JPH04126088A - Dna fragment having promoter activity - Google Patents
Dna fragment having promoter activityInfo
- Publication number
- JPH04126088A JPH04126088A JP2244779A JP24477990A JPH04126088A JP H04126088 A JPH04126088 A JP H04126088A JP 2244779 A JP2244779 A JP 2244779A JP 24477990 A JP24477990 A JP 24477990A JP H04126088 A JPH04126088 A JP H04126088A
- Authority
- JP
- Japan
- Prior art keywords
- gene
- base sequence
- dna fragment
- promoter activity
- promoter
- 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
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- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 13
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Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、植物細胞内等で異種蛋白遺伝子を発現させる
時、好適に使用されるプロモーター活性を保持するＤＮ
Ａ断片、該ＤＮＡ断片か導入されたベクター及び該ベク
ターにより形質転換された宿主に関する。Detailed Description of the Invention [Industrial Application Field] The present invention provides a DNA that retains promoter activity and is suitably used when expressing a heterologous protein gene in plant cells.
The present invention relates to a fragment A, a vector into which the DNA fragment has been introduced, and a host transformed with the vector.
〔従来技術及び発明が解決しようとする課題〕遺伝子組
換え技術の分野では、遺伝子の発現量を増大させるため
に強力なプロモーターにより、産生ｍＲＮＡ量を増大さ
せることが行われている。[Prior Art and Problems to be Solved by the Invention] In the field of genetic recombination technology, in order to increase the amount of gene expression, strong promoters are used to increase the amount of mRNA produced.
植物においては、その様な例としてカルフラワーモザイ
クウィルス３５ ｓ （ＣａＭＶ３５ ｓ）プロモータ
ーが多用されている。しかしながら、このプロモーター
を使用して異種蛋白遺伝子を発現させた場合、必ずしも
期待通りの発現量か得られないことが多い。そこで、更
に強力なプロモーターの開発か望まれていた。In plants, the caulflower mosaic virus 35s (CaMV35s) promoter is frequently used as such an example. However, when a heterologous protein gene is expressed using this promoter, the expected expression level is often not always obtained. Therefore, there was a desire to develop an even more powerful promoter.
ところで、ペルオキシダーゼには、多くのアイソザイム
が存在すると言われ、ペルオキシダーゼを高生産する西
洋ワサビの培養細胞か既に獲得されている［ Ｙａｍａ
ｄａ、 Ｙ、、 ｅｔ ａｌ、、 Ｊ、Ｃｈｅｍ、Ｔｅ
ｃｈ、Ｂｉｏｃｈ、、３８．３１（１９８７）］。また
、この西洋ワサビ培養細胞から得たベルオキシダーゼア
イソサイムのゲノム遺伝子の構造か決定され、その５°
上流にプロモーター配列が存在することか確認されてい
る（昭和６２年度日本醗酵工学会講演要旨集、 ｐ２＋
）。By the way, it is said that there are many isozymes of peroxidase, and cultured horseradish cells that produce high peroxidase have already been obtained [Yama
da, Y., et al., J. Chem. Te.
ch, Bioch, 38.31 (1987)]. In addition, the structure of the genome gene of peroxidase isocyme obtained from cultured horseradish cells was determined, and its 5°
It has been confirmed that there is a promoter sequence upstream (Collection of abstracts from the 1985 Japanese Society of Fermentation Engineering, p2+
).
本発明者らは、これらの事情に鑑み、各種研究を重ねた
結果、この西洋ワサビの培養細胞から、多種のペルオキ
シダーゼアイソザイム遺伝子のクローンを取得し、各ク
ローンのプロモーターを検索したところ、高いプロモー
ター活性を有するＤＮＡ断片を見い出し、本発明の完成
に至った。In view of these circumstances, the present inventors conducted various studies and obtained clones of various peroxidase isozyme genes from cultured cells of this horseradish, and searched for the promoter of each clone, and found that the promoter activity was high. A DNA fragment having the following was discovered, leading to the completion of the present invention.
即ち、本発明の特徴は、第１図に示す全部もしくは一部
の塩基配列、またはそれらと均等な塩基配列を有し、プ
ロモーター活性を有するＤＮＡ断片、該ＤＮＡが挿入さ
れたベクター及び該ベクターにより形質転換された宿主
に存する。That is, the features of the present invention include a DNA fragment having all or a part of the base sequence shown in FIG. 1 or a base sequence equivalent thereto and having promoter activity, a vector into which the DNA has been inserted, and present in the transformed host.
以下にこれを詳述する。This will be explained in detail below.
本発明のＤＮＡ断片は次のようにして調製できる。The DNA fragment of the present invention can be prepared as follows.
まず、本発明の出発遺伝子である各種ペルオキシダーゼ
アイソザイム遺伝子は西洋ワサビの培養細胞から常法に
従い、例えば、Ｍｏ１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ。First, various peroxidase isozyme genes, which are the starting genes of the present invention, are obtained from cultured horseradish cells according to conventional methods, for example, Molecular Cloning.
（１９８９）、 ｐ、８．３．（Ｃｏｌｄ Ｓｐｒｉｎ
ｇ Ｈａｒｂｏｒ Ｌａｂｓ、）に記載の方法に従い、
ｃＤＮＡライブラリーを調製し、Ｗｅｌ １ｎｄｅｒの
報告したＦＥＢＳ Ｌｅｔｔｅｒｓ、 （１９７６）。(1989), p, 8.3. (Cold Spring
According to the method described in G Harbor Labs, )
A cDNA library was prepared and the FEBS Letters reported by Wel 1nder (1976).
