JP2022029767A - Method for freely controlling heavy metal transport, and plant with controlled absorption of cadmium and manganese - Google Patents
Method for freely controlling heavy metal transport, and plant with controlled absorption of cadmium and manganese Download PDFInfo
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Abstract
Description
本発明は、例えばカドミウム及びマンガンの重金属輸送に関連する遺伝子及びタンパク質の変異を利用してその吸収を自在に制御する方法、並びにカドミウム及びマンガン吸収が制御された植物に関する。 The present invention relates to, for example, a method for freely controlling the absorption of cadmium and manganese by utilizing mutations in genes and proteins related to heavy metal transport, and plants in which the absorption of cadmium and manganese is controlled.
食品を通じて一定量を超えるカドミウムを長年にわたり摂取し続けると、腎機能障害等の健康への悪影響を及ぼす可能性がある。特にコメは日本を含め東アジアや東南アジアの国々において主食であるため、カドミウムの主要な摂取源になっている。また、ダイズやコムギ等の他の作物や野菜等も少なからずカドミウムを摂取しているため、カドミウムによる健康被害リスクを回避する上で、作物由来のカドミウム濃度を減らす必要がある。 Ingestion of more than a certain amount of cadmium through food for many years may have adverse health effects such as renal dysfunction. In particular, rice is a staple food in East Asian and Southeast Asian countries including Japan, so it is a major source of cadmium. In addition, since other crops such as soybeans and wheat and vegetables also ingest not a little cadmium, it is necessary to reduce the concentration of cadmium derived from crops in order to avoid the risk of health damage caused by cadmium.
従来において、コメのカドミウム濃度の低減方法として、以下の方法が知られている:
(1) 客土による土壌の浄化-農用地土壌汚染防止法に基づき、汚染米(0.4ppm以上)を産出する水田に対して現在も適用;
(2) 湛水管理を中心とした営農技術によるカドミウム吸収抑制-汚染の恐れがある水田に対して、特に出穂前後3週間の湛水管理をベースに広い地域で適用。現在の主要な対策になっている;
(3) カドミウム低吸収品種による吸収抑制-カドミウム低吸収遺伝子(イネNramp5(OsNramp5)の機能欠損型)を導入した品種の育成と導入(特許文献1)-農水省が発表したカドミウム低減指針の中で、今後の主要な対策として位置づけ。
Conventionally, the following methods are known as methods for reducing the cadmium concentration of rice:
(1) Purification of soil with soil dressing-Based on the Agricultural Land Soil Contamination Prevention Law, it is still applied to paddy fields that produce contaminated rice (0.4 ppm or more);
(2) Suppression of cadmium absorption by farming technology centered on flood management-Applicable to paddy fields where there is a risk of contamination, especially in a wide area based on flood management for 3 weeks before and after heading. It is currently the main measure;
(3) Suppression of absorption by low-absorption varieties of cadmium-Cultivation and introduction of varieties into which a low-absorption cadmium gene (function-deficient type of rice Nramp5 (OsNramp5)) has been introduced (Patent Document 1) -In the guidelines for reducing cadmium published by the Ministry of Agriculture, Forestry and Fisheries. , Positioned as a major measure in the future.
しかしながら、これらの方法には以下の問題点がある:
(1) 客土による土壌の浄化では、コストが高く(500万円/10a)、また客土材の確保が困難である;
(2) 湛水管理では、管理が面倒であり、また用水の確保が困難である。さらに、効果が天候や土壌条件によって変動するため、カドミウムの低減効果が安定しない;
(3) カドミウム低吸収品種では、OsNramp5の機能欠損型遺伝子がカドミウムだけでなくマンガン吸収も大幅に抑制するため、特にマンガン濃度の低い土壌では、当該カドミウム低吸収品種は、ごま葉枯病に罹病しやすく、玄米収量や品質が低下する。
However, these methods have the following problems:
(1) Purification of soil with soil dressing is expensive (5 million yen / 10a), and it is difficult to secure soil dressing materials;
(2) In flood management, management is troublesome and it is difficult to secure water. Furthermore, the effect of reducing cadmium is not stable because the effect varies depending on the weather and soil conditions;
(3) In low-absorption varieties of cadmium, the function-deficient gene of OsNramp5 significantly suppresses not only cadmium but also manganese absorption. It is easy to do, and the yield and quality of brown rice are reduced.
本発明は、上述の実情に鑑み、カドミウム及びマンガン吸収が制御されたイネ等の植物を提供することを目的とする。 In view of the above circumstances, it is an object of the present invention to provide a plant such as rice whose absorption of cadmium and manganese is controlled.
上記課題を解決するため鋭意研究を行った結果、OsNramp5タンパク質において特定の部位にアミノ酸置換を有するイネの変異体や酵母によれば、カドミウム及びマンガン吸収を自在に制御することができることを見出し、本発明を完成するに至った。 As a result of diligent research to solve the above problems, we found that the absorption of cadmium and manganese can be freely controlled by rice variants and yeasts that have amino acid substitutions at specific sites in the OsNramp5 protein. The invention was completed.
すなわち、本発明は、以下を包含する。
(1)配列番号12に示されるアミノ酸配列において、第6番目のグルタミン残基が他のアミノ酸に置換されたアミノ酸配列を含むタンパク質。
(2)以下の(i)又は(ii)のタンパク質である、(1)記載のタンパク質。
(i)配列番号2に示されるアミノ酸配列において、第337番目のグルタミン残基が他のアミノ酸に置換されたアミノ酸配列を含むタンパク質;
(ii)(i)記載のタンパク質のアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含み、且つ第337番目の前記他のアミノ酸を保持するタンパク質であって、イネにおいてカドミウム及びマンガンを輸送するトランスポーター機能を有する、前記タンパク質。
(3)以下の(a)又は(b)のタンパク質である、(2)記載のタンパク質。
(a)配列番号4に示されるアミノ酸配列を含むタンパク質;
(b)配列番号4に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含み、且つ第337番目のリシン残基を保持するタンパク質であって、該タンパク質をコードする遺伝子を有するイネにおいて、野生型イネ品種と比較してカドミウム及びマンガン吸収を抑制し、且つ該野生型イネ品種と同一の品種である、OsNramp5の機能欠損型遺伝子を有するイネと比較してカドミウム及びマンガン吸収を増加させる、前記タンパク質。
(4)(1)~(3)のいずれかに記載のタンパク質をコードする遺伝子。
(5)(4)記載の遺伝子を含む組換えベクター。
(6)(4)記載の遺伝子又は(5)記載の組換えベクターを有する形質転換体。
(7)(4)記載の遺伝子を発現するカドミウム吸収抑制植物。
(8)植物がイネである、(7)記載のカドミウム吸収抑制植物。
(9)(4)記載の遺伝子を含む植物を特定するための遺伝子マーカー。
(10)植物がイネである、(9)記載の遺伝子マーカー。
(11)(1)~(3)のいずれかに記載のタンパク質を利用する、植物のカドミウム及びマンガン吸収を制御する方法。
(12)植物がイネである、(11)記載の方法。
(13)(8)記載のカドミウム吸収抑制植物から得られたコメから製造された飲食品。
That is, the present invention includes the following.
(1) A protein containing an amino acid sequence in which the sixth glutamine residue is replaced with another amino acid in the amino acid sequence shown in SEQ ID NO: 12.
(2) The protein according to (1), which is the following protein (i) or (ii).
(i) A protein containing an amino acid sequence in which the glutamine residue at position 337 is replaced with another amino acid in the amino acid sequence shown in SEQ ID NO: 2;
(ii) A protein containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of the protein according to (i) and retaining the other amino acid at position 337, which contains cadmium and manganese in rice. The protein having a transporter function of transporting.
(3) The protein according to (2), which is the following protein (a) or (b).
(a) Protein containing the amino acid sequence shown in SEQ ID NO: 4;
(b) A protein containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having the lysine residue at position 337, and having a gene encoding the protein. In rice, it suppresses the absorption of cadmium and manganese as compared with the wild-type rice cultivar, and absorbs cadmium and manganese as compared with the rice having the function-deficient gene of OsNramp5, which is the same cultivar as the wild-type rice cultivar. The protein to increase.
(4) A gene encoding the protein according to any one of (1) to (3).
(5) A recombinant vector containing the gene described in (4).
(6) A transformant having the gene described in (4) or the recombinant vector described in (5).
(7) A cadmium absorption-suppressing plant expressing the gene described in (4).
(8) The cadmium absorption-suppressing plant according to (7), wherein the plant is rice.
(9) A genetic marker for identifying a plant containing the gene described in (4).
(10) The genetic marker according to (9), wherein the plant is rice.
(11) A method for controlling the absorption of cadmium and manganese in a plant using the protein according to any one of (1) to (3).
