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Cotton ghas1 gene, ghas1 protein, recombinant vector, recombinant bacteria and application thereof

A recombinant vector, recombinant bacteria technology, applied in the field of genetic engineering, can solve the problems of asymmetry, no obvious main leaf veins, abnormal pod shape and so on

Active Publication Date: 2020-07-03
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the normal wild-type Arabidopsis, the as1 and as2 mutants have many phenotypic defects: both leaves are curled downward and have appendages (leaf lobe); petioles and leaves are shorter, There is no obvious main vein, and the number of branches of the vein is small and asymmetric; when the mutant leaves are used for tissue culture in a hormone-free medium, the frequency of callus differentiation into roots is lower than that of wild-type leaves, but the differentiation into stems The frequency of the latter is higher than that of the latter; the degree of polar differentiation on the adaxial surface of the leaves is reduced, and the morphology of the pods is also abnormal
However, the cotton AS1 gene and its functions in salt stress resistance and epidermal hair development have not been reported yet.

Method used

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  • Cotton ghas1 gene, ghas1 protein, recombinant vector, recombinant bacteria and application thereof
  • Cotton ghas1 gene, ghas1 protein, recombinant vector, recombinant bacteria and application thereof
  • Cotton ghas1 gene, ghas1 protein, recombinant vector, recombinant bacteria and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Obtaining the full-length cDNA of cotton GhAS1

[0027] (1) GhAS1 gene-specific primer design

[0028] Use Arabidopsis thaliana AtAS1 gene sequence to NCBI to do Blast, and select the EST sequence (890bp) of GhAS1 gene with the highest similarity according to the comparison results. Using primer premier 5.0 software, a pair of forward primers and a pair of reverse primers were designed at the 3' end of the EST sequence and a pair of reverse primers were designed for the nested PCR process in the 5' and 3' RACE experiments. The distance between the two forward primers and the distance between the two reverse primers are about 50bp, and at the same time ensure that the known sequences at the 3' and 5' ends of 200bp to 300bp can be amplified. After the sequencing is completed, it is used to determine the sequence and stitching. After obtaining the full length of the GhAS1 gene, design primers containing the entire coding region for confirmation. The designed primer ...

Embodiment 2

[0097] Sequence analysis and expression analysis of cotton GhAS1 gene.

[0098] After obtaining the full-length cDNA of the cotton GhAS1 gene, the cDNA sequence was translated into an amino acid sequence using DNAMAN software, and then compared with homologous genes and homologous proteins in other crops. The alignment results of the coding region sequence of the cotton GhAS1 gene and the gene coding region sequence of Arabidopsis thaliana AtAS1 (Arabidopsis TAIR database, http: / / www.arabidopsis.org / index.jsp, AT2G37630) are as follows figure 1As shown, the similarity is 64.02%. The amino acid sequence of cotton GhAS1 was compared with homologous proteins in other plants, namely AtAS1 in Arabidopsis thaliana and PHAN in snapdragon (Morimoto, R., Nishioka, E., Murai, K. and Takumi, S. (2009 ) Functional conservation of wheat orthologs of maize rough sheath1 and rough sheath2 genes. Plant molecular biology, 69, 273-285.), RS2 in corn (Timmermans, M.C., Hudson, A., Becraft, P.W....

Embodiment 3

[0103] Obtaining of plants with overexpression of GhAS1 gene.

[0104] Using the cDNA of cotton leaf tissue as a template, the full-length cDNA of the GhAS1 gene was amplified with the gene-specific primer pair XbaⅠ-880E-F: 5'-GTCTCTAGATGTCATTCCGTCTATCTTATTTG-3' and SacⅠ-880E-R: 5'-AATGAGCTCCAACAACTTCAAATTCACATACC-3', Purified by 1% agarose gel electrophoresis, the recovered cDNA fragment and pCAMBIA1301 plasmid were digested overnight with XbaI and SacI respectively, and then purified by agarose gel electrophoresis after enzyme digestion. Mix the digested cDNA fragment and the pCAMBIA1300 plasmid in a ratio of 5:1 (such as Figure 4 shown), and ligated overnight at 16°C using T4 ligase. The ligation product was transformed into DH5α-competent Escherichia coli, and after selection by resistant LB medium containing kanamycin (50 μg / ml), a positive single clone was picked and inoculated in 10 ml of LB culture medium containing kanamycin, Shake overnight at 37°C at 200 rpm, and...

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Abstract

The invention relates to a cotton GhAS1 gene, a GhAS1 protein, a recombinant vector, a recombinant bacterium and application thereof, and belongs to the technical field of genetic engineering. ThroughRACE, the cDNA overall length of the cotton GhAS1 gene is obtained, and it is discovered that the cotton GhAS1 gene is similar to the arabidopsis AtAS1 gene by 67%; the protein sequence of gene coding is obtained, and it is discovered that the conservative DNA binding domain and calcium ion binding domain and the arabidopsis AtAS1 gene, antirrhinum majus PHAN, corn RS2 and wheat WRS2 have high homology. The GhAS1 gene is conversed into arabidopsis mutant as1-101, and a transgenic plant is obtained; by studying the transgenic plant, it is discovered that the cotton GhAS1 gene can recover the phenotypic defects of the Atas1 mutant, can promote the development of the epidermal hair of the transgenic plant, and has the effect of improving the salinity resistance inhibition of the plant.

Description

technical field [0001] The invention relates to cotton GhAS1 gene, GhAS1 protein, recombinant vector, recombinant bacteria and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Cotton is one of the most important fiber crops and occupies an important position in my country's national economy. As the area of ​​cultivated land continues to decrease, the competition for land for grain and cotton has intensified, and the cotton planting area is gradually approaching areas with poor soil or even salinized areas. Therefore, improving the salt stress resistance of cotton varieties has important production and application value. Arabidopsis ASYMMETRIC LEAVES1(AT2G37630) and AtAS2(AT1G65620) genes are involved in the process of leaf polarity establishment. Compared with the normal wild-type Arabidopsis, the as1 and as2 mutants have many phenotypic defects: both leaves are curled downward and have appendages (leaf lobe); peti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415A01H6/20
Inventor 王中王姗姗杨军李锋武明珠谢小东罗朝鹏
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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