He Pro Ala 115 120 125Arg Asp Ser Tyr Asn Leu His Pro Gly Asp Ala Gin Arg He Pro Ala 115 120 125

[SI lie He Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 28 201144438 145 150 155 160[SI lie He Lys Leu Ala lie Pro Val Asn Lys Pro Gly Arg Tyr Asp Asp 28 201144438 145 150 155 160

Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190Ser His Asn lie Leu Glu Thr Ser Phe His Ser Glu Phe Glu Glu lie 180 185 190

Ala Lys Ser Ser Gin Val Val Tyr Pr-o Lys Val Val Tyr Pro Lys Arg 225 230 235 240Ala Lys Ser Ser Gin Val Val Tyr Pr-o Lys Val Val Tyr Pro Lys Arg 225 230 235 240

Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val 245 250 255Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val Val Tyr Pro Lys Val 245 250 255

Val Tyr Pro Lys Val Val Tyr Pro Lys Thr lie Ser Ser Glu Asp Glu 260 265 270Val Tyr Pro Lys Val Val Tyr Pro Lys Thr lie Ser Ser Glu Asp Glu 260 265 270

Pro Phe Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly 275 280 285Pro Phe Asn Leu Arg Ser Arg Asn Pro lie Tyr Ser Asn Asn Phe Gly 275 280 285

Lys Phe Phe Glu He Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu 290 295 300Lys Phe Phe Glu He Thr Pro Glu Lys Asn Pro Gin Leu Arg Asp Leu 290 295 300

Asp He Phe Leu Ser Ser Val Asp lie Asn Glu Gly Ala Leu Leu Leu 305 310 315 320Asp He Phe Leu Ser Ser Val Asp lie Asn Glu Gly Ala Leu Leu Leu 305 310 315 320

Pro His Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly 325 330 335Pro His Phe Asn Ser Lys Ala lie Val lie Leu Val lie Asn Glu Gly 325 330 335

Asp Ala Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin 340 345 350Asp Ala Asn lie Glu Leu Val Gly lie Lys Glu Gin Gin Gin Lys Gin 340 345 350

Lys Gin Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu 355 360 365Lys Gin Glu Glu Glu Pro Leu Glu Val Gin Arg Tyr Arg Ala Glu Leu 355 360 365

Ser Glu Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val 370 375 380Ser Glu Asp Asp Val Phe Val lie Pro Ala Ala Tyr Pro Phe Val Val 370 375 380

Asn Ala Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu 385 390 395 400Asn Ala Thr Ser Asn Leu Asn Phe Leu Ala Phe Gly lie Asn Ala Glu 385 390 395 400

Asn Asn Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg 405 410 415Asn Asn Gin Arg Asn Phe Leu Ala Gly Glu Lys Asp Asn Val Val Arg 405 410 415

Gin lie Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin 420 425 430Gin lie Glu Arg Gin Val Gin Glu Leu Ala Phe Pro Gly Ser Ala Gin 420 425 430

Asp Val Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp 435 440 445Asp Val Glu Arg Leu Leu Lys Lys Gin Arg Glu Ser Tyr Phe Val Asp 435 440 445

Ala Gin Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro 450 455 460Ala Gin Pro Gin Gin Lys Glu Glu Gly Ser Lys Gly Arg Lys Gly Pro 450 455 460

Phe Pro Ser lie Leu Gly Ala Leu Tyr Leu Glu His His His His His 465 470 475 480Phe Pro Ser lie Leu Gly Ala Leu Tyr Leu Glu His His His His His 465 470 475 480

His m 29 201144438 <210〉 13 <211〉 <212〉 <213> 23 DNA 人工序列 <220〉 <223> Gyl-23A <400〉 13 agtcatcacc atggccaagc tag <210> 14 <211〉 <212> <213> 29 DNA 人工序列 # <220〉 <223> Gyl-30B <400> 14 cccgagctcc taagccacag ctctcttctHis m 29 201144438 <210> 13 <211> <212><213> 23 DNA artificial sequence <220> <223> Gyl-23A <400> 13 agtcatcacc atggccaagc tag <210> 14 &lt ;211> <212><213> 29 DNA artificial sequence # <220〉 <223> Gyl-30B <400> 14 cccgagctcc taagccacag ctctcttct

