JPH04341187A - New dna sequence and fused gene containing the same sequence - Google Patents
New dna sequence and fused gene containing the same sequenceInfo
- Publication number
- JPH04341187A JPH04341187A JP2419212A JP41921290A JPH04341187A JP H04341187 A JPH04341187 A JP H04341187A JP 2419212 A JP2419212 A JP 2419212A JP 41921290 A JP41921290 A JP 41921290A JP H04341187 A JPH04341187 A JP H04341187A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- sequence
- beta
- glucuronidase
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 33
- 108091028043 Nucleic acid sequence Proteins 0.000 title claims description 5
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 29
- 108010060309 Glucuronidase Proteins 0.000 claims abstract description 25
- 102000053187 Glucuronidase Human genes 0.000 claims abstract description 12
- 230000035945 sensitivity Effects 0.000 claims abstract description 6
- 230000014621 translational initiation Effects 0.000 claims description 12
- 230000004927 fusion Effects 0.000 claims description 8
- 230000000694 effects Effects 0.000 abstract description 20
- 210000001938 protoplast Anatomy 0.000 abstract description 11
- 108020004414 DNA Proteins 0.000 abstract description 10
- 239000013612 plasmid Substances 0.000 abstract description 10
- 102000004190 Enzymes Human genes 0.000 abstract description 9
- 108090000790 Enzymes Proteins 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 6
- 244000068988 Glycine max Species 0.000 abstract description 5
- 235000010469 Glycine max Nutrition 0.000 abstract description 5
- 108090000128 Lipoxygenases Proteins 0.000 abstract description 5
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 5
- 235000009566 rice Nutrition 0.000 abstract description 5
- 108091008146 restriction endonucleases Proteins 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 108020004511 Recombinant DNA Proteins 0.000 abstract description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 abstract description 2
- 210000004748 cultured cell Anatomy 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 230000002463 transducing effect Effects 0.000 abstract 2
- 241000193830 Bacillus <bacterium> Species 0.000 abstract 1
- 240000007594 Oryza sativa Species 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000000977 initiatory effect Effects 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 230000001131 transforming effect Effects 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- 241000208125 Nicotiana Species 0.000 description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 4
- 241000209094 Oryza Species 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000594 mannitol Substances 0.000 description 4
- 235000010355 mannitol Nutrition 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 108091035707 Consensus sequence Proteins 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- ARQXEQLMMNGFDU-JHZZJYKESA-N 4-methylumbelliferone beta-D-glucuronide Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ARQXEQLMMNGFDU-JHZZJYKESA-N 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000008844 regulatory mechanism Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108010029182 Pectin lyase Proteins 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は、新規ＤＮＡ配列及びそ
れを含有する融合遺伝子に関するものであり、更に詳細
には、該ＤＮＡ配列からなる翻訳開始部位配列及びそれ
とレポーター遺伝子とを結合してなる融合遺伝子に関す
るものである。[Industrial Application Field] The present invention relates to a novel DNA sequence and a fusion gene containing the same, and more specifically, a translation initiation site sequence consisting of the DNA sequence and a fusion gene formed by combining the same with a reporter gene. It concerns fusion genes.
【０００２】本発明に係る融合遺伝子は、レポーター遺
伝子として例えばベーターグルクロニダーゼ遺伝子を使
用した場合、ベーターグルクロニダーゼ遺伝子単用に比
してその感度が大幅に上昇する。したがって、一般的に
転写効率が低い一般の遺伝子のプロモーターの検定ない
し活性測定を行うに当り、このように感度の高いレポー
ター遺伝子はきわめて有用である。[0002] When the fusion gene according to the present invention uses, for example, the beta-glucuronidase gene as a reporter gene, its sensitivity is greatly increased compared to when the beta-glucuronidase gene is used alone. Therefore, such a highly sensitive reporter gene is extremely useful for assaying promoters or measuring activity of genes that generally have low transcription efficiency.
