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Application of CkGST gene

A gene and sequence technology, applied in the field of enhancing resistance to various adversities, can solve problems such as resistance that has not been reported yet

Active Publication Date: 2012-05-09
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the numerous reports on GST, resistance to ABA, glufosinate in herbicides, fungicides, and insecticides has not been reported

Method used

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  • Application of CkGST gene
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  • Application of CkGST gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Cloning and sequence structure analysis of Caragana CkGST gene

[0023] Caragana seedlings (seeds purchased from Xinjiang Turpan Botanical Garden) grown for 2-3 months were used as materials and induced overnight with 100 μM ABA (Sigma Company). After the leaves are washed, put them into a homogenizer treated with DEPC water and sterilized, add TRIzol (Invitrogen) to grind, homogenize, and stand at room temperature for 5 minutes. Chloroform was added to extract twice, and the supernatant was taken, and the total RNA was precipitated with isopropanol, air-dried, and dissolved in DEPC water. cDNA was obtained by reverse transcription with polyT primers, and RT-PCR was performed (see Kit DRR019A, Takara Biotech. (Dalian) Co. Ltd. for specific operations).

[0024] The primer sequence of polyT is as follows:

[0025] 5'-CGAGCGGCCGCCCGGGCAGGTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 1)

[0026] RT-PCR reaction conditions are as follows

[0027] DEPC water 85μL

[0028] ...

Embodiment 2

[0093] Example 2. CkGST Kinetic Detection

[0094] a) Construction of prokaryotic expression vector for transformation, prokaryotic expression and purification of CkGST

[0095] The CkGSTcDNA coding region was ligated into the prokaryotic expression vector pET28a (Novagen Company), and expressed in Escherichia coli by IPTG (see the pETsystem manual of Novagen Company for specific operations). In view of the gene cloned and expressed as CkGST, GST magnetic beads (MagneHis of Promega TM Protein Purification System, Cat.V8500) to separate and purify the CkGST protein expressed by bacteria ( figure 2 ).

[0096] The specific purification operation is as follows:

[0097] 1. Induce 500ml of bacterial liquid and collect the bacterial cells. Wash once or twice with sterile water.

[0098] 2. Quick-freeze the collected bacteria at -20°C for 15-20 minutes.

[0099] 3. Add 100ml of cell lysis reagent (cell lysis Reagent), and mix gently at room temperature for 30 minutes.

[01...

Embodiment 3

[0124] Embodiment 3. Containing the construction of the plant expression vector of CkGST gene

[0125] According to the cds sequence of CkGST, a primer (SEQ ID NO: 10, 5'-ATCT GGATCC ATGGCAAATGAGGTGGTTCTG-3'), with a Sac I restriction site primer on the right end (SEQ ID NO: 11, 5'-TCAT GAGCTC CTACTCAATGCCAAACCTCTTTC-3'), using the high-fidelity Pyrobest Taq enzyme from TaKaRa Company, using the pUCm-GST formed in Example 1 as a template, amplifying the fragment (680bp) with BamH I and Sac I restriction sites, using BamH I And Sac I double cut. At the same time, pBI121 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was double cut with BamH I and Sac I. Then, after high temperature inactivation of BamH I and Sac I of the two double cut products, T4 ligase was added for overnight ligation, transformed into DH5α, coated with LBK plate, randomly picked a single colony, and extracted the plasmid. Take the pGST plasmid connected to the GST fragment, which is the construct...

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Abstract

The invention provides application of a CkGST gene in improving stress resistance of a plant. The gene originates from Caragana korshinskii Kom and encodes glutathione S-transferase. The invention relates to resistance of CkGST gene-transformed plants to heavy metal, salts, herbicides, bactericides, pesticides and ABA.

Description

technical field [0001] The present invention relates to the use of CkGST gene, especially the use of enhancing resistance to various adversities. Specifically, the present invention relates to CkGST gene-transferred plants with enhanced resistance to heavy metals, NaCl, ABA, insecticides, fungicides and herbicides. technical background [0002] When plants are subjected to external adversity stress (such as salt stress, drought stress, pesticide spraying, heavy metal pollution, etc.), it will lead to reactive oxygen species (ROS) in plants (such as O 2 、H 2 o 2 , O 2 · , OH · etc.) generation. The reactive oxygen species (ROS) produced by these are highly destructive, and they mainly attack the unsaturated double bonds in the lipids on the cell membrane, resulting in a chain reaction of free radicals, resulting in the peroxidation, cracking, and toxic to cells [1] . [0003] Glutathione-S-transferase (GST, E.C.2.5.1.18) is a multifunctional enzyme that mainly cataly...

Claims

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Application Information

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IPC IPC(8): C12N15/54A01H5/00
Inventor 胡赞民李之国苏晓华尹维波陈宇红宋丽英
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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