Application of arabidopsis glutathione peroxidase gene HAD1 in anti-drought aspect for plant

A technology of glutathione peroxidase and anti-drought, applied in the field of genetic engineering

Inactive Publication Date: 2009-03-11
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Glutathine peroxidase (Glutathine Peroxidase, GPX) is an important defense enzyme against oxidative stress. Its function of scavenging reactive oxygen species in animals and yeast has been confirmed, but its application in plants has not yet been It has been reported, for example, how it responds to external stress, how to sense and clear the ROS produced by abiotic stress needs to be further explored

Method used

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  • Application of arabidopsis glutathione peroxidase gene HAD1 in anti-drought aspect for plant
  • Application of arabidopsis glutathione peroxidase gene HAD1 in anti-drought aspect for plant

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Embodiment 1

[0015] Identification of HAD1 Gene Protein Activity

[0016] The method of expressing HAD1 protein in vitro was used to investigate whether it had peroxidase activity. The expression vector expressing HAD1 fusion protein was constructed with BL21 as the carrier, and expressed in Escherichia coli. Oxidase activity analysis, it is known that the consumption of NADPH required for the conversion of the two states of oxidation and reduction of glutathione peroxidase, under the excitation of 340nm wavelength, the absorbance value of NADPH can be measured, and its consumption can be calculated, namely The more NADPH is consumed, the higher the enzymatic activity of HAD1. The results showed that the prokaryotic expression of HAD1, Trx, TR, NADPH and H 2 o 2 In case of co-existence, OD 340nm Decreases continuously over time. When recording for 16 minutes, the OD value decreased by about 0.4, while there was only a small decrease when GSH was used as the electron donor. In addition...

Embodiment 2

[0018] In vivo expression of HAD1 gene

[0019] The HAD1 gene promoter and GUS fusion (pCMBIA1381 carrier) transgenic positive plants obtained by PCR amplification technology were used to detect the expression of HAD1 in different tissues of Arabidopsis thaliana. Histochemical staining showed that the whole The plants all have strong expression, and the GUS activity was measured in different tissues such as leaves, flowers, roots, stems, and pods. The results showed that the expression levels in leaves and roots were the strongest, while those in fruits, pods, stems, and flowers The expression is relatively weak; RT-PCR analysis of the transcription of HAD1 mRNA in different tissues also confirmed that the expression level in roots and leaves is higher; in addition, the expression level of HAD1 gene in plant guard cells is significantly higher than that of other isozyme genes .

Embodiment 3

[0021] Overexpression of HAD1 gene and molecular phenotype identification of mutants

[0022] In order to prove the function of HAD1 protein, its T-DNA insertion mutant (SALK_071176) was obtained from SALK laboratory, and its insertion site was located in the first exon; SALK laboratory provided the designed LP and RP double primer pair SALK_071176 was identified, and wild-type Arabidopsis genomic DNA was used as a template. PCR amplification results showed that there was an obvious band at 900bp, but SALK_071176 did not appear. In order to further identify homozygotes, the primers LBa1 and LBb1 designed on the left border of the T-DNA were used to carry out PCR reactions with the RP on the genome respectively, and two distinct lines were amplified at about 500bp and 700bp using the SALK_071176 plant genomic DNA as a template. The bands differ in size by about 200bp. In addition, RT-PCR analysis showed that the transcription of the mRNA of potential mutant HAD1 was terminated...

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Abstract

The invention relates to application of a glutathione peroxidase gene HAD1 (High amount of water loss under drought stress) of arabidopsis in the aspect of drought resistance, wherein an HAD1 gene mutant (SALK_071176) is acquired from an arabidopsis resource center (ABRC, Ohio State University); and the gene No. is AT2g43350, and the length of cDNA of the gene is 625 bp, with proteins of 206 coded amino acids. The HAD1 gene can be used for improving the drought tolerance of crops, greatly reduces the cost of water irrigation during the growth process of the crops, improves the utilization rate of moisture of plants, can increase the yield of the crops, and has important economic value and wide application prospect.

Description

technical field [0001] The invention relates to the application of Arabidopsis glutathione peroxidase HAD1 gene in plant drought resistance, belonging to the field of genetic engineering. Background technique [0002] At present, my country's population is growing rapidly, and food production is facing increasingly severe challenges. Drought adversity seriously affects the growth and development of crops, and is the main factor limiting the improvement of crop yield and quality. According to statistics, the world's arid and semi-arid areas account for 1 / 3 of the world's total area, while my country accounts for 1 / 2, of which About 65 percent of the drylands are not irrigated. At present, there are about 1 billion mu of low- and medium-yield fields in my country, most of which are caused by drought and salinity. Improving the drought resistance of crops and establishing new strains with high drought resistance are urgent problems to be solved to increase grain production. Al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/82C12N9/08
Inventor 宋纯鹏苗雨晨郭敬功李坤代杰
Owner HENAN UNIVERSITY
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