【０００３】0003
【従来の技術】一般に、遺伝子発現の調節機構の研究は
、目的の遺伝子をレポーター遺伝子と接続させ、レポー
ター遺伝子の翻訳産物の活性を検定する方法が用いられ
ている。すなわち、ある遺伝子のプロモーターを調査す
る場合、本来のその遺伝子の構造遺伝子部分を用いてそ
の発現量を測定するのが最も良いのであるが、その発現
量が微少である等測定が困難な場合がしばしば生じる。
そこで、その遺伝子のプロモーターの発現量の測定が容
易な遺伝子（レポーター遺伝子）を構造遺伝子と取り替
えて接続し、その発現を調査して、いわば間接的な方法
で目的遺伝子のプロモーターの活性を測定し、遺伝子操
作に利用しているのである。BACKGROUND OF THE INVENTION Generally, research on the regulatory mechanism of gene expression involves connecting a gene of interest with a reporter gene and assaying the activity of the translation product of the reporter gene. In other words, when investigating the promoter of a gene, it is best to measure the expression level using the original structural gene part of the gene, but there are cases where the expression level is so small that it is difficult to measure. Occurs often. Therefore, we connect a gene (reporter gene) whose promoter expression level can be easily measured with the structural gene, investigate its expression, and measure the activity of the target gene's promoter using an indirect method. , and are used for genetic manipulation.
【０００４】現在、植物遺伝子の研究には、このレポー
ター遺伝子としてベーターグルクロニダーゼ遺伝子が広
く利用されている。ベーターグルクロニダーゼ遺伝子は
大腸菌由来であり本来の植物からは検出されていないこ
とから、植物の研究に適している。また、その検定の容
易さやコスト面などからも広く普及するようになった。[0004] Currently, beta-glucuronidase genes are widely used as reporter genes in plant gene research. The beta-glucuronidase gene is derived from Escherichia coli and has not been detected in native plants, making it suitable for plant research. It has also become widely popular due to its ease of testing and cost.
【０００５】しかし、ベーターグルクロニダーゼ遺伝子
は大腸菌由来であるため、その翻訳開始部位は植物の翻
訳開始部位のコンセンサス配列と異なっている。そのた
め植物細胞内でのベーターグルクロニダーゼ遺伝子のｍ
ＲＮＡの翻訳効率は低い。ベーターグルクロニダーゼ遺
伝子は、転写効率が高い３５Ｓプロモーターに接続した
場合は翻訳効率が低くても実用的には問題のないレポー
ター遺伝子として用いることができるが、一般の遺伝子
のプロモーターを接続した場合は転写効率が低いため検
定が困難になることが考えられる。[0005] However, since the beta-glucuronidase gene is derived from Escherichia coli, its translation initiation site differs from the consensus sequence of plant translation initiation sites. Therefore, m of the beta-glucuronidase gene in plant cells.
The translation efficiency of RNA is low. If the beta-glucuronidase gene is connected to the 35S promoter, which has high transcription efficiency, it can be used as a reporter gene with no practical problems even if the translation efficiency is low, but if it is connected to the promoter of a general gene, the transcription efficiency will be low. It is conceivable that the test will be difficult due to the low value.
【０００６】[0006]
【発明が解決しようとする課題】本発明が解決しようと
する課題は、感度の高いすぐれたレポーター遺伝子を開
発する点である。[Problem to be Solved by the Invention] The problem to be solved by the present invention is to develop an excellent reporter gene with high sensitivity.
【０００７】[0007]
【課題を解決するための手段】本発明は上記目的を達成
するためになされたものであって、各方面から検討した
結果、植物遺伝子の翻訳開始部位にコンセンサス配列が
見出されることに着目した。[Means for Solving the Problems] The present invention was made to achieve the above object, and as a result of various studies, it was noticed that a consensus sequence was found at the translation initiation site of a plant gene.
【０００８】そこで本発明者らは、大豆リポキシゲナー
ゼ遺伝子間で保持されている翻訳開始部位に含まれる配
列ＧＣＡＡＡＧＡＴＧＴＴＴに新たに着目した。[0008] Therefore, the present inventors newly focused on the sequence GCAAAGATGTTT contained in the translation initiation site that is conserved between soybean lipoxygenase genes.