(12) The method according to (11), wherein the plant is rice.
(13) A food or drink produced from rice obtained from the cadmium absorption-suppressing plant according to (8).
本発明に係るOsNramp5又はそのオーソログの特定部位のアミノ酸が変化したタンパク質をコードする遺伝子を有するイネ等の植物によれば、カドミウム及びマンガン吸収を自在に制御することができる。具体的には、本発明に係るカドミウム吸収抑制植物によれば、従来品種よりもカドミウム吸収が抑制されることで、得られるコメ等の作物におけるカドミウム濃度を低減することができる。特に、本発明に係るカドミウム吸収抑制イネによれば、OsNramp5機能欠損型遺伝子を有するイネに比べてマンガン吸収抑制が緩和されることで、マンガン欠乏による生育への影響や病気への罹病性を低減することができる。 According to a plant such as rice having a gene encoding a protein in which the amino acid of a specific site of OsNramp5 or its ortholog according to the present invention is changed, the absorption of cadmium and manganese can be freely controlled. Specifically, according to the cadmium absorption-suppressing plant according to the present invention, the cadmium concentration in the obtained crops such as rice can be reduced by suppressing the cadmium absorption as compared with the conventional varieties. In particular, according to the cadmium absorption-suppressing rice according to the present invention, the suppression of manganese absorption is alleviated as compared with the rice having the OsNramp5 function-deficient gene, thereby reducing the influence on growth and susceptibility to diseases due to manganese deficiency. can do.
以下、本発明を詳細に説明する。
本発明に係るタンパク質は、多くの生物種において保存性が高い配列番号12に示されるアミノ酸配列において、第6番目のグルタミン残基が他のアミノ酸に置換されたアミノ酸配列を含む、OsNramp5(イネNramp5)又はそのオーソログの変異体タンパク質である。
Hereinafter, the present invention will be described in detail.
The protein according to the present invention contains OsNramp5 (rice Nramp5), which contains an amino acid sequence in which the sixth glutamine residue is replaced with another amino acid in the amino acid sequence shown in SEQ ID NO: 12, which is highly conserved in many biological species. ) Or a variant protein of its ortholog.
OsNramp5タンパク質は、イネにおいてカドミウム及びマンガンを輸送するトランスポータータンパク質である。OsNramp5タンパク質により、イネは土壌中から根を通じてカドミウム及びマンガンを吸収する。 The OsNramp5 protein is a transporter protein that transports cadmium and manganese in rice. The OsNramp5 protein allows rice to absorb cadmium and manganese from the soil through the roots.
配列番号2に示されるアミノ酸配列から成るタンパク質は、コシヒカリ由来のOsNramp5タンパク質である。また、配列番号1に示される塩基配列から成る遺伝子は、コシヒカリ由来のOsNramp5タンパク質をコードする遺伝子(cDNA)である。 The protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is the OsNramp5 protein derived from Koshihikari. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 is a gene (cDNA) encoding the OsNramp5 protein derived from Koshihikari.
図8に示すように、配列番号12に示されるアミノ酸配列は、他生物種由来のOsNramp5のオーソログにも高度に保存されており、この配列はOsNramp5タンパク質に加えて、他生物種のOsNramp5のオーソログタンパク質にも包含されるものである。OsNramp5のアミノ酸配列と相同性が高く、配列番号12に示されるアミノ酸配列を保存している他生物種のOsNramp5のオーソログタンパク質としては、例えば図8に示す、コムギ(Triticum_aestivum)Nrampタンパク質(配列番号13のアミノ酸配列)、トウモロコシ(Zea_maize)Nrampタンパク質(配列番号14のアミノ酸配列)、ダイズ(Glycin_max)Nrampタンパク質(配列番号15のアミノ酸配列)、ジャガイモ(Solanum_tuberosum)Nrampタンパク質(配列番号16のアミノ酸配列)、ウシ(Bos_taurus)Nrampタンパク質(配列番号17のアミノ酸配列)、ニワトリ(Gallus_gallus)Nrampタンパク質(配列番号18のアミノ酸配列)、ブタ(Sus_scrofa_domesticus)Nrampタンパク質(配列番号19のアミノ酸配列)、タイセイヨウダラ(Gadus_morhua)Nrampタンパク質(配列番号20のアミノ酸配列)、タイセイヨウサケ(Salmo_salar)Nrampタンパク質(配列番号21のアミノ酸配列)、バナメイエビ(Penaeus_vannamei)Nrampタンパク質(配列番号22のアミノ酸配列)等が挙げられる。 As shown in FIG. 8, the amino acid sequence shown in SEQ ID NO: 12 is highly conserved in the ortholog of OsNramp5 derived from other organisms, and this sequence is highly conserved in the ortholog of OsNramp5 of other species in addition to the OsNramp5 protein. It is also included in proteins. As an ortholog protein of OsNramp5 of another organism species having high homology with the amino acid sequence of OsNramp5 and conserving the amino acid sequence shown in SEQ ID NO: 12, for example, wheat (Triticum_aestivum) Nramp protein (SEQ ID NO: 13) shown in FIG. (Amino acid sequence of SEQ ID NO: 15), corn (Zea_maize) Nramp protein (amino acid sequence of SEQ ID NO: 14), soybean (Glycin_max) Nramp protein (amino acid sequence of SEQ ID NO: 15), potato (Solanum_tuberosum) Nramp protein (amino acid sequence of SEQ ID NO: 16), Bos_taurus Nramp protein (amino acid sequence of SEQ ID NO: 17), chicken (Gallus_gallus) Nramp protein (amino acid sequence of SEQ ID NO: 18), pig (Sus_scrofa_domesticus) Nramp protein (amino acid sequence of SEQ ID NO: 19), Gadus_morhua ) Nramp protein (amino acid sequence of SEQ ID NO: 20), Salmo_salar Nramp protein (amino acid sequence of SEQ ID NO: 21), Penaeus_vannamei Nramp protein (amino acid sequence of SEQ ID NO: 22) and the like.
配列番号12に示されるアミノ酸配列から成る保存領域は、OsNramp5タンパク質(配列番号2に示されるアミノ酸配列)における第332番目~第343番目のアミノ酸配列に相当する(図8において四角で囲んだ部分)。当該保存領域は、イネと他生物種との間で高度に保存されている領域である。本発明者等は、OsNramp5タンパク質又はそのオーソログタンパク質中の当該保存領域における第6番目のグルタミン残基(配列番号2に示されるアミノ酸配列における第337番目のグルタミン残基に相当する)を他のアミノ酸に置換することで、当該OsNramp5変異体タンパク質を有するイネ及びそのオーソログの変異体タンパク質を有する生物種において、カドミウム及びマンガンの吸収性を変化させ、自在に制御することができる可能性を見出した。 The conserved region consisting of the amino acid sequence shown in SEQ ID NO: 12 corresponds to the 332nd to 343rd amino acid sequences in the OsNramp5 protein (amino acid sequence shown in SEQ ID NO: 2) (the part surrounded by a square in FIG. 8). .. The conserved area is a highly conserved area between rice and other species. The present inventors have set the 6th glutamine residue (corresponding to the 337th glutamine residue in the amino acid sequence shown in SEQ ID NO: 2) in the conserved region in the OsNramp5 protein or its ortholog protein to another amino acid. By substituting with, it was found that the absorption of cadmium and manganese can be changed and freely controlled in the rice having the OsNramp5 mutant protein and the organism having the orthologous variant protein thereof.
ここで、他のアミノ酸としては、グルタミン以外のいずれのアミノ酸であってもよいが、例えば、リシン、ヒスチジン、チロシン、アスパラギン酸、プロリン、アルギニンが好ましい。 Here, the other amino acid may be any amino acid other than glutamine, but for example, lysine, histidine, tyrosine, aspartic acid, proline, and arginine are preferable.
一実施形態において、本発明に係るタンパク質は、以下の(i)又は(ii)のOsNramp5変異体タンパク質である。 In one embodiment, the protein according to the present invention is the OsNramp5 mutant protein according to (i) or (ii) below.
(i)配列番号2に示されるアミノ酸配列において、第337番目のグルタミン残基が他のアミノ酸に置換されたアミノ酸配列を含むか又はそれから成るタンパク質;
(ii)(i)記載のタンパク質のアミノ酸配列と少なくとも80%、好ましくは少なくとも90%、特に好ましくは少なくとも95%の配列同一性を有するアミノ酸配列を含むか又はそれから成り、且つ第337番目の前記他のアミノ酸を保持するタンパク質であって、イネにおいてカドミウム及びマンガンを輸送するトランスポーター機能を有する、前記タンパク質。
(i) In the amino acid sequence shown in SEQ ID NO: 2, a protein containing or consisting of an amino acid sequence in which the 337th glutamine residue is replaced with another amino acid;
(ii) Containing or consisting of an amino acid sequence having at least 80%, preferably at least 90%, particularly preferably at least 95% sequence identity with the amino acid sequence of the protein according to (i), and consisting of the 337th statement. A protein that retains other amino acids and has a transporter function for transporting cadmium and manganese in rice.