７２、１９記載の西洋ワサビのペルオキシダーゼのアミ
ノ酸配列に基づき調製したＤＮＡプローブを用いて、遺
伝子を選択することができる。また、例えば、Ｍｏ１ｅ
ｃｕｌａｒ Ｃｌｏｎｉｎｇ、 （１９８９）、 ｐ、
９．４．（ＣｏｌｄＳｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａ
ｂｓ、）に記載の方法に従い、西洋ワサビの培養細胞か
らゲノムライブラリーを調製し、既に得られているアイ
ソザイム遺伝子をプローブにして、更に多くのペルオキ
シダーゼアイソザイム遺伝子を選択することができる。Genes can be selected using a DNA probe prepared based on the amino acid sequence of horseradish peroxidase described in 72 and 19. Also, for example, Mo1e
cular Cloning, (1989), p.
9.4. (ColdSpring Harbor La
A genomic library is prepared from cultured horseradish cells according to the method described in BS, ), and more peroxidase isozyme genes can be selected by using already obtained isozyme genes as probes.
なお、これらのペルオキシダーゼアイソザイム遺伝子の
選択方法は、既に本発明者等により発表されている。The method for selecting these peroxidase isozyme genes has already been published by the present inventors.
すなわち、Ｆｕｊｉｙａｍａ ｅｔ ａｌ、、 Ｅｕｒ
、Ｊ、Ｂｉｏｃｈｅｍ、 １７３゜（１９８８）、 ６
８１−６８７において、ｐｒＸｃｌａ及びｐｒｘｃｌｂ
と命名されたペルオキシダーゼアイソザイム遺伝子が単
離されたことが記載されており、またＦｕｊｉｙａｍａ
ｅｔ ａｌ、、 Ｇｅｎｅ、 ８９． （１９９０）
、 １６３−１６９において、ｐｒｘＣ２及びｐｒｘ
ｃ３と命名されたペルオキシダーゼアイソザイム遺伝子
か単離されたことが記載されている。Namely, Fujiyama et al.
, J.Biochem, 173° (1988), 6
81-687, prXcla and prxclb
It has been described that a peroxidase isozyme gene named
et al., Gene, 89. (1990)
, 163-169, prxC2 and prx
It has been described that a peroxidase isozyme gene named c3 was isolated.
本発明のＤＮＡ断片は、この様にして単離しただｐｒｘ
Ｃ！ａ、ｐｒｘｃｌｂ、ｐｒｘＣ２及びｐｒｘＣ３等の
クローンを種々の制限酵素によって切断し、異種蛋白を
発現させ、その量を測定することによって、最も高いプ
ロモーター活性を示した５′非翻訳領域の一部である。The DNA fragment of the present invention was isolated in this way, and
C! This is the part of the 5' untranslated region that showed the highest promoter activity by cutting clones such as a, prxclb, prxC2, and prxC3 with various restriction enzymes, expressing heterologous proteins, and measuring the amount. .
ｐｒｘｃｌａの５′非翻訳領域については、制限酵素Ｘ
ｂａＩ（塩基配列−５２５）と制限酵素ＥｃｏＲ１（塩
基配列−５）で切断した断片を調製した。For the 5' untranslated region of prxcla, restriction enzyme
A fragment was prepared by cutting with baI (base sequence -525) and restriction enzyme EcoR1 (base sequence -5).
ｐｒｘｃｌｂについては、制限酵素ＥｃｏＲＩ （塩基
配列−５３３）と制限酵素Ｎ５ｉＩ（塩基配列＋１）で
切断した断片を調製した。For prxclb, a fragment was prepared by cutting with restriction enzyme EcoRI (base sequence -533) and restriction enzyme N5iI (base sequence +1).
ｐｒｘＣ２については、制限酵素ＸｂａＩ（塩基配列−
５２７）と制限酵素Ｎ５ｉＩ（塩基配列＋５）で切断し
た断片を調製した。For prxC2, restriction enzyme XbaI (nucleotide sequence -
527) and restriction enzyme N5iI (base sequence +5) to prepare a fragment.
ｐｒｘＣ３については、制限酵素ＥｃｏＲ［（塩基配列
−４８４）と制限酵素Ｈｉｎｄｌ［Ｉ （塩基配列＋６
３）で切断し、更に修飾酵素Ｅｘｏｎｕｃｕｌｅａｓｅ
Ｉ［［でＨｉｎｄｎ［サイトから−５の位置まで分解し
た断片を調製した。For prxC3, restriction enzyme EcoR [(base sequence -484) and restriction enzyme Hindl[I (base sequence +6
3), and further modified with the modification enzyme Exonuclease.
A fragment digested from the Hindn[ site to the -5 position was prepared with I[[.
これらの各遺伝子の５“非翻訳領域のプロモーター活性
の検索は、レポータ遺伝子としてベーターグルクロニナ
ーゼ（ＧＵＳ）遺伝子を用い、それと５′非翻訳領域と
のキメラ遺伝子を作製し、それらとｐＵＣ系プラスミド
またはｐＢＩ系プラスミドとから発現プラスミドを構築
し、これらを植物プロトプラストに導入することにより
、または、植物形質転換体を作製し、ＧＵＳ遺伝子の発
現量を測定することにより行うことができる。その結果
、第１図に示すｐｒｘＣ２の５′非翻訳領域がＣａＭＶ
３５ｓプロモーターよりも高い活性を示し、更にｐｒｘ
ｃｌａ、ｐｒｘｃｌｂ、ｐｒｘＣ３の５′非翻訳領域よ
りも高い活性を示すことが見出された。To search for the promoter activity of the 5' untranslated region of each of these genes, we used the beta-glucuroninase (GUS) gene as a reporter gene, created a chimera gene between it and the 5' untranslated region, and inserted them into a pUC-based plasmid. Alternatively, it can be carried out by constructing an expression plasmid from the pBI-based plasmid and introducing these into plant protoplasts, or by producing a plant transformant and measuring the expression level of the GUS gene.As a result, The 5' untranslated region of prxC2 shown in Figure 1 is CaMV
It shows higher activity than the 35s promoter and also prx
It was found that it exhibited higher activity than the 5' untranslated regions of cla, prxclb, and prxC3.