【０００９】そこで、このコンセンサス配列を合成し、
ベーターグルクロニダーゼ遺伝子の上流に挿入した。こ
のプラスミドを調製し、タバコとイネのプロトプラスト
に電気的に導入しベーターグルクロニダーゼ活性を測定
した。その結果、両植物細胞内でともにベーターグルク
ロニダーゼ活性が改良前のプラスミドを使用した場合に
比べ、少なくとも１０倍以上上昇していることを確認し
、本発明の完成に至った。[0009] Therefore, this consensus sequence was synthesized,
It was inserted upstream of the beta-glucuronidase gene. This plasmid was prepared and electrically introduced into tobacco and rice protoplasts to measure beta-glucuronidase activity. As a result, it was confirmed that the beta-glucuronidase activity in both plant cells was at least 10 times higher than when using the plasmid before improvement, leading to the completion of the present invention.
【００１０】すなわち本発明は、ＧＣＡＡＡＧＡＴＧＴ
ＴＴからなる配列、及び該配列とレポーター遺伝子とを
結合してなる融合遺伝子に関するものである。以下、本
発明について詳述する。[0010] That is, the present invention provides GCAAAGATGT
This invention relates to a sequence consisting of TT and a fusion gene formed by linking the sequence and a reporter gene. The present invention will be explained in detail below.
【００１１】本発明に係るＤＮＡ配列、ＧＣＡＡＡＧＡ
ＴＧＴＴＴ、は大豆リポキシゲナーゼ遺伝子から制限酵
素等を用いて切り出してもよいし、ＤＮＡ合成機を用い
て合成してもよい。DNA sequence according to the invention, GCAAAGA
TGTTT may be excised from the soybean lipoxygenase gene using a restriction enzyme or the like, or may be synthesized using a DNA synthesizer.
【００１２】このようにして調製した翻訳開始部位配列
ＧＣＡＡＡＧＡＴＧＴＴＴは、レポーター遺伝子を含む
プラスミド内に導入する。レポーター遺伝子としては各
種のものが広範に使用できるが、例えばベーターグルク
ロニダーゼ遺伝子を使用する場合には、それを含有する
プラスミドが市販されており（ｐＢＩ２２１：クローン
テック社）、該プラスミドが有利に使用される。The translation initiation site sequence GCAAAGATGTTT thus prepared is introduced into a plasmid containing a reporter gene. A wide variety of reporter genes can be used; for example, when using the beta-glucuronidase gene, a plasmid containing it is commercially available (pBI221: Clontech), and this plasmid can be used advantageously. Ru.
【００１３】得られた組換え体ＤＮＡを用いて、大腸菌
等の宿主微生物を形質転換し、目的とする形質転換株を
選択し、翻訳開始部位配列とレポーター遺伝子とを結合
させた融合遺伝子をクローニングし、例えばベーターグ
ルクロニダーゼ遺伝子の場合にはｐＳＬＢＩ２２１クロ
ーンを得る。そこで、このクローンを培養し、常法にし
たがってＤＮＡを調製する。[0013] Using the obtained recombinant DNA, a host microorganism such as Escherichia coli is transformed, the desired transformed strain is selected, and a fusion gene in which the translation initiation site sequence and reporter gene are linked is cloned. For example, in the case of beta-glucuronidase gene, pSLBI221 clone is obtained. Therefore, this clone is cultured and DNA is prepared according to a conventional method.
【００１４】一方、タバコ、イネ等の培養細胞から酵素
処理等常法にしたがってプロトプラストを調製しておき
、これに上記で得たＤＮＡをエレクトロポーレーション
法等常法にしたがって導入する。[0014] On the other hand, protoplasts are prepared from cultured cells of tobacco, rice, etc. according to conventional methods such as enzyme treatment, and the DNA obtained above is introduced into them according to conventional methods such as electroporation.
【００１５】このプロトプラストを培養した後、これを
破砕して酵素液となし、その酵素の活性を蛍光法その他
既知の方法で測定する。After culturing the protoplasts, they are crushed to obtain an enzyme solution, and the activity of the enzyme is measured by a fluorescence method or other known method.
