Transmethylase gene in betaine synthesis process 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: 2012-07-25
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 ...

Method used

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  • Transmethylase gene in betaine synthesis process and application thereof
  • Transmethylase gene in betaine synthesis process and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: ApGSMT2 / ApDMT2 gene cloning and creation of maize transgenic stress-resistant materials

[0091] 1) ApGSMT2 / ApDMT2 gene cloning

[0092] 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 / L Tris-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% before preheating β-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 tra...

Embodiment 2

[0115] Example 2: Synthesis of ApGSMT2d and ApDMT2d genes and creation of maize transgenic stress-resistant materials

[0116] 1) Synthesis of ApGSMT2d and ApDMT2d genes and construction of plant expression constructs

[0117] 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.

[0118] 2) Obtaining sterile corn seedlings

[0119] 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

[0135] Example 3, construction of ApTpGSMT2d, ApTpDMTd2 genes and creation of maize transgenic stress-resistant materials

[0136] 1) Synthesis of ApTpGSMT2d and ApTpDMTd2 genes and construction of plant expression constructs

[0137] 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 transmethylase gene in a betaine synthesis process, and modification and application thereof. The method comprises the following steps: cloning glycine sarcosine N-transmethylase gene (ApGSMT2) and dimethyl glycine N-transmethylase gene (ApDMT2) from aphanothece halophytica, substituting codons with lower occurrence possibility in higher plants in the ApGSMT2 and the ApDMT2 with higher plant preferred codons to generate a gene ApGSMT2d and a gene ApDMT2d, and constructing the serial genes, of which the coded products are respectively positioned in the chloroplast, byprotein locating targeting sequence and transmethylase gene fusion. Glycine and sarcosine are used as the substrates, the methylation of the glycine is catalyzed, the methylation of the dimethyl glycine is catalyzed by ApDMT, and the glycinebetaine is synthesized under the concerted catalysis. The ApGSMT2/ApDMT2 or modifier genes thereof are synergically transferred into corn, wheat, cotton, soybean, tobacco and other crops, so high-concentration glycinebetaine is accumulated in the cells, thereby obviously enhancing the stress tolerance of the plant.

Description

[0001] This application is a divisional application of the publication number CN 101805744A. The application date of the original application: 2009.09.09, the application number: 200910018647.6, and the title of the invention: a methyltransferase gene in the betaine synthesis pathway and its modification and utilization. technical field [0002] 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 [0003] In betaine natural synthesis plants, glycine bet...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H1/02A01H5/00
Inventor 张举仁何影李坤朋何春梅
Owner SHANDONG UNIV
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