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Methyl transferase gene in betaine synthesis path and modification and application thereof

A technology of methyltransferase and synthetic pathway, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problem of unreported betaine accumulation level, etc.

Inactive Publication Date: 2010-08-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 1996, Lilius et al. transferred the betA gene into tobacco, and the salt tolerance of the transgenic plants was significantly improved compared with the control plants, but they did not report the accumulation level of betaine

Method used

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  • Methyl transferase gene in betaine synthesis path and modification and application thereof
  • Methyl transferase gene in betaine synthesis path and modification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: ApGSMT2 / ApDMT2 gene cloning and creation of maize transgenic stress-resistant materials

[0088] 1) ApGSMT2 / ApDMT2 gene cloning

[0089] Cultivate Aphanothece halophytica to OD600=1.2, take 30ml of the bacterial liquid into a 50ml centrifuge tube, and collect the bacterial cells by centrifugation. The cells were resuspended in STE (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0)), collected by centrifugation, and repeated once. Then add 20ml 2×CTAB (100mmol / LTris-HCl pH 8.0, 1.4mol / LNaCl, 20mmol / LEDTA pH 8.0, 2% CTAB) buffer, mix well, and incubate in a water bath at 65°C for 90min (add 1%β before preheating -mercaptoethanol), shake well 2-3 times in between. Then the centrifuge tube was taken out from the water bath, after cooling to room temperature, an equal volume of chloroform / isoamyl alcohol (24:1) was added, the centrifuge tube was gently inverted several times, and centrifuged at 10,000 rpm for 10 min at room temperature. Then transfer the sup...

Embodiment 2

[0112] Example 2: Synthesis of ApGSMT2d and ApDMT2d genes and creation of maize transgenic stress-resistant materials

[0113] 1) Synthesis of ApGSMT2d and ApDMT2d genes and construction of plant expression constructs

[0114] ApGSMT2d and ApDMT2d genes were artificially synthesized by substituting partial codons of ApGSMT2 and ApDMT2 with preferred codons of cereals and maize. Then it was connected with the RD29 promoter to construct the fusion gene, and the latter was recombined into the plant expression vector pCPE to generate pCPE-ApGSMT2d-ApDMT2d. Introduction of recombinant plasmids into Agrobacterium tumefaciens for genetic transformation.

[0115] 2) Obtaining sterile corn seedlings

[0116] Bagging to obtain the seeds of corn backbone inbred lines such as Zheng 58, Qi 319, Chang 7-2, etc., soaked with 70% ethanol for 10 minutes, then soaked with 0.1% mercuric chloride for 10-12 minutes, and then washed with sterile water for 3 -5 times. Shake the seeds constantly ...

Embodiment 3

[0132] Example 3, construction of ApTpGSMT2d, ApTpDMTd2 genes and creation of maize transgenic stress-resistant materials

[0133] 1) Synthesis of ApTpGSMT2d and ApTpDMTd2 genes and construction of plant expression constructs

[0134] In order to efficiently express ApGSMT2 and ApDMT2 in maize cells and realize the accumulation of glycine betaine in chloroplasts (plastids), the ApGSMT2d and ApDMT2d genes were respectively fused with the coding region of the transit peptide for positioning chloroplast proteins, so that ApGSMT2 and ApDMT2 proteins are localized in chloroplasts (plastids) after synthesis, and catalyze the synthesis of a large amount of glycine betaine in chloroplasts. PCR primers were designed based on the reported Arabidopsis thaliana RUBISCO small subunit gene sequence, and the guide peptide coding region of the RUBISCO small subunit was cloned from Arabidopsis thaliana, and connected to the open reading frames of the ApGSMT2d gene and ApDMT2d gene after sequen...

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Abstract

The invention discloses a methyl transferase gene in a betaine synthesis path and modification and application thereof. The method for synthesizing betaineb by using the methyl transferase gene comprises the following steps: colonizing glycine sarcosine N-methyl transferase gene (ApGSMT2) and dimethylglycine N-methyl transferase gene (ApDMT2) from aphanothece halophytica; generating ApGSMT2 and ApDMT2 genes by substituting codons which has lower occurring probability in higher plants in the ApGSMT2 and ApDMT2 with codons preferred by higher plants; and constructing series gene of a coding product, which are respectively positioned on a chloroplast and a mitochondria by using a method for fusing a protein positioning targeting sequence with the N-methyl transferase gene; the ApGSMT2 catalyzes glycine to be methylated by taking glycine and sarcosine as substrates; the ApDMT2 catalyzes dimethyl glycine to be methylated; the ApGSMT2 and the ApDMT2 carry out concerted catalysis to synthesize glycine betaine. ApGSMT2 / ApDMT2 or modifier genes thereof are transferred under the synergism action into maize, wheat, cotton, soy beans, tobacco and other crops and high-concentration glycine betaine is accumulated in cells, so that the stress resistance of plants can be remarkably improved.

Description

technical field [0001] The invention discloses two kinds of methyltransferase gene sequences in the synthesis pathway of cyanobacterium glycine betaine, the sequences of their modified genes and the protein sequences encoded by them respectively, and the two genes are used for stress resistance breeding of crops, belonging to field of genetic engineering. Specifically, it relates to the cloning and modification of a glycine sarcosine N-methyltransferase gene and a dimethylglycine N-methyltransferase gene involved in a glycine betaine biosynthesis pathway and utilization thereof. Background technique [0002] In betaine natural synthesis plants, glycine betaine, as an osmoprotectant, can improve the resistance of plants to various abiotic stresses (such as salinity, drought, low temperature, etc.), and the degree of resistance to a certain stress It is closely related to the accumulation level of betaine. Genetic engineering studies have shown that accumulating glycine beta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82C12N15/84A01H1/02C12R1/01
Inventor 张举仁何影李坤朋何春梅
Owner SHANDONG UNIV
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