【００１６】このようにしてレポーター遺伝子の発現、
例えばベーターグルクロニダーゼの酵素活性、を測定す
ることにより、結果として本来の遺伝子のプロモーター
の活性を測定することができるのである。そしてこの場
合、前記したようにベーターグルクロニダーゼの活性は
、翻訳開始部位配列を用いることによって、それを用い
ない場合に比して１０倍以上も上昇しているため、レポ
ーター遺伝子の感度の大幅上昇が達成され、その結果と
してプロモーターの活性の測定がきわめて容易に行われ
ることになるのである。[0016] In this way, the expression of the reporter gene,
For example, by measuring the enzyme activity of beta-glucuronidase, it is possible to measure the activity of the promoter of the original gene. In this case, as mentioned above, the activity of beta-glucuronidase is increased more than 10 times by using the translation initiation site sequence compared to when it is not used, so the sensitivity of the reporter gene is significantly increased. As a result, promoter activity can be measured very easily.
【００１７】[0017]
【００１８】[0018]
【Ａ．実験に供した試薬】制限酵素ＢａｍＨＩ，Ｘｂａ
Ｉは東洋紡から、ライゲーションキット、シークエンス
キットは宝酒造、〔α−３２Ｐ〕ｄＣＴＰはＡｍｅｒｓ
ｈａｍ社、ＤＮＡ合成用試験薬はＡＢＩ社よりそれぞれ
購入した。[A. Reagents used in the experiment] Restriction enzymes BamHI, Xba
I is from Toyobo, ligation kit and sequencing kit are from Takara Shuzo, [α-32P] dCTP is from Amers
The test drug for DNA synthesis was purchased from Ham and ABI.
【００１９】[0019]
【Ｂ．実験材料】ベーターグルクロニダーゼ遺伝子を含
むプラスミドｐＢＩ２２１は、Ｃｌｏｎｔｅｃｈ社より
購入した。[B. Experimental Materials: Plasmid pBI221 containing the beta-glucuronidase gene was purchased from Clontech.
【００２０】[0020]
【００２１】[0021]
【１．融合遺伝子の作成】大豆リポキシゲナーゼ遺伝子
の翻訳開始部位ＧＣＡＡＡＧＡＴＧＴＴＴはＤＮＡ合成
機を用いて合成した。ｐＢＩ２２１を制限酵素ＢａｍＨ
Ｉ及びＸｂａＩで消化してから１％アガロースゲル電気
泳動を行いゲルから回収した。これを上記の翻訳開始部
位ＤＮＡとライゲーションさせ、これを用いて大腸菌Ｈ
Ｂ１０１株を形質転換させた。目的のものが作成されて
いることを確認するために、ベーターグルクロニダーゼ
遺伝子から上流に向かって塩基配列を決定し、大豆リポ
キシゲナーゼ遺伝子の翻訳開始部位を含むものを選択し
た。このクローン（以下クローンと呼ぶ）をｐＢＩ２２
１／ｓｌと命名し、以下の実験に用いた。[1. Creation of fusion gene: The translation initiation site GCAAAGATGTTT of the soybean lipoxygenase gene was synthesized using a DNA synthesizer. pBI221 with restriction enzyme BamH
After digestion with I and XbaI, 1% agarose gel electrophoresis was performed and the residue was collected from the gel. This was ligated with the above translation start site DNA and used to infect E. coli H.
The B101 strain was transformed. To confirm that the desired product had been created, the base sequence was determined upstream from the beta-glucuronidase gene, and one containing the translation initiation site of the soybean lipoxygenase gene was selected. This clone (hereinafter referred to as clone) was used as pBI22
It was named 1/sl and used in the following experiment.
【００２２】[0022]
【２．プラスミドＤＮＡの調製】クローンを５０ｍｌの
５０μｇ／ｍｌのアンピシリンを含むＬＢ培地に接種し
３７℃で一晩培養した。この培養液から遠心分離で大腸
菌を回収し、アルカリリシス法でＤＮＡを調製した。こ
のＤＮＡを超遠心法で精製し以下の実験に用いた。[2. Preparation of plasmid DNA: The clone was inoculated into 50 ml of LB medium containing 50 μg/ml ampicillin and cultured overnight at 37°C. Escherichia coli was recovered from this culture solution by centrifugation, and DNA was prepared by alkaline lysis. This DNA was purified by ultracentrifugation and used in the following experiments.