本発明では、第１図に示すＤＮＡ塩基配列は、プロモー
ター活性を損なわない範囲内で一部を削除あるいは改変
してもよい。In the present invention, a portion of the DNA base sequence shown in FIG. 1 may be deleted or modified within a range that does not impair promoter activity.
本発明のプロモーター活性を有するＤＮＡ断片はｐＵＣ
系プラスミド、ｐＢＩ系プラスミド等のプラスミドベク
ター中に挿入され、異種蛋白遺伝子を発現させるための
ベクターとして使用される。The DNA fragment having promoter activity of the present invention is pUC
It is inserted into a plasmid vector such as a pBI-based plasmid or a pBI-based plasmid, and used as a vector for expressing a heterologous protein gene.
プロモーター活性を有するＤＮＡ断片の各種プラスミド
への挿入は、ＤＮＡ組換えの一般的方法、例えば、Ｍｏ
１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ、 （１９８９）、（
Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｓ、）
に記載の方法に従って行うことができる。例えばｐｒｘ
Ｃ２の５°非翻訳領域を含むＸｂａ Ｉ ／Ｎｓｉ Ｉ
断片はｐＵｃ１８のＸｂａ ＩとＰｓｔＩで切断したマ
ルチクローニングサイトとライゲーションすれば、プラ
スミドｐＵｃ１８に挿入することができる。Insertion of DNA fragments with promoter activity into various plasmids can be carried out using common methods of DNA recombination, such as Mo
1ecular Cloning, (1989), (
Cold Spring Harbor Labs,)
It can be carried out according to the method described in . For example prx
Xba I/Nsi I containing the 5° untranslated region of C2
The fragment can be inserted into plasmid pUc18 by ligating it with the multiple cloning site of pUc18 cut with Xba I and Pst I.
本発明のＤＮＡ断片を含有するベクターは、ベーターグ
ルクロニナーゼ（ＧＵＳ） 、ペルオキシダーゼ、アス
コルビン酸オキシダーゼ、ルシフェラーゼ等各種動植物
、酵母等真核生物由来の遺伝子を発現させるのに好適で
ある。Vectors containing the DNA fragments of the present invention are suitable for expressing genes derived from various animals and plants, yeast, and other eukaryotes, such as beta-glucuroninase (GUS), peroxidase, ascorbate oxidase, and luciferase.
また、本発明のベクターは、植物細胞または、酵母細胞
等の宿主を形質転換するのに好適に用いられる。Furthermore, the vector of the present invention is suitably used to transform hosts such as plant cells or yeast cells.
以下に実施例を示し、本発明を更に詳しく説明するが、
本発明はその要旨を越えない限り以下の実施例に於いて
限定されるものではない。Examples will be shown below to explain the present invention in more detail.
The present invention is not limited to the following examples unless it exceeds the gist thereof.
実施例１
［１］ペルオキシダーゼアイソザイム遺伝子の調製■
西洋ワサビゲノムＤＮＡライブラリーの調製西洋ワサビ
の茎から誘導された細胞培養（Ｆｕｊｉｙａｍａ ｅｔ
ａｌ、、 ５ｔｒｕｃｔｕｒｅ ｏｆ ｔｈｅ ｈｏ
ｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅ ｉｓｏｚｙ
ｍｅ ｃ ｇｅｎｅｓ、 Ｅｕｒ、、Ｊ、Ｂｉｏｃｈｅ
ｍ。Example 1 [1] Preparation of peroxidase isozyme gene■
Preparation of horseradish genomic DNA library Cell culture derived from horseradish stems (Fujiyama et al.
al,, 5structure of the ho
rseradishperoxidase isozy
me c genes, Eur,, J, Bioche
m.
１７３、 （１９８８）、 ６８］−６８７）から［Ｂ
１１ｎ ａｎｄ ５ｔａｆｆｏｒｄＮｕｃｌｅｉｃ Ａ
ｃ１ｄｓ Ｒｅｓ、３． （１９７６）、 ２３０３−
２３０８］の方法で全ＤＮＡを調製した。次にこれらを
Ｍａｎｉａｔｉｓの方法で制限酵素５ａｕ３Ａ Ｉで部
分分解した。この部分分解断片を１０〜４０％シュクロ
ース密度勾配遠心分離（３３，０００ｒｐｍ、 １６時
間）で分画し、１０〜２０ｋｂの長さのＤＮＡ断片を回
収した。これを更にアルカリフォスファターゼで処理し
、脱リン化した後、あらかじめＢａｍＨＩと５ａｌＩで
処理したλＥＭＢＬ４（プロメガ製）とライゲーション
し、Ｈｏｒｎ（１９７９）の方法によりパッケージング
した。組換えファージで感染のためＥ、ｃｏｌｉ ＮＭ
５３９を使用した。173, (1988), 68]-687) to [B
11n and 5taffordNucleic A
c1ds Res, 3. (1976), 2303-
Total DNA was prepared using the method described in [2308]. Next, these were partially digested with restriction enzyme 5au3A I according to the method of Maniatis. This partially degraded fragment was fractionated by 10-40% sucrose density gradient centrifugation (33,000 rpm, 16 hours), and DNA fragments with a length of 10-20 kb were recovered. This was further treated with alkaline phosphatase to dephosphorylate, and then ligated with λEMBL4 (manufactured by Promega) that had been previously treated with BamHI and 5alI, and packaged by the method of Horn (1979). E. coli NM for infection with recombinant phages
539 was used.