また、一実施形態において、本発明に係るタンパク質は、以下の(a)又は(b)のOsNramp5変異体タンパク質である。 Further, in one embodiment, the protein according to the present invention is the OsNramp5 mutant protein according to (a) or (b) below.
(a)配列番号4に示されるアミノ酸配列を含むか又はそれから成るタンパク質;
(b)配列番号4に示されるアミノ酸配列と少なくとも80%、好ましくは少なくとも90%、特に好ましくは少なくとも95%の配列同一性を有するアミノ酸配列を含むか又はそれから成り、且つ第337番目のリシン残基を保持するタンパク質であって、該タンパク質をコードする遺伝子を有するイネにおいて、野生型イネ品種と比較してカドミウム及びマンガン吸収を抑制し、且つ該野生型イネ品種と同一の品種である、OsNramp5の機能欠損型遺伝子を有するイネと比較してカドミウム及びマンガン吸収を増加させる、前記タンパク質。
(a) A protein comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4;
(b) Contains or consists of an amino acid sequence having at least 80%, preferably at least 90%, particularly preferably at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 4, and the lysine residue at position 337. OsNramp5, a protein that retains a group and has a gene encoding the protein, suppresses the absorption of cadmium and manganese as compared with the wild-type rice cultivar, and is the same cultivar as the wild-type rice cultivar. The protein that increases the absorption of cadmium and manganese as compared to rice with a function-deficient gene.
ここで、保持される第337番目の他のアミノ酸又はリシン残基には、OsNramp5変異体タンパク質のアミノ酸配列における他の部位での1以上のアミノ酸の欠失、置換、挿入及び/又は付加によってアミノ酸残基番号が第337番目から変更された当該他のアミノ酸又はリシン残基(すなわち、第337番目の他のアミノ酸又はリシン残基に対応するアミノ酸残基)も包含される。 Here, the retained amino acid at the 337th other amino acid or lysine residue is amino acid by deletion, substitution, insertion and / or addition of one or more amino acids at other sites in the amino acid sequence of the OsNramp5 mutant protein. The other amino acid or lysine residue whose residue number has been changed from the 337th position (that is, the amino acid residue corresponding to the other amino acid or the lysine residue at the 337th position) is also included.
配列番号4に示されるアミノ酸配列から成るタンパク質は、コシヒカリ由来のOsNramp5タンパク質のアミノ酸配列(配列番号2)において、第337番目のグルタミン(Q)残基がリシン(K)残基に置換されたアミノ酸配列から成るOsNramp5変異体タンパク質である。また、配列番号3に示される塩基配列から成る遺伝子は、当該OsNramp5変異体タンパク質をコードする遺伝子(cDNA)である。 The protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is an amino acid in which the 337th glutamine (Q) residue is replaced with a lysine (K) residue in the amino acid sequence (SEQ ID NO: 2) of the OsNramp5 protein derived from Koshihikari. An OsNramp5 variant protein consisting of a sequence. The gene consisting of the base sequence shown in SEQ ID NO: 3 is a gene (cDNA) encoding the OsNramp5 mutant protein.
野生型イネ品種は、例えばコシヒカリ等のOsNramp5遺伝子が変異されていない既存のイネ品種である。OsNramp5の機能欠損型遺伝子を有するイネは、OsNramp5遺伝子における突然変異、OsNramp5遺伝子の欠失等によりOsNramp5タンパク質の機能(トランスポーターとしての輸送機能)が完全に欠損したイネであり、カドミウム吸収が抑制されたイネである。このようなOsNramp5の機能欠損型遺伝子を有するイネとしては、例えばコシヒカリ環1号が知られている(特許文献1)。 Wild-type rice varieties are existing rice varieties such as Koshihikari in which the OsNramp5 gene is not mutated. Rice having a function-deficient gene of OsNramp5 is a rice in which the function of OsNramp5 protein (transporter function as a transporter) is completely deleted due to mutation in OsNramp5 gene, deletion of OsNramp5 gene, etc., and cadmium absorption is suppressed. It is rice. As a rice having such a function-deficient gene of OsNramp5, for example, Koshihikari ring No. 1 is known (Patent Document 1).
上記(a)又は(b)の本発明に係るOsNramp5変異体タンパク質をコードする遺伝子を有するイネでは、例えば、カドミウム吸収が野生型イネ品種のカドミウム吸収に対して50~60%抑制され、且つマンガン吸収が野生型イネ品種のマンガン吸収に対して30~80%抑制される一方で、カドミウム吸収がOsNramp5の機能欠損型遺伝子を有するイネのカドミウム吸収に対して2~30倍増加され、且つマンガン吸収がOsNramp5の機能欠損型遺伝子を有するイネのマンガン吸収に対して1.3~6倍増加される。このように、上記(a)又は(b)の本発明に係るOsNramp5変異体タンパク質をコードする遺伝子を有するイネでは、カドミウム吸収が抑制される一方で、生育等に必要なマンガンの吸収抑制が緩和される。 In rice having the gene encoding the OsNramp5 mutant protein according to the present invention of (a) or (b) above, for example, cadmium absorption is suppressed by 50 to 60% with respect to cadmium absorption of wild-type rice varieties, and manganese. Absorption is suppressed by 30-80% with respect to manganese absorption in wild-type rice varieties, while cadmium absorption is increased 2-30-fold with respect to cadmium absorption in rice with a function-deficient gene of OsNramp5, and manganese absorption. Is increased by 1.3 to 6-fold with respect to manganese absorption in rice with a function-deficient gene of OsNramp5. As described above, in the rice having the gene encoding the OsNramp5 mutant protein according to the present invention of (a) or (b) above, the absorption of cadmium is suppressed, while the suppression of the absorption of manganese necessary for growth is alleviated. Will be done.
イネにおけるカドミウム及びマンガン吸収特性は、それぞれイネ由来の茎葉部、根部、玄米、稲わら等のサンプルにおいて測定されるカドミウム濃度及びマンガン濃度を指標に決定することができる。 The cadmium and manganese absorption characteristics in rice can be determined by using the cadmium concentration and manganese concentration measured in samples such as foliage, root, brown rice, and rice straw derived from rice as indicators.
また、上記(a)又は(b)の本発明に係るOsNramp5変異体タンパク質をコードする遺伝子を有するイネでは、例えば、カドミウム汚染土壌での栽培において、玄米のカドミウム濃度が0.4mg/kg以下、玄米のマンガン濃度が10mg/kg以上、稲わらのカドミウム濃度が2.0mg/kg以下、稲わらのマンガン濃度が100mg/kg以上であることが好ましい。 Further, in the rice having the gene encoding the OsNramp5 mutant protein according to the present invention of (a) or (b) above, for example, in cultivation in soil contaminated with cadmium, the cadmium concentration of brown rice is 0.4 mg / kg or less, and brown rice. It is preferable that the manganese concentration of rice straw is 10 mg / kg or more, the cadmium concentration of rice straw is 2.0 mg / kg or less, and the manganese concentration of rice straw is 100 mg / kg or more.
以上に説明する本発明に係るOsNramp5又はそのオーソログの変異体タンパク質を利用し、植物で発現させることで、植物のカドミウム及びマンガン吸収を制御することができる。ここで、植物としては、例えばイネ、コムギ、トウモロコシ、ダイズ、ジャガイモ等が挙げられるが、イネが好ましい。 By using the mutant protein of OsNramp5 or its ortholog according to the present invention described above and expressing it in a plant, the absorption of cadmium and manganese in the plant can be controlled. Here, examples of the plant include rice, wheat, corn, soybean, potato and the like, but rice is preferable.
本発明に係る遺伝子は、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子である。 The gene according to the present invention is a gene encoding a mutant protein of OsNramp5 or an ortholog thereof according to the present invention.
