Double expression application of Arabidopis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene

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

Active Publication Date: 2015-07-15
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 Arabi

Method used

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  • Double expression application of Arabidopis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene
  • Double expression application of Arabidopis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene
  • Double expression application of Arabidopis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene

Examples

Experimental program
Comparison scheme
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 1μ...

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-re...

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Abstract

The invention discloses a double expression application of Arabidopis thaliana serine hydroxymethyltransferase gene and formate dehydrogenase gene. The cDNA of Arabidopis thaliana serine hydroxymethyltransferase SHMT gene is used to construct a plant expression vector, the plant expression vector constructed by the cDNA of Arabidopis thaliana serine hydroxymethyltransferase SHMT gene is transferred to tobacco through Agrobacterium mediation, and a plant expression vector constructed by the cDNA of Arabidopis thaliana formate dehydrogenase FDH gene is transferred to the tobacco through Agrobacterium mediation after detection correctness to obtain two exogenous genes SHMT/FDH inserted double expression transgenic tobacco capable of improving the formaldehyde absorption and tolerance ability of plants. Experiment results show that the double expression transgenic tobacco can change the content of chlorophyll and soluble sugar in tobacco to effectively reduce the toxicity of HCHO to plants in order to make the transgenic tobacco leaf maintain good HCHO absorption ability under the stress of high concentration HCHO; and the double expression transgenic tobacco can effectively reduce the oxidation stress and has a substantially better oxidation stress reduction effect 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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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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