<210〉 15 <211> <212> <213> 26 DNA 人工序列 <220> <223> • Gy5NcoI-26A <400〉 15 tcgtccatgg ggaagccctt cttcac <210> 16 <211> <212〉 <213> 28 DNA 人工序列 <220〉 <223> Gy5SacI-30B <400〉 16 ggcggagctc ttatgggttg accaaagg 201144438 <210〉 17 <211> 25 <212〉 DNA <213〉人工序列 <220> <223> Beta-25A <400> 17 atgatgagag tgcggtttcc tttgt <210> 18 <211> 26 <212> DNA <213〉人工序列<210> 15 <211><212><213> 26 DNA artificial sequence <220><223> • Gy5NcoI-26A <400> 15 tcgtccatgg ggaagccctt cttcac <210> 16 <211><212><213> 28 DNA artificial sequence <220> <223> Gy5SacI-30B <400> 16 ggcggagctc ttatgggttg accaaagg 201144438 <210> 17 <211> 25 <212> DNA <213 〉Artifical sequence<220><223> Beta-25A <400> 17 atgatgagag tgcggtttcc tttgt <210> 18 <211> 26 <212> DNA <213>

<220> <223> Beta-26B <400> 18 tcagtagaga gcacctaaga ttgaag <210> 19 <211〉 27 <212〉 DNA <213>人工序列 <220〉<220><223> Beta-26B <400> 18 tcagtagaga gcacctaaga ttgaag <210> 19 <211> 27 <212> DNA <213> Artificial sequence <220>

<223> 7S-SinaI<223> 7S-SinaI

<400> 19 cccgggatga tgagagtgcg gtttcct <210> 20 <211> 27 <212> DNA <213〉人工序列 <220> <223> 7S-SacI <400〉 20 gagctctcag tggtggtggt ggtggtg<400> 19 cccgggatga tgagagtgcg gtttcct <210> 20 <211> 27 <212> DNA <213> artificial sequence <220><223> 7S-SacI <400> 20 gagctctcag tggtggtggt ggtggtg