本発明に係る遺伝子には、(a)配列番号3に示される塩基配列を含むか又はそれから成る遺伝子、又は(b)配列番号3に示される塩基配列と少なくとも80%、好ましくは少なくとも90%、特に好ましくは少なくとも95%の配列同一性を有する塩基配列を含むか又はそれから成り、且つ配列番号4に示されるアミノ酸配列における第337番目のリシン残基に対応するコドン(例えば配列番号3に示される塩基配列における第1009~1011番目の「aag」)を保持する遺伝子であって、該遺伝子を有するイネにおいて、野生型イネ品種と比較してカドミウム及びマンガン吸収を抑制し、且つ該野生型イネ品種と同一の品種である、OsNramp5の機能欠損型遺伝子を有するイネと比較してカドミウム及びマンガン吸収を増加させるタンパク質をコードする遺伝子が含まれる。 The gene according to the present invention includes (a) a gene containing or consisting of the base sequence shown in SEQ ID NO: 3, or (b) at least 80%, preferably at least 90% of the base sequence shown in SEQ ID NO: 3. Particularly preferably, a codon containing or consisting of a base sequence having at least 95% sequence identity and corresponding to the 337th lysine residue in the amino acid sequence set forth in SEQ ID NO: 4 (eg, set forth in SEQ ID NO: 3). It is a gene that carries the 1009th to 1011th "aag") in the base sequence, and in rice having this gene, it suppresses the absorption of cadmium and manganese as compared with the wild type rice varieties, and the wild type rice varieties. Contains a gene encoding a protein that increases cadmium and manganese uptake compared to rice, which has a function-deficient gene for OsNramp5, which is the same cultivar as.
本発明に係る遺伝子を宿主に導入することで、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質を発現させることができる。 By introducing the gene according to the present invention into a host, a mutant protein of OsNramp5 or an ortholog thereof according to the present invention can be expressed.
宿主としては、特に限定されるものではないが、サッカロミセス・セレビシエ(Saccharomyces cerevisiae)等の酵母、大腸菌(Escherichia coli)等のエッシェリヒア属、バチルス・ズブチリス(Bacillus subtilis)等のバチルス属又はシュードモナス・プチダ(Pseudomonas putida)等のシュードモナス属等に属する細菌、COS細胞等の動物細胞、Sf9等の昆虫細胞、あるいはイネ科(イネ等)、アブラナ科等に属する植物が挙げられる。 The host is not particularly limited, but yeast such as Saccharomyces cerevisiae, Escherichia coli such as Escherichia coli, Bacillus subtilis and the like, or Pseudomonas petitda ( Bacteria belonging to the genus Pseudomonas such as Pseudomonas putida), animal cells such as COS cells, insect cells such as Sf9, or plants belonging to the family Rice (rice, etc.), Bacillus, etc. can be mentioned.
先ず、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子を準備する。当該遺伝子は、例えば、野生型遺伝子が由来するイネ等の生物種のゲノムDNA等を鋳型として、該領域の両端の塩基配列に相補的なプライマーを用いたPCRによって容易に得ることができる。ただし、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質は、OsNramp5又はそのオーソログのアミノ酸配列においてアミノ酸置換を有するものであるので、上述のように得られたPCR産物に、部位特異的突然変異誘発法やCRISPR-Cas9をベースにした一塩基編集法(Endo M, Mikami M, Endo A, Kaya H, Itoh T, Nishimasu H, Nureki O, Toki S (2019) Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM. Nature Plants 5: 14-17;Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 551: 464-471;Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353: aaf8729)等によって変異をさらに導入することによって、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子を得ることができる。 First, a gene encoding a mutant protein of OsNramp5 or its ortholog according to the present invention is prepared. The gene can be easily obtained, for example, by PCR using genomic DNA of an organism such as rice from which a wild-type gene is derived as a template and primers complementary to the base sequences at both ends of the region. However, since the variant protein of OsNramp5 or its ortholog according to the present invention has an amino acid substitution in the amino acid sequence of OsNramp5 or its ortholog, site-specific mutagenesis is induced in the PCR product obtained as described above. Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM. Nature Plants 5: 14-17; Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of AT to GC in genomic DNA without DNA cleavage. Nature 551 : 464-471; Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A (2016) Targeted nucleotide editing using hybrid prokaryotic and By further introducing the mutation by vertebrate adaptive immune systems. Science 353: aaf8729) or the like, a gene encoding a mutant protein of OsNramp5 or its ortholog according to the present invention can be obtained.
一旦、塩基配列が確定されると、その後は化学合成によって、又はクローニングされたプローブを鋳型としたPCRによって、あるいは該塩基配列を有するDNA断片をプローブとしてハイブリダイズさせることによって、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子を得ることができる。さらに、部位特異的突然変異誘発法やCRISPR-Cas9をベースにした一塩基編集法等によって本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子の変異型であって変異前と同等の機能を有するものを合成することができる。なお、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質をコードする遺伝子に変異を導入するには、公知の手法を採用することができる。 Once the base sequence is determined, OsNramp5 according to the present invention is subsequently hybridized by chemical synthesis, PCR using a cloned probe as a template, or a DNA fragment having the base sequence as a probe. Alternatively, a gene encoding a mutant protein of the ortholog can be obtained. Furthermore, it is a mutant type of the gene encoding the mutant protein of OsNramp5 or its ortholog according to the present invention by a site-specific mutagenesis method, a single nucleotide editing method based on CRISPR-Cas9, etc., and is equivalent to that before the mutation. Those having a function can be synthesized. A known method can be adopted for introducing a mutation into a gene encoding a mutant protein of OsNramp5 or its ortholog according to the present invention.
本発明に係る遺伝子を含む組換えベクターは、適当なベクターに本発明に係る遺伝子を挿入することにより得ることができる。使用するベクターは、宿主中で複製可能なものであれば特に限定されず、例えばプラスミド、シャトルベクター、ヘルパープラスミド、バイナリーベクター等が挙げられる。また該ベクター自体に複製能がない場合には、宿主の染色体に挿入すること等によって複製可能となるDNA断片であってもよい。 A recombinant vector containing the gene according to the present invention can be obtained by inserting the gene according to the present invention into an appropriate vector. The vector to be used is not particularly limited as long as it can be replicated in the host, and examples thereof include a plasmid, a shuttle vector, a helper plasmid, and a binary vector. If the vector itself does not have replication ability, it may be a DNA fragment that can be replicated by inserting it into a host chromosome or the like.
さらに、本発明に係る遺伝子又は本発明に係る遺伝子を含む組換えベクター(以下、「本発明に係る組換えベクター等」という)を宿主中に導入することにより形質転換体を作製する。 Further, a transformant is prepared by introducing a gene according to the present invention or a recombinant vector containing the gene according to the present invention (hereinafter referred to as "recombinant vector according to the present invention") into a host.
植物を宿主とする場合は、植物体全体、植物器官(例えば葉、花弁、茎、根、種子等)、植物組織(例えば表皮、師部、柔組織、木部、維管束等)、植物培養細胞等が用いられる。植物への本発明に係る組換えベクター等の導入方法としては、例えばエレクトロポレーション法、アグロバクテリウム法、パーティクルガン法、PEG法等が挙げられる。 When using a plant as a host, the whole plant, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg, epidermis, phloem, soft tissues, xylem, vascular bundles, etc.), plant culture Cells and the like are used. Examples of the method for introducing the recombinant vector or the like according to the present invention into a plant include an electroporation method, an Agrobacterium method, a particle gun method, a PEG method and the like.
酵母への本発明に係る組換えベクター等の導入方法は、酵母にDNAを導入する方法であれば特に限定されず、例えば電気穿孔法(エレクトロポレーション法)、スフェロプラスト法、酢酸リチウム法等が挙げられる。 The method for introducing the recombinant vector or the like according to the present invention into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, for example, an electroporation method, a spheroplast method, a lithium acetate method. And so on.
細菌への本発明に係る組換えベクター等の導入方法は、細菌にDNAを導入する方法であれば特に限定されるものではない。例えばカルシウムイオンを用いる方法、エレクトロポレーション法等が挙げられる。 The method for introducing the recombinant vector or the like according to the present invention into a bacterium is not particularly limited as long as it is a method for introducing DNA into the bacterium. For example, a method using calcium ions, an electroporation method and the like can be mentioned.
動物細胞を宿主とする場合は、サル細胞COS-7、Vero、チャイニーズハムスター卵巣細胞(CHO細胞)、マウスL細胞等が用いられる。動物細胞への本発明に係る組換えベクター等の導入方法としては、例えばエレクトロポレーション法、リン酸カルシウム法、リポフェクション法等が挙げられる。 When animal cells are used as a host, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells and the like are used. Examples of the method for introducing the recombinant vector or the like according to the present invention into animal cells include an electroporation method, a calcium phosphate method, a lipofection method and the like.
昆虫細胞を宿主とする場合は、Sf9細胞等が用いられる。昆虫細胞への本発明に係る組換えベクター等の導入方法としては、例えばリン酸カルシウム法、リポフェクション法、エレクトロポレーション法等が挙げられる。 When an insect cell is used as a host, Sf9 cells or the like are used. Examples of the method for introducing the recombinant vector or the like according to the present invention into insect cells include a calcium phosphate method, a lipofection method, an electroporation method and the like.