【００２３】[0023]
【３．プロトプラストの調製】タバコ及びイネの培養細
胞を遠心分離で集め、マンニトール溶液を加え懸濁する
。さらに遠心分離した後、酵素セルラーゼ、ペクトリア
ーゼを含むマンニトール溶液を加え、３０℃で１時間酵
素処理する。処理後、遠心分離で酵素液を除き、マンニ
トール溶液を加え懸濁する。マンニトール溶液で細胞を
洗浄後、細胞濃度を測定し、遺伝子導入実験に用いた。[3. Preparation of protoplasts: Cultured tobacco and rice cells are collected by centrifugation, and a mannitol solution is added to suspend them. After further centrifugation, a mannitol solution containing the enzymes cellulase and pectolyase is added, and enzyme treatment is performed at 30°C for 1 hour. After treatment, the enzyme solution is removed by centrifugation, and a mannitol solution is added and suspended. After washing the cells with a mannitol solution, the cell concentration was measured and used in gene transfer experiments.
【００２４】[0024]
【４．プロトプラストへの導入及びベーターグルクロニ
ダーゼ活性の測定】プロトプラストをエレクトロポーレ
ーション用バッファーに懸濁し、導入するプラスミドＤ
ＮＡ溶液を加える。これらに電気パルスをかけ、プロト
プラストにＤＮＡを導入する。このプロトプラストをＬ
ＳＤ培地で２４時間培養後、プロトプラストを集めベー
ターグルクロニダーゼ活性測定用バッファーに懸濁し、
超音波破砕する。この粗酵素液に基質である４−メチル
ウンベリフェリル グルクロナイド（ＭＵＧ）を
加え、３７℃で１時間酵素反応を行なわせ、炭酸ナトリ
ウム溶液を加え反応を停止させた後、蛍光光度計で活性
を測定した。その結果、タバコとイネの双方の細胞内で
ともにベーターグルクロニダーゼ活性が改良前のプラス
ミドを使用した場合に比べ、少なくとも１０倍以上上昇
していることが確認された。[4. Introduction into protoplasts and measurement of beta-glucuronidase activity] Plasmid D to be introduced into protoplasts by suspending them in electroporation buffer
Add NA solution. Electric pulses are applied to these to introduce DNA into protoplasts. This protoplast is L
After culturing in SD medium for 24 hours, protoplasts were collected and suspended in beta-glucuronidase activity measurement buffer.
Ultrasonic crush. The substrate 4-methyl umbelliferyl glucuronide (MUG) was added to this crude enzyme solution, the enzyme reaction was carried out at 37°C for 1 hour, the reaction was stopped by adding sodium carbonate solution, and the activity was measured using a fluorometer. It was measured. As a result, it was confirmed that the beta-glucuronidase activity in both tobacco and rice cells was increased by at least 10 times compared to when the plasmid before improvement was used.
【００２５】[0025]
【発明の効果】本発明にしたがって翻訳開始部位配列を
レポーター遺伝子の直前に挿入することにより、レポー
ター遺伝子を改良してその感度を大幅に上昇せしめるこ
とができる。Effects of the Invention According to the present invention, by inserting a translation initiation site sequence immediately before a reporter gene, the reporter gene can be improved and its sensitivity can be greatly increased.
【００２６】したがって、発現の測定が容易に行えるレ
ポーター遺伝子と該配列とを結合しておき、この融合遺
伝子をある遺伝子の構造遺伝子部分と置換してこのレポ
ーター遺伝子の発現を測定することにより、当該遺伝子
のプロモーターの活性を測定することができ、その結果
、遺伝子発現の調節機構の研究や遺伝子操作技術の開発
に大いに役立つものである。[0026] Therefore, by combining a reporter gene whose expression can be easily measured with the sequence, replacing this fused gene with the structural gene part of a certain gene, and measuring the expression of this reporter gene, the expression of the reporter gene can be easily measured. The activity of a gene's promoter can be measured, and as a result, it is of great use in research on regulatory mechanisms of gene expression and in the development of gene manipulation techniques.
【００２７】また本発明によればベーターグルクロニダ
ーゼ遺伝子のほか各種の遺伝子をレポーター遺伝子とし
て広く使用することができるので、上記効果が更に増進
されるだけでなく、植物体内においてレポーター遺伝子
自体の発現量が大幅に上昇するので、ベーターグルクロ
ニダーゼ等レポーター遺伝子に由来する蛋白質やペプチ
ドの大量生産も可能であるし、更にまた本発明はプロモ
ーター活性の測定方法としても有用である。[0027] Furthermore, according to the present invention, various genes other than the beta-glucuronidase gene can be widely used as reporter genes, so that not only the above effects are further enhanced, but also the expression level of the reporter gene itself is reduced in the plant body. Since the amount increases significantly, it is possible to mass-produce proteins and peptides derived from reporter genes such as beta-glucuronidase, and the present invention is also useful as a method for measuring promoter activity.