Claims

201144438 七、申請專利範圍： 1. -種用於降血脂的重組大豆儲藏性蛋白，其係於大丑儲藏性蛋白的非核心'構造區域予以插人或置換成至少— vvyp活性胜肽，其中該大豆儲藏性蛋白包括選自列蛋白質所構成的群組：p_伴大豆料白β次單元、大豆球蛋白Gy5以及大豆球蛋白Gyl。 2. 如申請專利範圍帛1項所述之用於降血脂的重組大豆，藏性蛋白’其中該大豆儲藏性蛋白係β-伴大豆球蛋白次單元’並且具有一如SEQ m Ν〇: 1〇所示序列，且於其介於第229至230位置之間的胺基酸序列置換成ννγρ活性胜肽。 ' 3·如申請專利範圍第1項所述之用於降血脂的重組大豆儲藏性蛋白，其中該大豆儲藏性蛋白係一大豆球蛋白 Gy5並且具有一如SEQ ID NO: 6所示序列，且於其至少一選自於由下列者所構成的群組之胺基酸序列，置換成 VVYP活性胜肽：介於第2〇3至2〇7位置之間、第至 212位置之間以及第213至218位置之間的胺基酸序列。 4 ·如申凊專利範圍第1項所述之用於降血脂的重組大豆儲藏性蛋白，其中該大豆儲藏性蛋白係大豆球蛋白Gyi，並且具有一如SEQ ID NO: 2所示序列，且於其至少一選自於由下列者所構成的群組之胺基酸序列，置換成ννγρ活性胜肽：介於第119至126位置之間、第286至290位置之間以及第292至300位置之間的胺基酸序列。 5.如申請專利範園第1至4項任一項所述之用於降血脂的重組大豆儲藏性蛋白，其中該VVYP活性胜肽包含纈[” 1 201144438 胺酸-纈胺酸-酪胺酸-脯胺酸（valine_valine_tyr〇sinprc)line， VVYP)序列以及至少一酵素辨識切點。 6. 如申請專利範圍第5項所述之用於降血脂的重組大丑儲藏性蛋白，其中該酵素辨識切點係為消化道酵素辨識切點。 7. 如申請專利範圍第6項所述之用於降血脂的重組大豆儲藏性蛋白，其中該至少一消化道酵素辨識切點係選自於下列所構成的群組：胰蛋白酶（trypsin)的辨識切點、羧狀酶B (Carb〇Xypeptidase B)的辨識切點、胰凝乳蛋白酶 (chymotrpsin)的辨識切點或者它們的組合。 8·如申4專利範圍第1至4項任一項所述之用於降血脂的重組大豆儲藏性蛋白，其中該ννγρ活性胜肽係包含如下列公式所示的胺基酸序列： Xaa,-(Val-Val-Tyr-Pro-Xaa2)n > 其中Xaa,、Xaaz分別與相鄰的纈胺酸（Val)以及脯胺酸（Pro)形成酵素辨識切點；以及^至少為j。 9·如申凊專利範圍第8項所述之用於降企脂的重組大豆儲藏性蛋白，其中η為1。 10. 如申請專利範園第9項所述之用於降血脂的重組大豆儲藏性蛋白，其中Xaaig精胺酸（Arg);以及以心為離胺酸（Lys)。 11. 如申請專利範圍第1項所述之用於降血脂的重組大豆儲藏性蛋白，其具有一選自於由下列者所構成之群組的胺基酸序列：SEQIDNO: 12、SEQIDN0: 8 以及 SEQmN〇: 201144438 12. —種重組載體，其包含一控制序列以及一與該控制序列可操作的相連接之重組儲藏性蛋白基因序列，該重組儲藏性蛋白基因序列編碼一如申請專利範圍第i至丨1項任一項所述之用於降血脂的重組大豆儲藏性蛋白。 13. 如申請專利範圍第12項所述之重組載體，其中該控制序列係花椰菜嵌紋病毒35S啟動子或大豆16_kD種_^ 成熟蛋白GmPM9基因之啟動子。 14 ·如申請專利範圍第12項所述之重組載體，其係選 # 自於下列者所構成的群組··寄存於財團法人食品工業研究發展所生物資源保存暨研究中心之pET21b Gy5nsp_v2535，其寄存編號為BCRC 940584 ;寄存於財團法人食品工業研究發展所生物資源保存暨研究中心之pET21_7Sbsnp V57，其寄存編號為 BCRC 940585; pCa-p-V57; pGm-p-V57 以及 pCAMBCAP，Gy5-l〇〇 15. —種基因轉殖植物細胞，其係具有一重組儲藏性蛋白基因序列’該重組儲藏性蛋白基因序列編碼一如申請專 • 利範圍第1至11項任一項所述之用於降血脂的重組大豆儲藏性蛋白。 16. 如申請專利範圍第15項所述之基因轉殖植物細胞，其係屬於大豆、稻米或玉米細胞。 17. —種製備用於降血脂的重組大豆儲藏性蛋白之方法’其包括：將一種如申請專利範圍第12至14項中任一項所述之重組載體導入一植物細胞内；以及令該植物細胞生長’藉以表現出用於降血脂的重組大 3 201144438 豆儲藏性蛋白。201144438 VII. Patent application scope: 1. A recombinant soybean storage protein for lowering blood fat, which is inserted or replaced into at least a vvyp active peptide in the non-core 'structural region of the large ugly storage protein, wherein The soy storage protein comprises a group consisting of a column of proteins: p_conglyzed white beta subunit, glycinin Gy5, and glycinin Gyl. 2. The recombinant protein for use in lowering blood fat as described in claim 1 of the patent application, wherein the soy storage protein is a β-conglycinin subunit and has a SEQ m Ν〇: 1 The sequence shown is 置换, and the amino acid sequence between its positions 229 to 230 is replaced with a ννγρ-active peptide. 3. The recombinant soybean storage protein for lowering blood fat according to claim 1, wherein the soybean storage protein is a glycinin Gy5 and has a sequence as shown in SEQ ID NO: 6, and Substituting at least one amino acid sequence selected from the group consisting of: VVYP active peptide: between positions 2〇3 to 2〇7, between positions 212 and The amino acid sequence between positions 213 to 218. 4. The recombinant soybean storage protein for lowering blood fat according to claim 1, wherein the soybean storage protein is glycinin Gyi, and has a sequence as shown in SEQ ID NO: 2, and The at least one amino acid sequence selected from the group consisting of: ννγρ active peptide: between positions 119 to 126, between positions 286 to 290, and from 292 to 300 The amino acid sequence between the positions. 