本発明に係る組換えベクター等が宿主に組み込まれたか否かの確認は、PCR法、サザンハイブリダイゼーション法、ノーザンハイブリダイゼーション法等により行うことができる。例えば、形質転換体からDNAを調製し、DNA特異的プライマーを設計してPCRを行う。その後は、増幅産物についてアガロースゲル電気泳動、ポリアクリルアミドゲル電気泳動、キャピラリー電気泳動等を行い、臭化エチジウム、SYBR Green液等により染色し、そして増幅産物をバンドとして検出することにより、形質転換されたことを確認する。また、予め蛍光色素等により標識したプライマーを用いてPCRを行い、増幅産物を検出することもできる。さらに、マイクロプレート等の固相に増幅産物を結合させ、蛍光、酵素反応等により増幅産物を確認する方法も採用してもよい。 Whether or not the recombinant vector or the like according to the present invention has been incorporated into the host can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like. For example, DNA is prepared from transformants, DNA-specific primers are designed, and PCR is performed. After that, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and transformed by detecting the amplified product as a band. Confirm that. It is also possible to detect the amplification product by performing PCR using a primer labeled in advance with a fluorescent dye or the like. Further, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence, enzymatic reaction or the like may also be adopted.
また、得られた形質転換体を生育可能な条件下で培養することで、本発明に係るOsNramp5又はそのオーソログの変異体タンパク質を当該形質転換体より得ることができる。 Further, by culturing the obtained transformant under viable conditions, the mutant protein of OsNramp5 or its ortholog according to the present invention can be obtained from the transformant.
特に、本発明は、植物への本発明に係る組換えベクター等の導入により得られた形質転換体である、本発明に係る遺伝子を発現するカドミウム吸収抑制植物に関する。また、本発明に係るカドミウム吸収抑制植物には、形質転換体に加えて、例えば薬剤処理等の変異処理やゲノム編集によって得られた本発明に係る遺伝子を発現する植物も包含される。 In particular, the present invention relates to a cadmium absorption-suppressing plant expressing a gene according to the present invention, which is a transformant obtained by introducing a recombinant vector or the like according to the present invention into a plant. Further, the cadmium absorption-suppressing plant according to the present invention includes, in addition to the transformant, a plant expressing the gene according to the present invention obtained by mutation treatment such as drug treatment or genome editing.
ここで、植物としては、例えばイネ、コムギ、トウモロコシ、ダイズ、ジャガイモ等が挙げられるが、イネが好ましい。 Here, examples of the plant include rice, wheat, corn, soybean, potato and the like, but rice is preferable.
また、本発明に係るカドミウム吸収抑制植物と既存の植物品種とを交配し、効率良く新たな低カドミウム吸収植物品種を作出することが可能である。 In addition, it is possible to efficiently produce a new low-cadmium-absorbing plant cultivar by mating the cadmium-absorbing plant according to the present invention with an existing plant cultivar.
イネにおける当該作出方法としては、例えば、以下のような手順が挙げられる:
1) 本発明に係るカドミウム吸収抑制イネ(A植物)と既存のイネ品種(B植物)とを交配し、F1を作出する;
2) F1とB植物を交雑させる;
3) 交雑後の植物から本発明に係る遺伝子を有する個体を選抜する;
4) B植物に交雑を重ねていく「戻し交雑」によって、A植物が有する本発明に係る遺伝子を意図的にB植物に導入する;
5)その際、多数の戻し交雑個体の中から、本発明に係る遺伝子を有する個体のみを選抜する。
6) 前記個体選抜をする際は、幼植物段階の茎葉や根から抽出したゲノムDNAを使用すればよい;
7) B植物に交雑を繰り返し、本発明に係る遺伝子が入った個体のみを選抜することで、ゲノム構造のほとんどがB植物であるが、カドミウム及びマンガン吸収のみA植物の遺伝形質の入った新たな品種が作出できる;
8) B植物はジャポニカ種でもインディカ種でもかまわない。
Examples of the production method in rice include the following procedures:
1) Cadmium absorption-suppressing rice (plant A) according to the present invention is crossed with an existing rice variety (plant B) to produce F1;
2) Cross F1 and B plants;
3) Individuals having the gene according to the present invention are selected from the crossed plants;
4) By "backcrossing" in which crosses are repeated on plant B, the gene according to the present invention possessed by plant A is intentionally introduced into plant B;
5) At that time, only individuals having the gene according to the present invention are selected from a large number of backcross individuals.
6) When selecting the individual, genomic DNA extracted from the foliage and roots of the seedling stage may be used;
7) By repeating crossing with B plant and selecting only individuals containing the gene according to the present invention, most of the genomic structure is B plant, but only cadmium and manganese absorption are new with the genetic trait of A plant. Can produce various varieties;
8) The B plant may be either Japonica or Indica.
上述の本発明に係る遺伝子を有するイネ等の植物個体のみを選抜(又は特定)するためには、遺伝子マーカーを活用することができる。本発明に係る遺伝子マーカーは、本発明に係る遺伝子とOsNramp5又はそのオーソログの遺伝子との塩基配列の違い(例えば、上述のOsNramp5タンパク質における第337番目のアミノ酸に対応するコドンの違い)に基づいて、本発明に係る遺伝子を有するイネ等の植物個体とそれ以外の個体とを識別することができる。 In order to select (or specify) only individual plants such as rice having the above-mentioned gene according to the present invention, a gene marker can be utilized. The gene marker according to the present invention is based on the difference in the base sequence between the gene according to the present invention and the gene of OsNramp5 or its ortholog (for example, the difference in the codon corresponding to the 337th amino acid in the above-mentioned OsNramp5 protein). It is possible to distinguish between a plant individual such as rice having a gene according to the present invention and an individual other than that.
本発明に係る遺伝子マーカーは、本発明に係る遺伝子が交配や形質転換によって他の植物品種に導入されたか否か、塩基配列の違いから判断するための目印となる。植物から抽出したゲノムDNAを鋳型にし、ゲノムDNAの塩基配列に従って適当に選択された塩基配列を持つ合成オリゴヌクレオチドをプライマーとして用い、これらを混合させた反応液中においてPCR増幅反応を行い、その生成物を含む反応液を例えばアガロース電気泳動に供することで、増幅された各種のDNA断片が分画されて、そのDNA断片が本発明に係る遺伝子に対応することを確認できる。 The gene marker according to the present invention serves as a marker for determining whether or not the gene according to the present invention has been introduced into another plant variety by mating or transformation based on the difference in the base sequence. Using genomic DNA extracted from plants as a template and synthetic oligonucleotides having a base sequence appropriately selected according to the base sequence of genomic DNA as primers, PCR amplification reaction is performed in a mixed reaction solution to generate the same. By subjecting the reaction solution containing the substance to, for example, agarose electrophoresis, various amplified DNA fragments can be fractionated, and it can be confirmed that the DNA fragments correspond to the genes according to the present invention.
例えば、下記の実施例4に示すように、本発明に係るカドミウム吸収抑制イネと既存のイネ品種とからゲノムDNAを抽出し、開始コドンのアデニン(A)から数えて3658番目の塩基から3'方向に19bsを標的としたプライマー(配列番号5と6)及び3796番目の塩基から5'方向に25bsを標的としたプライマー(配列番号7)で、抽出物のPCRを行う。カドミウム吸収抑制イネの遺伝子型には配列番号5と7のプライマーが反応して138bsの長さのDNA増幅断片が生成し、既存のイネ品種の遺伝子型には配列番号6と7のプライマーが反応して178bsの長さのDNA増幅断片が生成するため、電気泳動に供するとそれぞれのサイズに分画され、各個体の識別に利用できる。 For example, as shown in Example 4 below, genomic DNA was extracted from the cadmium absorption-suppressing rice according to the present invention and existing rice varieties, and 3'from the 3658th base counted from the start codon adenine (A). PCR of the extract is performed with primers targeting 19 bs in the direction (SEQ ID NOs: 5 and 6) and primers targeting 25 bs in the 5'direction from the 3796th base (SEQ ID NO: 7). Primers of SEQ ID NOs: 5 and 7 react with genotypes of cadmium absorption-suppressed rice to generate DNA amplification fragments with a length of 138 bs, and primers of SEQ ID NOs: 6 and 7 react with genotypes of existing rice varieties. As a result, a DNA amplified fragment with a length of 178 bs is generated, and when subjected to electrophoresis, it is fractionated into each size and can be used for identification of each individual.
