CN1597968A

CN1597968A - Choline mono oxidase of controlling betaine synthsizing and expression carrier of betaine aldehyde dehydrogenase dual gene thereof

Info

Publication number: CN1597968A
Application number: CN 200410057271
Authority: CN
Inventors: 韩烈保; 刘君; 何宇锋; 陈其军; 信金娜
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2004-08-30
Filing date: 2004-08-30
Publication date: 2005-03-23

Abstract

The invention realizes the resistance of grasses in lawn by constructing plant double-gene carrier and applying transgenic technique to transfer the carrier in the grasses. The two carriers contain different starters to make the double-gene carrier able to convert both monocotyledon and dicotyledon. It includes the steps: 1. obtaining CMO and BADH genes from existing plasmid in my lab; 2. applying existing carrier in my lab and the two genes to construct single-gene carrier with different starters; 3. making different enzyme cutting and linking many times, thus obtaining two double-gene plant expression carriers containing different starter; 4. tranferring the obtained plant expression carrier in Escherichia coli and Agrobacterium Tumefacies to lay a foundation of gene conversion of the monocotyledon and dicotyledon.

Description

Control trimethyl-glycine synthetic choline mono-oxygenase, betaine-aldehyde dehydrogenase double gene expression vector

Technical field:

The present invention relates to make up the dual-gene carrier of plant, and expect to utilize transgenic technology that carrier is changed in the plant, improve drought resisting, the salt resistance ability of plant.

Background technology:

Occurring in nature extensively exists the nontoxic micromolecular compound that a class is called as osmotic protection agent or Osmolyte regulator; when there is unfavorable factor in external environment; for example arid, salt damage, when cold and hot; just can accumulate certain density osmotic protection agent in many organisms; play and regulate osmotic pressure in the born of the same parents, the active effect of protection intracellular enzyme.Trimethyl-glycine is a kind of important osmotic protection agent, and its accumulation makes the important enzyme in many metabolism can keep active under osmotic stress.In more than the 150 kind of metabolite of studying, trimethyl-glycine is best Osmolyte regulator,

The trimethyl-glycine route of synthesis is simple, carries out genetic manipulation conveniently, and the trimethyl-glycine synthase gene is that the most important and most promising side of body chases after one of resistant gene.Trimethyl-glycine is synthetic via the two-step oxidation reaction by choline: choline → betaine aldehyde chloride → trimethyl-glycine, wherein by the catalytic the first step oxidizing reaction of choline-monooxygenase (cmo) (CMO), betaine-aldehyde dehydrogenase (BADH) catalysis second step oxidizing reaction, so CMO and BADH are two key genes of control trimethyl-glycine synthetic.Use the transfer-gen plant of CMO and BADH single-gene carrier all to improve certain drought resistance.In order further to improve the drought-resistant ability of transfer-gen plant, we study with CMO and two gene integrations of BADH the possibility in same expression vector, and in the successfully back possibility of studying dual-gene carrier transformed plant of vector construction, the resistance that will observe transfer-gen plant simultaneously changes.

Summary of the invention:

The present invention has not only considered the conversion of dual-gene carrier aspect monocotyledons, but also considered conversion aspect dual-gene plant, made up two kinds of carriers that contain different promoters, dual-gene carrier can all can be transformed in single, double cotyledon plant, specifically comprised:

1, respectively from the existing pBI-CMO in this laboratory, obtains CMO and BADH gene on the pBI-BADH plasmid;

2, the carrier that is had with gene that obtains in above-mentioned 1 and laboratory, make up the CMO single-gene carrier and the BADH single-gene carrier (two kinds of CMO single-gene carriers that band Ubi and 35S promoter are arranged, BADH single-gene carrier band 35S promoter) that have different promoters respectively;

3, with the single-gene carrier that has different promoters that obtains in 2, carry out enzyme and cut, the gene fragment that will have promotor and BADH is linked on the CMO single-gene carrier, obtains the dual-gene plant expression vector of two kinds of different promoters;

4, change intestinal bacteria and Agrobacterium over to the dual-gene plant expression vector that obtains, for future unifacial leaf plant and dicotyledonous plant gene transformation lays the first stone.

Wherein the gene of CMO cuts back to close by enzyme and obtains, and the gene of BADH obtains by the PCR method amplification, and wherein the primer of BADH has added BamH I restriction enzyme site sequence.

Other steps of experiment such as the structure of plant expression vector, the conversion of plant, the screening of resistance seedling is all undertaken by currently known methods.

Brief Description Of Drawings:

The dual-gene vector plasmid figure of BADH (pCUC35SB is the name to this carrier, and other restriction enzyme site is to utilize in the building process) that Fig. 1 represents to have the CMO of Ubi promotor and has 35S promoter;

After Fig. 2 represents the carrier that Fig. 1 makes up is transformed into Agrobacterium, the bacterial plaque that the screening through the resistance plate is grown carry out PCR identify (from left to right, swimming lane 1 to 12 usefulness be the primer of CMO, what other swimming lanes were used is the primer of BADH);

Fig. 3 represents that the carrier that makes up is carried out enzyme cuts evaluation, with restriction enzyme BamH I carrier carried out enzyme and cuts, and the leakage of electricity swimming, obtain restriction enzyme digestion and electrophoresis image as shown in the figure;

Fig. 4 represents the dual-gene vector plasmid figure of CMO-BADH (pC35SC35SB is the name to this carrier, and other restriction enzyme site is to utilize in the building process) that two promotors all are 35S;