5. The recombinant soybean storage protein for lowering blood fat according to any one of claims 1 to 4, wherein the VVYP active peptide comprises 缬[" 1 201144438 Aminic acid-proline-tyramine The acid-proline (valine_valine_tyr〇sinprc) line, VVYP) sequence and at least one enzyme recognizes the cut point. 6. The reconstituted large ugly storage protein for lowering blood fat as described in claim 5, wherein the enzyme is identified The cut point is a digestive enzyme identification cut point. 7. The recombinant soybean storage protein for reducing blood fat according to claim 6, wherein the at least one digestive enzyme identification cut point is selected from the group consisting of the following Group: trypsin identification cut point, identification point of Carb〇Xypeptidase B, identification cut point of chymotpsin or a combination thereof. The recombinant soybean storage protein for reducing blood fat according to any one of the items 4, wherein the ννγρ-active peptide comprises an amino acid sequence as shown by the following formula: Xaa,-(Val-Val-Tyr-Pro- Xaa2)n > where X Aa, Xaaz and the adjacent proline (Val) and proline (Pro) form an enzyme identification cut-point; and ^ at least j. 9 · as described in claim 8 of the patent scope for the enterprise a recombinant soy storage protein of a lipid, wherein η is 1. 10. A recombinant soybean storage protein for lowering blood fat as described in claim 9 of the patent application, wherein Xaaig arginine (Arg); 11. A lysine-reserving protein for lowering blood lipids according to claim 1, which has an amino acid sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 8 and SEQmN〇: 201144438 12. A recombinant vector comprising a control sequence and a recombinant storage protein gene sequence operably linked to the control sequence, the recombinant storage protein gene sequence The recombinant soybean storage protein for use in lowering blood lipids according to any one of claims 1 to 3, wherein the recombinant carrier is a broccoli as described in claim 12, wherein the control sequence is broccoli. Mosaic virus 35S promoter or Soybean 16_kD species _^ Promoter of mature protein GmPM9 gene. 14 · Recombinant vector as described in claim 12 of the patent application, which is selected from the group consisting of: · deposited in the food industry research pET21b Gy5nsp_v2535, the Bioresource Conservation and Research Center of the Institute for Development, with the registration number BCRC 940584; pET21_7Sbsnp V57 deposited in the Bioresource Conservation and Research Center of the Food Industry Research and Development Institute, with the registration number BCRC 940585; pCa-p-V57 ; pGm-p-V57 and pCAMBCAP, Gy5-l〇〇15. A gene transfer plant cell, which has a recombinant storage protein gene sequence. The recombinant storage protein gene sequence encodes a patent application scope. The recombinant soybean storage protein for lowering blood fat according to any one of items 1 to 11. 16. The gene transfer plant cell of claim 15, which belongs to soybean, rice or corn cells. 17. A method of preparing a recombinant soybean storage protein for lowering blood fat, comprising: introducing a recombinant vector according to any one of claims 12 to 14 into a plant cell; Plant cell growth 'to show the recombinant large 3 201144438 bean storage protein for lowering blood fat.

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