また、本発明は、上記のカドミウム吸収抑制イネから得られたコメから製造された飲食品に関する。当該飲食品によれば、飲食品由来のカドミウム摂取量を減らすことができる。飲食品としては、例えばライスミルク、せんべい、米粉パン等のコメ加工製品が挙げられる。 The present invention also relates to foods and drinks produced from rice obtained from the above-mentioned cadmium absorption-suppressing rice. According to the food and drink, the intake of cadmium derived from the food and drink can be reduced. Examples of foods and drinks include processed rice products such as rice milk, rice crackers, and rice flour bread.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to these examples.
〔実施例1〕薬剤処理(DEB2回処理)による水稲変異体の獲得
コシヒカリ種子(M0)を28℃により18時間逆浸透水を吸水させ、同温度にて0.09%DEB(1,2,3,4-ジエポキシブタン)水溶液で12時間処理した後、播種し、温室でM1世代の植物を栽培した。
[Example 1] Acquisition of paddy rice mutants by chemical treatment (DEB twice treatment) Koshihikari seeds (M 0 ) were allowed to absorb reverse osmosis water at 28 ° C for 18 hours, and 0.09% DEB (1,2,3) at the same temperature. , 4-Diepoxybutane) After treatment with an aqueous solution for 12 hours, the seeds were sown and M 1 generation plants were cultivated in a greenhouse.
M1植物体に実ったM2種子をバルク化し、5,000粒の種子を28℃により18時間逆浸透水を吸水させ、同温度にて0.03%DEB水溶液で12時間処理した後、播種し、圃場(つくば市)においてM2世代の植物を栽培した。 M 2 seeds grown on M 1 plants are bulked, 5,000 seeds are allowed to absorb reverse osmosis water at 28 ° C for 18 hours, treated with 0.03% DEB aqueous solution at the same temperature for 12 hours, then sown and fielded. M2 generation plants were cultivated in (Tsukuba City).
得られたM3種子を1個体から2粒ずつ採取し、3,072個体を栽培し、ゲノムDNAを珪藻土法により抽出し、下記のTILLING法によりOsNramp5遺伝子に変異を持つ系統の選抜を行った。 Two of the obtained M 3 seeds were collected from one individual, 3,072 individuals were cultivated, genomic DNA was extracted by the diatomaceous earth method, and strains having a mutation in the OsNramp5 gene were selected by the following TILLING method.
〔実施例2〕Tilling法によるOsNramp5変異個体の選抜
実施例1において得られた上記DNA(DEB2回処理変異体集団)を2個ずつを混合した2個体バルクを作製し、PrimeSTAR GXL(タカラバイオ)によりOsNramp5のゲノムDNAを数種類のプライマーを用いてPCR法により増幅した。熱変性(98℃)により二本鎖DNAを乖離させ、温度を下げて再会合(アニーリング)させた後、セロリより抽出したCel-Iヌクレアーゼによる処理、アガロース電気泳動により、ミスマッチ部位で切断されたPCR産物を検出した。
[Example 2] Selection of OsNramp5 mutant individuals by the Tilling method A two-individual bulk was prepared by mixing two of the above DNAs (DEB double-treated mutant population) obtained in Example 1 to prepare PrimeSTAR GXL (Takara Bio). The genomic DNA of OsNramp5 was amplified by the PCR method using several types of primers. Double-stranded DNA was dissociated by heat denaturation (98 ° C), and after lowering the temperature and reassociating (annealing), it was cleaved at the mismatch site by treatment with Cel-I nuclease extracted from celery and agarose gel electrophoresis. PCR products were detected.
さらに、バルク化されたDNAのどちらが、変異体であるかを調べるために、単独のDNA及び単独のDNAとコシヒカリDNAを1対1で混合したDNAをテンプレートとして、上記と同じようにPCR法で増幅し、Cel-Iヌクレアーゼ処理し、アガロース電気泳動によって標的DNAに変異が生じた個体を特定した。OsNramp5の変異位置はシーケンス解析により、特定した。 Furthermore, in order to investigate which of the bulked DNA is a mutant, a single DNA or a DNA obtained by mixing a single DNA and a Koshihikari DNA in a one-to-one manner is used as a template by the PCR method as described above. Individuals with mutations in the target DNA were identified by amplification, Cel-I nuclease treatment, and agarose gel electrophoresis. The mutation position of OsNramp5 was identified by sequence analysis.
なお、薬剤処理を用いた変異処理により、OsNramp5のアミノ酸配列に非同義置換が認められた個体は17、塩基の欠損やスプライシング部位に変異が生じた個体は2つあり、その中で発芽が認められた15個体について、カドミウム、マンガン等吸収性評価に利用した(図1)。 In addition, 17 individuals had non-synonymous substitutions in the amino acid sequence of OsNramp5 by mutation treatment using drug treatment, and 2 individuals had base defects or mutations in the splicing site, and germination was observed among them. The 15 individuals were used for evaluation of absorption of cadmium, manganese, etc. (Fig. 1).
〔実施例3〕OsNramp5変異系統のカドミウム、マンガン等重金属吸収性評価
(1)水耕栽培による幼植物での吸収性評価
実施例2におけるOsNramp5に変異が生じた系統(世代はM3)の催芽種子を底面に3mm口径の穴を開けた96穴のPCRプレートに播種し、スタイロホームで作製した浮遊台にプレートを乗せ、木村B水耕液(1/2濃度)の入った20L容コンテナ容器に置いた。
[Example 3] Evaluation of absorption of heavy metals such as cadmium and manganese of OsNramp5 mutant line ( 1 ) Evaluation of absorption of seedlings by hydroponics. Seeds are sown on a 96-hole PCR plate with a hole of 3 mm caliber on the bottom, the plate is placed on a floating table made by Stylo Home, and a 20 L container container containing Kimura B hydroponic solution (1/2 concentration). I put it in.
播種後14日経過した幼苗を、0.05ppm カドミウム(Cd)濃度を含む木村B溶液に移し、2日間処理した。処理後、茎葉部をサンプリングし、70℃で3日間乾燥させた。乾燥物を濃硝酸で分解し、その分解液を誘導結合プラズマ質量分析装置(ICP-MS)(PerkinElmer社製、商品名ELAN DRC-e)にてCdとマンガン(Mn)濃度を測定した。 Seedlings 14 days after sowing were transferred to Kimura B solution containing 0.05 ppm cadmium (Cd) concentration and treated for 2 days. After the treatment, the foliage was sampled and dried at 70 ° C. for 3 days. The dried product was decomposed with concentrated nitrate, and the Cd and manganese (Mn) concentrations were measured with an inductively coupled plasma mass spectrometer (ICP-MS) (trade name: ELAN DRC-e, manufactured by PerkinElmer).
選抜した15の変異系統の中で、8系統はコシヒカリ(KSH)と3系統はコシヒカリ環1号(KK1)と同等のCdやMn濃度を示した。残りの3系統(A30T, D260V, Q337K)のCdやMn濃度はコシヒカリとコシヒカリ環1号の中庸に位置したが、Q337K以外、生育抑や形質が不安定であったため、評価の対象から外し、Q337K(リシン)に着目した(図2)。 Of the 15 mutant strains selected, 8 strains showed Koshihikari (KSH) and 3 strains showed the same Cd and Mn concentrations as Koshihikari ring No. 1 (KK1). The Cd and Mn concentrations of the remaining 3 strains (A30T, D260V, Q337K) were located in the middle of Koshihikari and Koshihikari Ring No. 1, but they were excluded from the evaluation because of unstable growth suppression and traits other than Q337K. We focused on Q337K (lysine) (Fig. 2).
種子の世代を進め、Q337Kの変異を持つM4系統(3018W)について、上記と同様に14日幼苗をCdを含む水耕液で2日間処理し、乾燥・酸分解後、ICP-MSでCdとMn濃度を測定して形質の安定性を評価した。 For the M4 line ( 3018W ) with the Q337K mutation, the seedlings were treated with a hydroponic solution containing Cd for 2 days in the same manner as above for 2 days, dried and acid-decomposed, and then Cd with ICP-MS. And Mn concentration were measured to evaluate the stability of the trait.
その結果、3018Wはコシヒカリ環1号に比べてCdとMn濃度の増加が認められたが、有害元素であるCdはコシヒカリの半分以下に抑えられた(図3)。 As a result, 3018W was found to have increased Cd and Mn concentrations compared to Koshihikari Ring No. 1, but the harmful element Cd was suppressed to less than half that of Koshihikari (Fig. 3).
(2)カドミウム汚染圃場での吸収性評価
播種後約1ヶ月経過したQ337Kの変異を持つM4系統(3018W)の幼苗を、Cd汚染水田土壌(土壌のCd濃度:1.8mg/kg)に移植した。移植1ヶ月後に中干しを行うことで土壌を酸化的し、その後登熟期まで間断灌漑の水管理を行うことで、水稲のCd吸収が高まりやすい土壌環境で栽培した。
(2) Evaluation of absorbability in cadmium-contaminated fields Transplant M4 line ( 3018W ) seedlings with Q337K mutation about 1 month after sowing into Cd-contaminated paddy soil (Cd concentration of soil: 1.8 mg / kg). did. One month after transplanting, the soil was oxidized by mid-drying, and then water management of intermittent irrigation was performed until the ripening period, so that the paddy rice was cultivated in a soil environment where Cd absorption was likely to increase.