Claims (4)
ＮＡ配列。[Claim 1] D consisting of GCAAAGATGTTT
NA sequence.
ポーター遺伝子の感度上昇用翻訳開始部位配列。2. A translation initiation site sequence for increasing the sensitivity of a reporter gene consisting of GCAAAGATGTTT.
ター遺伝子とを結合してなる融合遺伝子。3. A fusion gene obtained by linking the DNA sequence according to claim 1 and a reporter gene.
ニダーゼである請求項２又は３に記載の配列又は遺伝子
。4. The sequence or gene according to claim 2 or 3, wherein the reporter gene is beta-glucuronidase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2419212A JP2987943B2 (en) | 1990-12-14 | 1990-12-14 | Novel DNA sequence and fusion gene containing it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2419212A JP2987943B2 (en) | 1990-12-14 | 1990-12-14 | Novel DNA sequence and fusion gene containing it |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04341187A true JPH04341187A (en) | 1992-11-27 |
| JP2987943B2 JP2987943B2 (en) | 1999-12-06 |
Family
ID=18526864
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2419212A Expired - Fee Related JP2987943B2 (en) | 1990-12-14 | 1990-12-14 | Novel DNA sequence and fusion gene containing it |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2987943B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058075A (en) * | 2017-06-20 | 2017-08-18 | 商丘师范学院 | A kind of plant protoplast purifying instrument and purification process |
-
1990
- 1990-12-14 JP JP2419212A patent/JP2987943B2/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107058075A (en) * | 2017-06-20 | 2017-08-18 | 商丘师范学院 | A kind of plant protoplast purifying instrument and purification process |
| CN107058075B (en) * | 2017-06-20 | 2023-10-20 | 商丘师范学院 | Plant cell protoplast purification instrument and purification method |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2987943B2 (en) | 1999-12-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shi et al. | Transcriptional repression by YY1, a human GLI-Krüippel-related protein, and relief of repression by adenovirus E1A protein | |
| Lam et al. | Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. | |
| CN109477115B (en) | Expression system for eukaryotes | |
| CA2332628C (en) | Seed-preferred promoters | |
| Selleck et al. | Photofootprinting in vivo detects transcription-dependent changes in yeast TATA boxes | |
| Strömvik et al. | A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs | |
| US6177613B1 (en) | Seed-preferred promoter | |
| JP3409079B2 (en) | Peptides having the function of suppressing gene transcription | |
| JP3407035B2 (en) | Peptides having the function of suppressing gene transcription | |
| JP2001269178A (en) | Peptide having function to suppress transcription of gene | |
| CN1795267B (en) | Stress-induced promoter and method of using the same | |
| JP2001269177A (en) | Peptide having function to suppress transcription of gene | |
| WO2008138579A1 (en) | Archaeal plasmid vector system | |
| Schallau et al. | Phylogenetic footprints in fern spore‐and seed‐specific gene promoters | |
| Anderson | Expression and purification of the adenovirus proteinase polypeptide and of a synthetic proteinase substrate | |
| JPH04341187A (en) | New dna sequence and fused gene containing the same sequence | |
| ZA200503562B (en) | Auxin-repressed, dormancy-associated promoter and uses thereof | |
| CA2809828A1 (en) | Regulatory element for heterologous protein production in the fruiting body of filamentous fungi | |
| EP0938572B1 (en) | Promoter from tobacco | |
| JPH06311884A (en) | Plasmid and escherichia coli transformed with the same | |
| JPH06228193A (en) | Trans-acting factor i | |
| US11913002B2 (en) | Modified plant endosperm specific promoter and use thereof | |
| CN116751792B (en) | Transcription factor downstream gene screening method | |
| US20030033628A1 (en) | Floral homeotic genes for manipulation of flowering in poplar and other plant species | |
| JPH08103278A (en) | Production of active human alt |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |