Application of Dual Expression of Serine Hydroxymethyltransferase Gene and Formate Dehydrogenase Gene in Arabidopsis

A technology of Arabidopsis serine hydroxymethyl and serine hydroxymethyl, applied in the field of plant genetic engineering

Active Publication Date: 2017-11-10
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serine (Ser) is one of the important products of formaldehyde metabolism in Arabidopsis, but Ser is not produced in the formaldehyde metabolism process of tobacco

Method used

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  • Application of Dual Expression of Serine Hydroxymethyltransferase Gene and Formate Dehydrogenase Gene in Arabidopsis
  • Application of Dual Expression of Serine Hydroxymethyltransferase Gene and Formate Dehydrogenase Gene in Arabidopsis
  • Application of Dual Expression of Serine Hydroxymethyltransferase Gene and Formate Dehydrogenase Gene in Arabidopsis

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Example 1: SHMT / FDH PCR Amplification and TA Cloning of Gene cDNA

[0072] Using TRIzoL Reagent (Invitrogen) from Arabidopsis ( Arabidopsis thaliana ) to extract total RNA from seedlings, take about 0.1g of plant tender leaves, add 1ml of TRIzoL extract, grind in a mortar, let stand at room temperature for 5min, then transfer to a centrifuge tube, then add 0.2ml of chloroform, shake and mix, and centrifuge for 15min (12000rpm ), transfer the supernatant to a new tube, add 0.5ml isopropanol, mix well and place at room temperature for 10min, centrifuge at 4°C for 10min (12000rpm), discard the supernatant, wash the precipitate with 1ml of 75% ethanol, and centrifuge at 4°C for 5min (7500rpm) , Discard ethanol and vacuum-dry the precipitate or dry it naturally, and dissolve RNA with 20 μl diethyl pyrocarbonate (DEPC) in water. Use M-MuLV Reverse Transcriptase Kit (TaKaRa) for cDNA synthesis, take about 0.1μg-5μg of total plant RNA, oligo(dT) 50ng, 10mM dNTP mix 1μl, ...

Embodiment 2

[0076] Example 2: Construction of entry vector pENTR-PrbcS- SHMT with pENTR-PrbcS-T*- FDH

[0077] use Sph I and xho I double enzyme cut pMD19- SHMT and pENTR-PrbcS- ADH , separated the excised vector and insert by agarose gel electrophoresis, and recovered pMD19- SHMT produced after being cut SHMT The cDNA fragment of the gene (1.6kb) and pENTR-PrbcS- ADH The vector fragment pENTR-PrbcS generated after being cleaved, and then pENTR-PrbcS and SHMT The cDNA fragment of the gene produces the entry vector pENTR-PrbcS- SHMT ( figure 2 ). Conversion of high efficiencies (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with kanamycin (Km, 50 μg / ml), and cultivate overnight at 37°C. Screen the Km-resistant recombinant colony, extract the plasmid from the Km-resistant recombinant colony, and select the successfully connected plasmid vector pENTR-PrbcS- ...

Embodiment 3

[0079] Embodiment 3: Construction of plant expression vector pK 2 -PrbcS- SHMT and pH 2 -PrbcS-T*- FDH

[0080] Through the LR response of Gateway technology, the SHMT Subcloning into the plant expression vector pK 2 GW 7 middle( image 3 ). Use the plasmid extraction kit to purify Gateway's target vector pK 2 GW 7 , add pENTR-PrbcS- to Gateway's LR reaction system SHMT and pK 2 GW 7 150ng each, 1μl LR Clonase II Enzyme Mix (Invitrogen), mixed well and reacted overnight at 25°C. SHMT Integrate into pK 2 GW 7 Plant expression vector plasmid pK obtained from SHMT 2 -PrbcS- SHMT . The reaction mixture was used to convert the high efficiency (10 8 ) Escherichia coli competent cells (DH5α, purchased from Tiangen Biochemical Technology Co., Ltd.), spread the transformed Escherichia coli on a plate added with spectinomycin (Spe, 50 μg / ml), culture overnight at 37°C, and screen for Spe resistance. Sexual recombinant colonies. Extract plasmids from Spe-resistant re...

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Abstract

The invention discloses the dual expression application of Arabidopsis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene. The invention uses Arabidopsis thaliana serine hydroxymethyltransferase SHMT gene cDNA to construct a plant expression vector, and mediates it through Agrobacterium Transform into tobacco, after the detection is correct, the plant expression vector constructed by Arabidopsis formate dehydrogenase FDH gene cDNA is transferred into it through Agrobacterium-mediated, and the double-expression transgenic tobacco with two exogenous genes SHMT / FDH inserted can be obtained. Improve the plant's ability to absorb and tolerate formaldehyde; the experimental results show that double-expression transgenic tobacco can effectively reduce the toxicity of HCHO to plants by changing the content of tobacco chlorophyll and soluble sugar, so that the transgenic tobacco leaves can maintain better under the stress of higher concentration of HCHO HCHO absorption energy; double-expression transgenic tobacco can effectively reduce the occurrence of oxidative stress, and this effect is more significant than single-expression transgenic tobacco.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to Arabidopsis serine hydroxymethyltransferase SHMT gene and formate dehydrogenase FDH The application of the plant expression carrier of the gene in the preparation of double expression transgenic plants with enhanced formaldehyde absorption capacity. Background technique [0002] Formaldehyde (HCHO) is widely used in the chemical and other industrial fields involved in chemical synthesis, industrial manufacturing, pharmaceutical synthesis, etc., especially formaldehyde plays a pivotal role in the synthesis of chemical pesticides and their intermediates. In the production process of modern chemical pesticides, it is inevitable to produce a large amount of formaldehyde-containing pesticide production wastewater. These pesticide production wastewater generally contain a large amount of formaldehyde and alcohol, benzene, phenol, paraformaldehyde, methylal and other substance...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/54C12N15/53A01H5/00
Inventor 陈丽梅刘婷曾智东
Owner KUNMING UNIV OF SCI & TECH
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