登熟した3018W変異体を玄米と稲わらに分け、乾燥後、上述した通り、濃硝酸で分解し、ICP-MSにて、CdやMn等重金属元素を測定した。比較のため、コシヒカリ及びコシヒカリ環1号も同時に栽培し、同様な方法で分析した。 The ripened 3018W mutant was divided into brown rice and rice straw, dried, decomposed with concentrated nitrate as described above, and measured for heavy metal elements such as Cd and Mn by ICP-MS. For comparison, Koshihikari and Koshihikari Ring No. 1 were also cultivated at the same time and analyzed by the same method.
フレームシフト変異により、OsNramp5の機能が完全に欠失したコシヒカリ環1号は、玄米や稲わらのCd濃度が極端に低下した。加えて、Mn濃度も著しく低下した。一方、OsNramp5にミスセンス変異を持つ3018Wは玄米や稲わらのCd濃度がコシヒカリの半減となった。3018WのMn濃度はコシヒカリよりは低レベルだが、コシヒカリ環1号に比べて明らかに高まっていた。玄米や稲わらの鉄(Fe)濃度が3018Wでやや高まる傾向にあるが、亜鉛(Zn)や銅(Cu)の濃度にはほとんど影響がなかった(図4)。 Due to the frameshift mutation, Koshihikari ring No. 1, which completely lacked the function of OsNramp5, had an extremely low Cd concentration in brown rice and rice straw. In addition, the Mn concentration was also significantly reduced. On the other hand, 3018W, which has a missense mutation in OsNramp5, reduced the Cd concentration of brown rice and rice straw to half that of Koshihikari. The Mn concentration of 3018W was lower than that of Koshihikari, but clearly higher than that of Koshihikari Ring No. 1. The iron (Fe) concentration of brown rice and rice straw tended to increase slightly at 3018 W, but there was almost no effect on the zinc (Zn) and copper (Cu) concentrations (Fig. 4).
〔実施例4〕Q337Kを検出するためのDNAマーカーの開発
実施例3におけるQ337Kの変異を持つ3018W系統のOsNramp5遺伝子変異を図5Aに示す。
[Example 4] Development of DNA marker for detecting Q337K The OsNramp5 gene mutation of the 3018W strain having the mutation of Q337K in Example 3 is shown in FIG. 5A.
図5Aに示すように、Q337Kの変異を持つ3018W系統のOsNramp5遺伝子変異は、第10エクソンにある337番目アミノ酸のコドンCAG(グルタミン:Q)がAAG(リシン:K)に変化したものであった(コシヒカリ由来のOsNramp5のDNA配列及びアミノ酸配列をそれぞれ配列番号1及び2に示す。また、3018W由来のOsNramp5のDNA配列及びアミノ酸配列をそれぞれ配列番号3及び4に示す)。 As shown in FIG. 5A, the OsNramp5 gene mutation of the 3018W strain with the mutation of Q337K was a change of the codon CAG (glutamine: Q) of the 337th amino acid in the 10th exson to AAG (lysine: K). (The DNA sequence and amino acid sequence of OsNramp5 derived from Koshihikari are shown in SEQ ID NOs: 1 and 2, respectively. The DNA sequence and amino acid sequence of OsNramp5 derived from 3018W are shown in SEQ ID NOs: 3 and 4, respectively).
Wild type(WT:コシヒカリ)と3018W系統の遺伝子型を判別するために、葉からゲノムDNAをXu et al.(2005, Plant Molecular Biology Reporter 23,291-295)の方法に従って抽出し、3種類のプライマー:OsNR5Q337K_F3658wtN40G3A[5'-(N)40GCACTGTTGGCATCTGAGC-3':配列番号5]、OsNR5Q337K_F3658mtG3T[5'-GCACTGTTGGCATCTGTGA-3':配列番号6]、OsNR5Q337K_R3796cmn[5'-TTTAATTACGCGCTAAGCTAGCTGG-3':配列番号7]を用いてゲノムDNAをPCRで増幅させた。 To distinguish between wild type (WT: Koshihikari) and 3018W strain genotypes, genomic DNA was extracted from the leaves according to the method of Xu et al. (2005, Plant Molecular Biology Reporter 23,291-295), and three types of primers: OsNR5Q337K_F3658wtN40G3A [5'-(N) 40 GCACTGTTGGCATCTGAGC-3': SEQ ID NO: 5], OsNR5Q337K_F3658mtG3T [5'-GCACTGTTGGCATCTGTGA-3': SEQ ID NO: 6], OsNR5Q337K_R3796cmn [5'GC The genomic DNA was amplified by PCR.
ゲル電気泳動により、増幅産物はWTで178bs、3018Wで138bs(図5B)付近にバンドが確認されるため、遺伝子型の判別は可能であった。また、WTと3018Wとの交配により、両方に遺伝子型(ヘテロ)が確認できれば、交配が成功したことになる。 By gel electrophoresis, bands were confirmed in the vicinity of 178bs for WT and 138bs for 3018W (Fig. 5B), so it was possible to determine the genotype. In addition, if the genotype (hetero) can be confirmed in both by mating with WT and 3018W, the mating is successful.
〔実施例5〕遺伝子組換え酵母を用いたカドミウム・マンガンの吸収試験
OsNramp5の337番目のアミノ酸(Q)をKを含め他のアミノ酸に置換させた場合のCdとMn吸収性の変化を調べた。
[Example 5] Absorption test of cadmium / manganese using recombinant yeast
The changes in Cd and Mn absorption when the 337th amino acid (Q) of OsNramp5 was replaced with other amino acids including K were investigated.
実施例3におけるQ337Kを持つ3018W系統のcDNAを鋳型として、プライマーセットOsNramp5_EcoRI_atgF(5'- AAGAGGAATTCCAACAATGGAGATTGAGAGAGAGA-3':配列番号8)及びOsNramp5_stp_XhoI_R(5'- ATCTTCCTCGAGTCTACCTTGGGAGCGGGATGTCG-3':配列番号9)(ユーロフィン社製)を用いてPCRを行い、全長ORFを含む増幅断片を増幅した。 Primer set OsNramp5_EcoRI_atgF (5'-AAGAGGAATTCCAACAATGGAGATTGAGAGAGAGA-3': SEQ ID NO: 8) and OsNramp5_stp_XhoI_R (5'-ATCTTCCGAGTCTACCTTG CG The amplified fragment containing the full-length ORF was amplified by PCR.
さらに、変異体3018Wが持つOsNRAMP5の337番目のアミノ酸を人為的にその他のアミノ酸に変化させたクローンの作製には、配列番号8のプライマーとプライマーOsNR5_Q337Xmt_R1025(5'- GTAATAGTGGAGCTNNNACCAGATGC-3':配列番号10)及びプライマーOsNR5_Q337Xmt_F995(5'- TGTTGGCATCTGGTnnnAGCTC-3':配列番号11)と配列番号9のプライマーで、OsNramp5ORFの変異箇所から前半・後半部分配列をPCRで増幅した(配列番号10及び11のプライマー名に示す「X」はオリジナルのQ(グルタミン)、変異体3018Wの遺伝子型K(リシン)以外のアミノ酸一文字表記となる。各アミノ酸(X)に対応するプライマー配列で、配列番号11の「nnn」及び配列番号10の「NNN」には、それぞれA(アラニン)ではGCA及びTGC、C(システイン)ではTGT及びACA、D(アスパラギン酸)ではGAT及びATC、E(グルタミン酸)ではGAA及びTTC、F(フェニルアラニン)ではTTT及びAAA、G(グリシン)ではGGT及びACC、H(ヒスチジン)ではCAT及びATG、I(イソロイシン)ではATT及びAAT、L(ロイシン)ではCTG及びGAC、M(メチオニン)ではATG及びCAT、N(アスパラギン)ではAAC及びGTT、P(プロリン)ではCCA及びTGG、R(アルギニン)ではAGA及びTCT、S(セリン)ではTCT及びAGA、T(スレオニン)ではACT及びAGT、V(バリン)ではGTT及びAAC、W(トリプトファン)ではTGG及びCCA、Y(チロシン)ではTAT及びATAの配列を用いた)。 Furthermore, for the preparation of a clone in which the 337th amino acid of OsNRAMP5 possessed by the mutant 3018W was artificially changed to another amino acid, the primer and primer OsNR5_Q337Xmt_R1025 (5'-GTAATAGTGGAGCTNNNACCAGATGC-3': SEQ ID NO: 10) ) And the primer OsNR5_Q337Xmt_F995 (5'-TGTTGGCATCTGGTnnnAGCTC-3': SEQ ID NO: 11) and the primer of SEQ ID NO: 9, the first half and the second half partial sequences were amplified by PCR from the mutation site of OsNramp5ORF (to the primer names of SEQ ID NOs: 10 and 11). The "X" shown is a one-letter notation for amino acids other than the original Q (glutamine) and genotype K (lysine) of mutant 3018W. Primer sequences corresponding to each amino acid (X), "nnn" of SEQ ID NO: 11 and "NNN" of SEQ ID NO: 10 includes GCA and TGC for A (alanine), TGT and ACA for C (cysteine), GAT and ATC for D (aspartic acid), and GAA, TTC and F (glutamic acid) for E (glutamic acid). TTT and AAA for phenylalanine), GGT and ACC for G (glycine), CAT and ATG for H (histidine), ATT and AAT for I (isoleucine), CTG and GAC for L (leucine), ATG and GAC for M (methionine) and AAC and GTT for CAT and N (asparagin), CCA and TGG for P (proline), AGA and TCT for R (arginine), TCT and AGA for S (serine), ACT and AGT for T (threonine), V (valine) ) Used GTT and AAC, W (tryptophan) used TGG and CCA, and Y (tyrosine) used TAT and ATA sequences).