■ 西洋ワサビペルオキシダーゼゲノムの選択西洋ワサ
ビペルオキシダーゼのｃＤＮＡクローンをｐｓＫｌ （
上記の文献の、Ｐｓｔｌで切り出した１、３ｋｂのＤＮ
Ａ）をマルチプライムＤＮＡラベリング系（Ａｍｅｒｓ
ｈａｍ）を使用して０Ｐでラベルした。■ Selection of the horseradish peroxidase genome A cDNA clone of horseradish peroxidase was extracted from psKl (
1.3 kb DN of the above document extracted with Pstl
A) using the multi-prime DNA labeling system (Amers
ham) and labeled with 0P.
Ｅ、ｃｏｌｊ ＮＭ５３９に感染させて生じたプラーク
をナイロン膜に移した後、上記のラベルしたｃＤＮＡプ
ローブでプラークハイブリダイゼーションを行った。ポ
ジティブクローンを精製後、ファージＤＮＡの制限酵素
切断断片をサザーンブロツティングを行い、ｃＤＮＡと
ハイブリダイズするＤＮＡ断片をｐＵｃ１９にサブクロ
ーンした。After the plaques generated by infection with E. colj NM539 were transferred to a nylon membrane, plaque hybridization was performed using the labeled cDNA probe described above. After purifying the positive clone, the restriction enzyme-cleaved fragment of the phage DNA was subjected to Southern blotting, and the DNA fragment that hybridized with the cDNA was subcloned into pUc19.
ｐｓＫｌに挿入されている″！Ｐ−ラベルｃＤＮＡとハ
イブリダイズする９個の組換えλＥＭＢ Ｌ４ファージ
を６Ｘ１０’の組換えファージの中から分離した。これ
らのファージのハイブリダイゼーションの強度は、３ク
ラスに分離された。Nine recombinant λEMB L4 phages that hybridized with the ``!P-labeled cDNA inserted into psKl were isolated from among 6 x 10'' recombinant phages.The hybridization intensities of these phages were divided into three classes. Separated.
ハイブリダイゼーションの強度が最も強いクローン（ク
ラスｌ）のうち、ｌクローンがｐｒｘＣｌａ、ｐｒｘｃ
Ｉｂ遺伝子をランダムに含んでいることがわかった。ハ
イブリダイゼーションの強度が余り強くないクローン（
クラス２）の４個の組換えクローンはｃＤＮＡプローブ
とハイブリダイズするＥｃｏＲＩ制限断片（４ｋｂ）を
含んでいた。Among the clones with the strongest hybridization intensity (class I), the l clones are prxCla and prxc.
It was found that the Ib gene was randomly included. Clones with less intense hybridization (
Four recombinant clones of class 2) contained an EcoRI restriction fragment (4 kb) that hybridized with the cDNA probe.
また、ハイブリダイゼーション強度が最も弱いクラス（
クラス３）は、プローブとハイブリダイズするＥｃｏＲ
Ｉ （１，５ｋｂ、 ０．８ ｋｂ）を含んでいた。こ
れらのＥｃｏＲＩ断片及びクラス２、クラス３のクロー
ンから得られる他の制限酵素切断断片とオーバーラツプ
する断片をｐｕｃ Ｉ ９にサブクローンし、塩基配列
を決定した。Also, the class with the weakest hybridization intensity (
Class 3) is EcoR that hybridizes with the probe.
I (1.5 kb, 0.8 kb). These EcoRI fragments and fragments overlapping with other restriction enzyme-cleaved fragments obtained from class 2 and class 3 clones were subcloned into puc I 9 and their nucleotide sequences were determined.
これらの塩基配列をｐｒｘｃｌａ及びｐｒｘＣｌｂの塩
基配列とそれから予測されるアミノ酸配列比較した結果
、ペルオキシダーゼアイソザイム遺伝子ｐｒｘＣ２及び
ｐｒｘＣ３と命名した［２］ ＤＮＡ断片ノｗＲ製
前記［１］で得たｐｒｘＣ２を制限酵素Ｘｂａ Ｉ（塩
基配列−５２７）とＮｓｉ！（塩基配列＋５）で切断し
、第１図に示すＤＮＡ断片を得た。As a result of comparing these nucleotide sequences with the nucleotide sequences of prxcla and prxClb and the amino acid sequences predicted therefrom, the peroxidase isozyme genes were named prxC2 and prxC3. Xba I (base sequence -527) and Nsi! (nucleotide sequence +5) to obtain the DNA fragment shown in FIG.
［３］タバコプロトプラストでのｐｒｘＣ２の５′非翻
訳領域のプロモーター活性の確認
ＤＮＡ組換えの為の一連の遺伝子操作は、基本的には、
Ｍｏ１ｅｃｕｌａｒ Ｃｌｏｎｉｎｇ、 （１９８９）
、（Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｓ
、）に従って行った。前記［２］で得たｐｒｘＣ２の５
′非翻訳領域５３２ｂｐ （第１図）とＧＵＳ遺伝子と
のキメラ遺伝子を作製し、更にそれらをｐＵｃｌＢにつ
なぎ、発現プラスミドを構築した。比較のために、ｐｒ
ｘＣ２の５′非翻訳領域Ｘｂａ Ｉ ／ Ｎｓｉ Ｉ断
片の代わりに、ＣａＭＶ３５ｓプロモーターを用いた発
現プラスミドも構築した。これらの発現プラスミドをエ
レクトロポレーション法によりタバコプロトプラストに
導入し、２４時間後のＧＵＳ活性を測定した。ＧＵＳ活
性は以下のようにして測定した。酵素抽出液３００μｌ
に、２ＩＩＩＭメチルウンベリフェリルグルクロライド
を含む抽出液（５０ｍＭリン酸緩衝液。[3] Confirmation of the promoter activity of the 5' untranslated region of prxC2 in tobacco protoplasts A series of genetic manipulations for DNA recombination basically consists of:
Molecular Cloning, (1989)
, (Cold Spring Harbor Labs
,) was followed. 5 of prxC2 obtained in [2] above
A chimeric gene consisting of a 532 bp untranslated region (Fig. 1) and the GUS gene was constructed, and this was further ligated to pUclB to construct an expression plasmid. For comparison, pr
An expression plasmid was also constructed using the CaMV35s promoter in place of the 5′ untranslated region Xba I/Nsi I fragment of xC2. These expression plasmids were introduced into tobacco protoplasts by electroporation, and GUS activity was measured 24 hours later. GUS activity was measured as follows. Enzyme extract 300μl
An extract containing 2IIIM methylumbelliferyl gluchloride (50 mM phosphate buffer).
ｐＨ７，０，１０ｍＭ ＥＤＴＡ ＳＯ，１％トリトン
Ｘ−１０００，１％ラウリザルコシン、ＩＯＩＩＩＭ
β−メルヵブトエ名ノール）を３００μ！加え、３７
℃、１時間酵素反応を行った。０．２ Ｍ ＮａｔＣＯ
＊ ２．４−を加えて反応を停止させ、生成物４−メチ
ルウンベリフェロンの蛍光量を蛍光光度計を用いて測定
（ＥＸ、 ３６５ｎｍ、 Ｅｍ、 ４５５ｎｍ）した。pH 7, 0, 10mM EDTA SO, 1% Triton X-1000, 1% laurisarcosine, IOIIIM
300μ of β-mercabutenol! In addition, 37
The enzyme reaction was carried out at ℃ for 1 hour. 0.2M NatCO
*2.4- was added to stop the reaction, and the amount of fluorescence of the product 4-methylumbelliferone was measured using a fluorometer (EX, 365 nm, Em, 455 nm).
４−メチルウンベリフェロン量は４−メチルウンベリフ
ェロンを用いて作製した標準曲線より求め、それを基に
ＧＵＳ活性を算出した。蛋白質濃度はＢｉｏ−Ｒａｄ社
のプロティンアッセイキットを用いて測定した。その結
果を第１表に示す。この結果より、ｐｒｘＣ２の５°非
翻訳領域にはプロモーター活性があり、しかも汎用され
ているＣａＭＶ３５ｓプロモーターよりも約４倍以上の
強い活性を存することかわかる。The amount of 4-methylumbelliferone was determined from a standard curve prepared using 4-methylumbelliferone, and the GUS activity was calculated based on it. Protein concentration was measured using a protein assay kit from Bio-Rad. The results are shown in Table 1. From this result, it can be seen that the 5° untranslated region of prxC2 has promoter activity, and the activity is about 4 times or more stronger than that of the widely used CaMV35s promoter.
第１表
プロモーター領域 ＧＵＳ
比活性
なし ８
ＣａＭＶ３５ ｓ １００”ｐｒｘＣ２
４４４
す２７．９ ｐｍｏｌ／ｍｉｎ ■ 蛋白［４１トラ
ンスジエニツクタバコ細胞でのｐｒｘＣ２の非翻訳領域
のプロモーター活性の確認ｐｒｘＣ２の５′非翻訳領域
とＧＵＳ遺伝子とのキメラ遺伝子のプラスミドｐＢ１１
０１へのつなぎ換えは第２図に示した。即ち、ｐｒｘＣ
２の５゛非翻訳領域、ＧＵＳ遺伝子、及びｐＵｃｌＢか
ら成る組換プラスミドからｐｒｘＣ２の５′非翻訳領域
とＧＵＳ遺伝子部分を含むＰｖｕＩ［／ Ｅｃ。Table 1 Promoter region GUS No specific activity 8 CaMV35 s 100”prxC2
444 27.9 pmol/min ■ Protein [41 Confirmation of promoter activity of untranslated region of prxC2 in transgenic tobacco cells Plasmid pB11 of chimeric gene of 5' untranslated region of prxC2 and GUS gene
The connection to 01 is shown in Figure 2. That is, prxC
A recombinant plasmid consisting of the 5' untranslated region of prxC2, the GUS gene, and pUclB was used to generate PvuI[/Ec] containing the 5' untranslated region of prxC2 and the GUS gene portion.
Ｒ［断片を切り出した。一方、ｐＢＩＩＯＩはＨｉ’ｎ
ｄ１［［／ ＥｃｏＲＩで切断後、ＨｉｎｄＩ［［切断
末端をクレノー酵素で修復し平滑末端とし、ＰｖｕｌＩ
／ ＥｃｏＲＩ断片を挿入した。R [The fragment was cut out. On the other hand, pBIIOI is Hi'n
d1[[/ After cutting with EcoRI, the cut ends were repaired with Klenow enzyme to make blunt ends, and PvulI
/EcoRI fragment was inserted.
トランスジェニックタバコの作製は、目的のキメラ遺伝
子をＴｒｉｐａｒｅｎｔａｌ Ｍａｉｔｉｎｇ法により
アグロバクテリウム（Ａｇｒｏｂａｃｔｅｒｉｕｍ）に
導入し、このキメラ遺伝子の入ったアグロバクテリウム
（Ａｇｒｏｂａｃｔｅｒｉｕｍ）を用いてＬｅａｆ Ｄ
ｉｓｃ法によりタバコ細胞を形質転換した。To produce transgenic tobacco, a chimeric gene of interest is introduced into Agrobacterium using the Triparental Mating method, and the Agrobacterium containing this chimeric gene is used to incubate Leaf D.
Tobacco cells were transformed by the isc method.
■ キメラ遺伝子のアグロバクテリウム（Ａｇｒｏｂａ
ｃｔｅｒｉｕｍ）への導入
ｐｒｘＣ２の５°非翻訳領域とＧＵＳ遺伝子及びｐＢＴ
＋０１から構築された組換プラスミドで形質転換された
Ｅ、ｃｏｌｉＣ６００（ドナーと称する）、ｐＲＫ２０
１３を保持するＥ、ｃｏｌｉＨＢ １０１ （ヘルパー
と称する）をそれぞれ５０μｇ／ｄカナマイシン含有Ｌ
Ｂ培地で３７°Ｃ１−夜培養した。アグロバクテリウム
ツメファシェンス（ＡｇｒｏｂａｃｔｅｒｉｕｍＴｕｍ
ｅｆａｃｉｅｎｓ）ＬＢＡ ４４０４株は、ＬＢ培地５
−で２５〜２８°Ｃ１約３６時間培養した。■ Chimeric gene Agrobacterium (Agroba
5° untranslated region of prxC2, GUS gene and pBT
E. coli C600 (referred to as donor), pRK20 transformed with the recombinant plasmid constructed from +01
E. coli HB 101 (referred to as helper) containing 50 μg/d kanamycin, respectively.
Cultured in medium B at 37°C for 1 night. Agrobacterium tumefaciens
efaciens) LBA 4404 strain, LB medium 5
- Cultured at 25-28°C for about 36 hours.
各培養液は、１．５ｍｔ’容エッペンドルフチューブに
とり、遠心により集菌した。菌体は０．５〜ｌｄの１０
ｍＭ Ｍｇ５Ｏ，で３回はと洗浄した後、約ｓｏμｐの
１０ｍＭ Ｍｇ５Ｏ，に懸濁した。各菌体懸濁液は、ド
ナー、ヘルパー、アグロバクテリウムツメファシェンス
（Ａ、 Ｔｕｍｅｆａｃｉｅｎｓ）の順でＬＢプレート
上にスポットし、２５℃、−夜混合培養した。混合培養
菌体は、白金耳等でかき取り、ｌ−の１０ｍＭ Ｍｇ５
Ｏ，に懸濁し、１０！〜ｌＯ″倍に希釈し、その５０μ
ｌを４００μｇ／ｄ カナマイシン含有Ｍｉ ｎＡプ
レー） ［ＭｉｎＡ培地（第２表参照）の入ったシャー
レ〕に蒔き、２５〜２８°Ｃ１２〜３日培養し、キメラ
遺伝子が導入されたアグロバクテリウムツメファシェン
ス（Ａ、 Ｔｕｍｅｆａｃｉｅｎｓ）の単一コロニーを
取得した。Each culture solution was placed in a 1.5 mt' volume Eppendorf tube, and bacteria were collected by centrifugation. Bacterial cells are 0.5 to 10 ld
After washing three times with mM Mg5O, it was suspended in approximately sop of 10mM Mg5O. Each bacterial cell suspension was spotted on an LB plate in the order of donor, helper, and Agrobacterium tumefaciens (A, Tumefaciens), and mixed culture was carried out at 25° C. overnight. The mixed culture bacterial cells were scraped off with a platinum loop, etc., and added with l-10mM Mg5.
Suspended in O, 10! Dilute to ~lO'' times and 50μ
Agrobacterium tumefacia into which the chimeric gene had been introduced was sown in a Petri dish containing MinA medium (see Table 2)] and cultured at 25-28°C for 12-3 days. A single colony of Tumefaciens (A, Tumefaciens) was obtained.
第２表 ＾、 ＭｉｎＡ培地 に、ＨＰＯ。Table 2 ^, MinA medium In, HPO.
ＫＨ，ＰＯ。K.H., P.O.
（ＮＨ４）＊５０４
Ｍｇ５Ｏａ・７８．０”
１０．５ ｇ／１
４．５ｇ／１
１．０ｇ／１
０．２ｇ／ｌ
クエン酸ナトリウム ０．５ｇ／ｌ
グルコースリ ２．０ｇ／ｌ
寒天 １２ ｇ／１
０′別途殺菌
Ｂ、 除菌用培地
ＬＳ培地（ｐＨ５，６〜５．８）
クラフオラン ０．５ｇ／ｌ
カナマイシン ０．１ｇ／Ｉ
Ｃ０シュート形成用培地
ＬＳ培地（ｐＨ５，６〜５．８）
ベンジルアデニン １ ■／１
ナフタレン酢酸 ０．１■／１
クラフオラン ０．５ ｇ／ｌ
カナマイシン ０．１ｇ／Ｉ
Ｄ、 カルス誘導培地
ＬＳ培地（ｐＨ５，６〜５．８）
ベンジルアデニン ０．８■／ｌ
ナフタレン酢酸 ０．５■／１
カナマイシン ０．１ｇ／ｌ
■Ｌｅａｆ Ｄｉｓｃ法によるタバコの形質転換キメラ
遺伝子を保持するアグロバクテリウムツメファシェンス
（Ａ、 Ｔｕｍｅｆａｃｉｅｎｓ）を５０μｇ／ｒｄカ
ナマイシン含有ＬＢ培地５ｒｄ中、２５〜２８°Ｃ１夜
培養したものを、シャーレに移し、そこに５１１１ｍ角
に切った無菌タバコの葉を表皮を下に、気孔を上にして
３分間浸汐した。余分の菌液をキムワイブで除き、ＬＳ
培地プレートに置床し、２５°Ｃで培養した。２日後、
除菌用培地プレート（第２表）に移植、３日後、シュー
ト形成用培地（第２表）へ移植した。その後、５〜７日
間隔でシュート形成用培地へ移植し続けた。茎葉部の発
達したシュートが出てきたら切取り、除菌用培地に移植
した。(NH4)*504 Mg5Oa・78.0” 10.5 g/1 4.5 g/1 1.0 g/1 0.2 g/l Sodium citrate 0.5 g/l Glucose 2.0 g/l Agar 12 g /1 0' Separate sterilization B, sterilization medium LS medium (pH 5,6-5.8) Clafuolan 0.5 g/l Kanamycin 0.1 g/I C0 shoot formation medium LS medium (pH 5,6-5.8 ) Benzyl adenine 1 ■/1 Naphthalene acetic acid 0.1 ■/1 Clafuoran 0.5 g/l Kanamycin 0.1 g/ID, Callus induction medium LS medium (pH 5,6-5.8) Benzyl adenine 0.8 ■ /l Naphthaleneacetic acid 0.5■/1 Kanamycin 0.1g/l ■Transformation of tobacco by Leaf Disc method LB medium containing Agrobacterium tumefaciens (A, Tumefaciens) carrying chimeric gene at 50 μg/rd kanamycin 5rd, cultured at 25-28°C for 1 night was transferred to a petri dish, and sterile tobacco leaves cut into 5111 m squares were soaked there for 3 minutes with the epidermis facing down and the stomata facing up.Excess bacterial solution is removed by Kimwaibu, LS
It was placed on a medium plate and cultured at 25°C. 2 days later,
The cells were transplanted to a sterilization medium plate (Table 2), and 3 days later, they were transplanted to a shoot formation medium (Table 2). Thereafter, transplantation into medium for shoot formation was continued at intervals of 5 to 7 days. When shoots with developed stems and leaves appeared, they were cut and transplanted to a sterilization medium.
成育した幼植物体は、バーミキュライトとピートモスを
１＝１に混合し、これに約１ ｇ／ｌのハイボネックス
溶液を充分混合したものを含む植木鉢へ移し、−日の内
１４時間は１０．０００ルツクスの光を照射し、２５°
Ｃで栽培した。なお、水は毎日与え、ハイボネックス等
の肥料は週に一回与えた。The grown seedlings were transferred to a flower pot containing a 1:1 mixture of vermiculite and peat moss, mixed well with a Hyvonex solution of approximately 1 g/l, and heated to 10,000 g/l for 14 hours during the day. Irradiate Lux light, 25°
Cultivated in C. In addition, water was given every day, and fertilizer such as Hyvonex was given once a week.
■ トランスジェニックタバコの各器官でのＧＵ活性
丈が約１０ｃｍまで成長した形質転換タバコを葉、茎、
根に分けて液体窒素で凍結させ、３００〜１０００■を
エッペンドルフチューブに分取した。抽出緩衝液（５０
ｍＭリン酸緩衝液、 ｐＨ７，０，１０ｍＭ ＥＤＴＡ
、０．１％トリトンＸ−１００，０，１％ラウリルザル
コン、１０ｍＭ β−メルカプトエタノール）３００
〜６００μｌと海砂を加え、ガラス棒で粉砕し、遠心分
離により上溝を回収した。ＧＵＳ活性及び蛋白量の測定
は前述（３）記載の方法に従った。その結果を第３表に
示した。プロモーターが存在しないとＧＵＳ遺伝子の発
現はほとんど認められないが、ｐｒｘＣ２のプロモータ
ーが存在すると葉、茎、根、いずれの器官でも高いＧＵ
Ｓ活性が認められ、ｐｒｘＣ２のプロモーターがいずれ
の器官でも高く発現しうることが示された。またｐｒｘ
Ｃ２のプロモーターは、同様に測定したｐｒＸｃｌａお
よびｐｒｘｃｔｂのプロモーターよりも高いＧＵＳ活性
を示した。■ GU activity in each organ of transgenic tobacco Transgenic tobacco that has grown to a height of approximately 10 cm is processed into leaves, stems,
The roots were divided and frozen in liquid nitrogen, and 300 to 1000 square centimeters were aliquoted into Eppendorf tubes. Extraction buffer (50
mM phosphate buffer, pH 7,0, 10mM EDTA
, 0.1% Triton X-100, 0.1% lauryl sarcone, 10mM β-mercaptoethanol) 300
~600 μl and sea sand were added, crushed with a glass rod, and the upper groove was collected by centrifugation. GUS activity and protein amount were measured according to the method described in (3) above. The results are shown in Table 3. In the absence of the promoter, almost no GUS gene expression is observed, but in the presence of the prxC2 promoter, high GU expression is observed in all organs, including leaves, stems, and roots.
S activity was observed, indicating that the prxC2 promoter can be highly expressed in any organ. Also prx
The C2 promoter showed higher GUS activity than the prXcla and prxctb promoters, which were similarly measured.
第３表
野性型 ＜２０ ＜２０ ＜２０なし
１ ＜２０ ２１ ２６２
＜２０ ＜２０ ＜２０ＣａＭＶ３５ｓ
１ ３８０ １７０ １１００ｐｒｘ
ｃ１ａ ｌ ＜２０ ２４０ ３
３０２ ＜２０ ４５ ２８ｐ
ｒｘｃ１ｂ １ ＜ ２０ ７５
６８０ｐｒｘＣ２１５３０１１００２８００
（” ｐｍｏｌ／ｍｉｎ ■ 蛋白）［５］ ）
−ランスジェニックタバコ植物体のカルス化によるＧＵ
Ｓ活性の誘導
各種形質転換タバコ植物体の東部を５Ｍ〜１ ｃｍ角に
切取り、これをカルス誘導培地（第２表）に置床し、２
５°Ｃ１暗所下で２〜３週間培養し形成された各カルス
のＧＵＳ活性を前述［３］記載の方法に従い測定した。Third surface wild type <20 <20 <20 none 1 <20 21 262
<20 <20 <20CaMV35s
1 380 170 1100prx
c1a l <20 240 3
302 <20 45 28p
rxc1b 1 < 20 75
680prxC2153011002800 (" pmol/min ■ Protein) [5] )
-GU by callus formation of transgenic tobacco plants
Induction of S activity The eastern part of various transformed tobacco plants was cut into 5M to 1 cm squares, placed on a callus induction medium (Table 2), and
The GUS activity of each callus formed by culturing in the dark at 5° C. for 2 to 3 weeks was measured according to the method described in [3] above.
その結果を第４表に示した。ＣａＭＶ３５ｓプロモータ
ーを導入したものではカルス化してもＧＵＳ活性は誘導
されず、比活性はむしろ低下した。他方、ｐｒｘＣ２の
プロモーターを導入したものではカルス化により更に約
４０倍活性が上昇し、ｐｒＸＣ２のプロモーターがカル
ス化に伴い、より誘導的に強く発現することが示された
。また、ｐｒｘＣ２のプロモーターは、同様に測定した
ｐｒｘｃｌａおよびｐｒｘｃｌｂのプロモーターよりも
カルス化に伴い、より誘導的に強く発現することが示さ
れた。The results are shown in Table 4. In the cells into which the CaMV35s promoter was introduced, GUS activity was not induced even after callus formation, and the specific activity was rather reduced. On the other hand, in those into which the prxC2 promoter was introduced, the activity further increased by about 40 times due to callus formation, indicating that the prXC2 promoter was more strongly expressed in an inducible manner with callus formation. Furthermore, it was shown that the promoter of prxC2 was more strongly expressed in an inducible manner with callus formation than the promoters of prxcla and prxclb, which were similarly measured.
第４表
プロモーター ＧＵＳ活性１ 誘導性葉 カルス
なし ６ １０ １．７ＣａＭＶ３
５ｓ ４７５ １０４ ０．２２ｐｒｘ
ｃ１ａ １６ ２５７ １６ｐｒｘ
ｃｌｂ ９ ２５０ ２８ｐｒｘＣ
２２２０９７００４４
（” ｐｍｏｌ／ｌｌ１ｉｎ ｗｇ 蛋白）〔発明の
効果〕
本発明のＤＮＡ断片は、植物細胞内等で良好なプロモー
ター活性を有するので、各種異種遺伝子を発現し得る発
現ベクターに有利に使用し得る。Table 4 Promoter GUS activity 1 Inducible leaf No callus 6 10 1.7CaMV3
5s 475 104 0.22prx
c1a 16 257 16prx
clb 9 250 28prxC
2220970044 ("pmol/ll1in wg protein) [Effects of the Invention] Since the DNA fragment of the present invention has good promoter activity in plant cells, etc., it can be advantageously used in expression vectors capable of expressing various heterologous genes.
本発明のベクターは、種々の有用なポリペプチドを宿主
内で効率よく発現させることができる。The vector of the present invention can efficiently express various useful polypeptides in a host.
第１図は、本発明のＤＮＡ断片の塩基配列を示す。
第２図は、ｐｒｘＣ２の５′非翻訳領域をＧＵＳ遺伝子
のｐＢｌｌｏｌへの組換えの方法を示す。
なお、図中において、Ｃ２はｐｒｘＣ２を、ＧＵＳはベ
ーターグルクロニナーゼを、ＮＰＴＩＩはネオマイシン
リン酸転移酵素の遺伝子を表わしている。FIG. 1 shows the base sequence of the DNA fragment of the present invention. FIG. 2 shows a method for recombining the 5' untranslated region of prxC2 into the GUS gene pBllol. In the figure, C2 represents prxC2, GUS represents beta-glucuroninase, and NPTII represents the neomycin phosphotransferase gene.
Claims (9)
それらと均等な塩基配列を有し、プロモーター活性を有
するＤＮＡ断片。 【遺伝子配列があります】(1) A DNA fragment having all or part of the base sequence shown below, or a base sequence equivalent thereto, and having promoter activity. [There is a gene sequence]
ソザイムの遺伝子に由来する請求項（１）記載のＤＮＡ
断片。(2) The DNA according to claim (1), wherein the DNA fragment is derived from a horseradish peroxidase isozyme gene.
piece.
する請求項（１）または（２）記載のＤＮＡ断片。(3) The DNA fragment according to claim (1) or (2), which has promoter activity in plants or yeast.
ター。(4) A vector into which the DNA fragment according to claim (1) is inserted.
求項（４）記載のベクター。(5) The vector according to claim (4), wherein the heterologous gene is under the transcriptional control of a promoter.
ードする遺伝子である請求項（５）記載のベクター。(6) The vector according to claim (5), wherein the heterologous gene is a gene encoding a physiologically active polypeptide.
キダーゼ、アスコルビル酸オキシダーゼ、ルシフェラー
ゼの少なくとも一種から選ばれる遺伝子である請求項（
５）記載のベクター。(7) A claim in which the heterologous gene is a gene selected from at least one of beta-glucuroninase, peroxidase, ascorbyl oxidase, and luciferase (
5) Vectors described.
た宿主。(8) A host transformed with the vector according to claim (4).
宿主。(9) The host according to claim (8), wherein the host is a plant or yeast.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24477990A JP3259178B2 (en) | 1990-09-14 | 1990-09-14 | DNA fragment having promoter activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24477990A JP3259178B2 (en) | 1990-09-14 | 1990-09-14 | DNA fragment having promoter activity |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11196728A Division JP2000041688A (en) | 1999-07-09 | 1999-07-09 | Dna fragment having promoter activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04126088A true JPH04126088A (en) | 1992-04-27 |
| JP3259178B2 JP3259178B2 (en) | 2002-02-25 |
Family
ID=17123797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24477990A Expired - Fee Related JP3259178B2 (en) | 1990-09-14 | 1990-09-14 | DNA fragment having promoter activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3259178B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998056921A1 (en) * | 1997-06-12 | 1998-12-17 | Dow Agrosciences Llc | Regulatory sequences for transgenic plants |
| US7354390B1 (en) | 1996-09-30 | 2008-04-08 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | Seed coat specific nucleotide sequence encoding peroxidase |
-
1990
- 1990-09-14 JP JP24477990A patent/JP3259178B2/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7354390B1 (en) | 1996-09-30 | 2008-04-08 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | Seed coat specific nucleotide sequence encoding peroxidase |
| WO1998056921A1 (en) * | 1997-06-12 | 1998-12-17 | Dow Agrosciences Llc | Regulatory sequences for transgenic plants |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3259178B2 (en) | 2002-02-25 |
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