After the carrier that Fig. 5 swimming lane 1 to 4 expression makes up Fig. 4 is transformed into Agrobacterium, the bacterial plaque that the screening through the resistance plate is grown carry out PCR detect (1 and 3 usefulness be the primer of BADH, 2 and 4 usefulness be the primer of CMO); Swimming lane 5,6 expressions are carried out enzyme to the carrier that makes up and are cut evaluation, and with restriction enzyme BamH I carrier is carried out enzyme and cut, and the leakage of electricity swimming, obtain restriction enzyme digestion and electrophoresis image as shown in the figure;

Embodiment

Embodiment one

1, the structure of the dual-gene dicotyledons expression vector of CMO-BADH

(1) structure of expression of plants intermediate carrier pCUC1303

With Sma I and Sac I double digestion plasmid pBI-CMO, reclaim 1.2kb CMO gene fragment, and through Sma I and Sac I double digestion, the big fragment of the pCU1303 plasmid of recovery connects, and obtains recombinant plasmid pCUC1303.

(2) structure of expression of plants intermediate carrier pC35S B1391

Design has the PCR primer of BamH I restriction enzyme site sequence, with the pfu polysaccharase pBI-BADH is carried out BDAH high-fidelity pcr amplification, reclaim amplified production, be connected with PMD-18T body carrier, the company of delivering checks order, and uses BamH I and BstE II double digestion PMD-18T-BADH then, reclaim the BADH gene fragment, with this gene fragment and through BamH I and BstE II double digestion, the big fragment of the pC35S1391 of recovery connects, and obtains recombinant plasmid pC35S B1391.

(3) structure of plant expression vector pCUC35SB

With BstE II and HindIII double digestion intermediate carrier pC35S B1391, reclaim band 35S promoter and the BADH gene fragment of 2.4kb, with through BstE II and HindIII double digestion, the big fragment of the intermediate carrier pCUC1303 of recovery connects, and obtains plant double gene expression vector pCUC35SB.

2, the structure of the dual-gene monocotyledons expression vector of CMO-BADH

(1) structure of expression of plants intermediate carrier pC35S B1391 is identical with structure dicotyledons expression vector

(2) structure of expression of plants intermediate carrier pC35SC1303

With BamH I and HindIII double digestion pC35S B1391 and pCU1303, electrophoresis reclaims small segment and big fragment respectively, connects, and detects and obtains to express intermediate carrier pC35SC1303.

(3) structure of plant expression vector pC35S C35SB

With BstE II and HindIII double digestion intermediate carrier pC35S B1391, reclaim band 35S promoter and the BADH gene fragment of 2.4kb, with through BstE II and HindIII double digestion, the big fragment of the intermediate carrier pC35SC1303 of recovery connects, and obtains plant double gene expression vector pC35SC35SB.

Embodiment two

1, Agrobacterium conversion tobacco plant

(1) aseptically process of tobacco leaf and section

Get the tobacco leaf of greenhouse growth, the flush away surface irregularities is carried out sterilising treatment with 70% ethanol and 2.5% clorox respectively, washs with sterilized water at last; Aseptic tobacco leaf cuts edge and main vein, is cut into the nail cover size.

(2) Agrobacterium is infected and cultivates

The explant that cuts soaked 10 minutes in Agrobacterium bacterium liquid, blotted the bacterium liquid on vegetable material surface with aseptic filter paper, changed the MS minimum medium over to, 28 ℃ of dark cultivations.After three days, material forwarded to contain in the antibiotic division culture medium cultivate.

2, particle gun transforms turfgrass

(1) sets up the turfgrass high frequency regenerating system

Adopt turfgrass mature seed, embryo and plumular axis etc. as explant, induce the embryo callus of turfgrass, set up the turfgrass high frequency regenerating system.

(2) bronze is handled and DNA bag quilt

Adopt the U.S. PDS-1000/He of Bio-Rad company type particle gun, handle bronze and bag by foreign DNA according to the method on its specification sheets.

(3) particle gun transforms

The target material that the embryo callus of inducing 2-5 month transforms as particle gun, method is to specifications bombarded callus.Callus after the bombardment changes the callus recovery media over to, after change over to and contain antibiotic inducing culture and further screen, last screening resistant plant in containing antibiotic division culture medium at last.

(4) acquisition of transfer-gen plant

The resistant plant that obtains is carried out PCR and Southern hybridization analysis, finally obtain transfer-gen plant.

Claims

1. plant transgene breeding technique that improves the plant drought salt resistance ability comprises:

The double gene expression vector of a, dicotyledons makes up;

B, monocotyledonous double gene expression vector make up;

The transgenic breeding of c, single, double cotyledon plant.

2. in accordance with the method for claim 1, it is characterized in that described plant expression vector is pCAMBIA1303.

3. in accordance with the method for claim 1, it is characterized in that, in the structure of described monocotyledons expression vector, dual-gene Ubi promotor-choline mono-oxygenase encoding gene (CMO) and the 35S promoter-betaine-aldehyde dehydrogenase encoding gene (BADH) of being respectively.

4. in accordance with the method for claim 1, it is characterized in that, in the structure of described dicotyledons expression vector, dual-gene 35S promoter-choline mono-oxygenase encoding gene (CMO) and the 35S promoter-betaine-aldehyde dehydrogenase encoding gene (BADH) of being respectively.

5. in accordance with the method for claim 1, it is characterized in that described monocotyledonous transgenic breeding comprises annual ryegrass, English ryegrass, English grass, Japanese lawn grass.

6. in accordance with the method for claim 1, it is characterized in that the transgenic breeding of described dicotyledons comprises tobacco, Stem or leaf of Shrub Lespedeza, sweet clover, clover.