各DNA断片を、In-Fusionクローニング(タカラバイオ社製、商品名In-Fusionクローニングキット)によって図6に示すpSTV28ベクターのマルチクローニングサイトを導入した酵母発現用ベクターpGADに挿入した。 Each DNA fragment was inserted into a yeast expression vector pGAD into which the multi-cloning site of the pSTV28 vector shown in FIG. 6 was introduced by In-Fusion cloning (manufactured by Takara Bio Inc., trade name In-Fusion cloning kit).
次いで、各コンストラクト及びpGAD(ベクターコントロール)を酢酸リチウム法によって(1)Cd感受性変異株Δycf1(MATa;ura3Δ0;leu2Δ0;his3Δ1;met15Δ0;YDR135c::kanMX4)、(2)Mn要求性変異株Δsmf1(MATa;ura3Δ0;leu2Δ0;his3Δ1;met15Δ0;YOL122c::kanMX4)にそれぞれ導入した。 Then, each construct and pGAD (vector control) were subjected to (1) Cd-sensitive mutant Δycf1 (MATa; ura3Δ0; leu2Δ0; his3Δ1; meta15Δ0; YDR135c :: kanMX4), (2) Mn-requiring mutant Δsmf1 (2) It was introduced into MATa; ura3Δ0; leu2Δ0; his3Δ1; met15Δ0; YOL122c :: kanMX4).
各酵母のアミノ酸要求性に合わせてSD液体培地で培養後、通常のSD寒天培地(-Cd,+Mn)あるいは(1)12.5μM又は15μM CdCl2を含む(+Cd)、(2)20mM又は25mM EGTAを含みMnを利用しにくくした(-Mn)SD寒天培地に希釈系列(OD600= 10-1,10-2,10-3,10-4)を作製してスポットして、30℃で2日間培養した。各遺伝子を導入したCd感受性株(Δycf1)、Mn要求性変異株(Δsmf1)の寒天培地上における±Cd及び±Mn処理による増殖程度から、OsNRAMP5タンパク質の337番目のアミノ酸変異によるMn及びCd輸送能の違いを図7に示した。 After culturing in SD liquid medium according to the amino acid requirement of each yeast, normal SD agar medium (-Cd, + Mn) or (1) containing 12.5 μM or 15 μM CdCl 2 (+ Cd), (2) 20 mM or A diluted series (OD600 = 10 -1 , 10 -2 , 10 -3 , 10 -4 ) was prepared and spotted on an SD agar medium containing 25 mM EGTA and making it difficult to use Mn, and at 30 ° C. Incubated for 2 days. From the degree of proliferation of Cd-sensitive strains (Δycf1) and Mn-requiring mutants (Δsmf1) into which each gene was introduced by treatment with ± Cd and ± Mn on agar medium, the Mn and Cd transport ability by the 337th amino acid mutation of OsNRAMP5 protein The difference between the above is shown in FIG.
図7Aから、Mn輸送能が欠損した酵母株Δsmf1においてOsNramp5を導入した株では-Mn処理区でベクターコントロール(VC)よりも高い増殖が認められ、Mn吸収が回復したことを示す。定性的な評価になるが、OsNRAMP5タンパク質の337番目のアミノ酸変異でもオリジナルのグルタミン(Q)と同等の増殖を示したアミノ酸型は、グルタミン酸(E)、フェニルアラニン(F)、メチオニン(M)、セリン(S)であった。一方で、最も細胞増殖が低かったアミノ酸はアスパラギン酸(D)、プロリン(P)、アルギニン(R)であったがVCと比べると僅かに増殖しており、極弱いながらもMn吸収能を持っていると思われた。それ以外のアミノ酸型は中程度の吸収を示し、その中でもヒスチジン(H)、リシン(K)、チロシン(Y)は酵母の増殖程度が小さいことから、中程度の吸収を示すアミノ酸でもその種類によって、Mn吸収が変化することがわかった。この傾向は、図7Bに示すCd感受性酵母株Δycf1を用いた実験でも確認され、アミノ酸の種類によってCdの吸収(酵母の増殖)は大きく変化していた。 FIG. 7A shows that in the yeast strain Δsmf1 lacking the Mn transport ability, the strain in which OsNramp5 was introduced showed higher proliferation than the vector control (VC) in the -Mn-treated group, indicating that Mn absorption was restored. Although it is a qualitative evaluation, the amino acid types that showed the same growth as the original glutamine (Q) even at the 337th amino acid mutation of the OsNRAMP5 protein were glutamic acid (E), phenylalanine (F), methionine (M), and serine. It was (S). On the other hand, the amino acids with the lowest cell proliferation were aspartic acid (D), proline (P), and arginine (R), but they proliferated slightly compared to VC, and although they were extremely weak, they had Mn absorption capacity. It seemed to be. Other amino acid types show moderate absorption, and among them, histidine (H), lysine (K), and tyrosine (Y) have a small degree of yeast growth. , Mn absorption was found to change. This tendency was also confirmed in an experiment using the Cd-sensitive yeast strain Δycf1 shown in FIG. 7B, and the absorption of Cd (growth of yeast) changed significantly depending on the type of amino acid.
従って、OsNRAMP5タンパク質の337番目のアミノ酸を人工的に変異させることで、Mn及びCd吸収能を自在にコントールできる可能性が示された。 Therefore, it was shown that the ability to absorb Mn and Cd could be freely controlled by artificially mutating the 337th amino acid of the OsNRAMP5 protein.
Claims (13)
(i)配列番号2に示されるアミノ酸配列において、第337番目のグルタミン残基が他のアミノ酸に置換されたアミノ酸配列を含むタンパク質;
(ii)(i)記載のタンパク質のアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含み、且つ第337番目の前記他のアミノ酸を保持するタンパク質であって、イネにおいてカドミウム及びマンガンを輸送するトランスポーター機能を有する、前記タンパク質。 The protein according to claim 1, which is the protein of (i) or (ii) below.
(i) A protein containing an amino acid sequence in which the glutamine residue at position 337 is replaced with another amino acid in the amino acid sequence shown in SEQ ID NO: 2;
(ii) A protein containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence of the protein according to (i) and retaining the other amino acid at position 337, which contains cadmium and manganese in rice. The protein having a transporter function of transporting.
(a)配列番号4に示されるアミノ酸配列を含むタンパク質;
(b)配列番号4に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含み、且つ第337番目のリシン残基を保持するタンパク質であって、該タンパク質をコードする遺伝子を有するイネにおいて、野生型イネ品種と比較してカドミウム及びマンガン吸収を抑制し、且つ該野生型イネ品種と同一の品種である、OsNramp5の機能欠損型遺伝子を有するイネと比較してカドミウム及びマンガン吸収を増加させる、前記タンパク質。 The protein according to claim 2, which is the protein of (a) or (b) below.
(a) Protein containing the amino acid sequence shown in SEQ ID NO: 4;
(b) A protein containing an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having the lysine residue at position 337, and having a gene encoding the protein. In rice, it suppresses the absorption of cadmium and manganese as compared with the wild-type rice cultivar, and absorbs cadmium and manganese as compared with the rice having the function-deficient gene of OsNramp5, which is the same cultivar as the wild-type rice cultivar. The protein to increase.
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