| Drought is a natural disaster that seriously restricts the quality and yield of cotton.Therefore,to improve the quality of cotton,improve cotton production,we must improve the drought resistance of cotton,cultivate new varieties of cotton,which is the cotton breeding project need to solve the problem.Xinjiang,as one of the main producing areas of cotton in China,is greatly affected by drought stress.Drought as a primary factor in abiotic stress has seriously affected the production of cotton in Xinjiang.Therefore,it is of great significance to cultivate and study the cotton drought resistance mechanism by means of genetic engineering technology and molecular biology method,and to further explore and clone genes which are closely related to drought resistance of cotton.In this study,the HSP70 and V-H~+-ATPase A subunits proteins were differentially expressed after drought stress,which would be related to plant drought resistance.In this study,the two total length of drought stress-related genes were obtained by RT-PCR,named as GhHSP70-26 and GhVHA-A in upland cotton KK1543.The expression patterns of the two genes were analyzed by fluorescence quantitative PCR,and the functions of the two genes were further analyzed by Agrobacterium tumefaciens transformation technique and VIGS technique.Explain the possible mechanism of action of the two genes from the molecular level.The main results obtained in this study are as follows:(1)The cloned GhHSP70-26 gene is a family gene,then using bioinformatics to analyze the GhHSP70gene family of upland cotton.The results of conserved domain sequence analysis showed that HSP70protein was highly conserved and contained three typical HSP70 signature motif sequences.Chromosome mapping analysis showed that the HSP70 gene family members were distributed unevenly in the 13chromosomes of subgroup A and D subgroups;The phylogenetic tree analysis showed that the the HSP70family members were divided into four subfamilies:Plastid Stroma,Mitochondrial Matrix,Endoplasmic Reticulum and Cytosol.The secondary structure of GhHSP70-26 protein was composed of?-helix,?-sheet,corners and irregular curl,except transmembrane region.(2)The expression pattern of GhHSP70-26 has a constitutive expression in cotton tissues,highest in the leaves.The expression of GhHSP70-26 in the leaves was up-regulated under drought stress,high temperature,high salt stress and ABA treatment.The expression of GhHSP70-26 gene in different cotton leaves was analyzed by real-time fluorescence quantitative PCR.The results showed that the expression level of GhHSP70-26 gene was positively correlated with cotton drought resistance.The results of prokaryotic expression showed that GhHSP70-26 protein was obtained at different time and different concentrations of IPTG inducer.(3)After drought treatment,the cotton plants with GhHSP70-26 silencing by VIGS technique showed that the water-loss rate of detached leaves was higher than that of the control plants,and the content of MDA and the increase of the conductivity were higher than those of the control plants.The chlorophyll content and the activities of total antioxidant enzymes lower in the VIGS plants than in control plants under drought stress.Which indicated that the silence of GhHSP70-26 gene reduced the drought resistance of VIGS plants and was more susceptible to drought persecution than the control plants.(4)Overexpression of GhHSP70-26 could increase the survival rate of transgenic tobacco under drought stress,which could reduce the water loss of transgenic tobacco leaves and enhance the drought tolerance of transgenic tobacco.At the same time overexpression of GhHSP70-26 increased the drought resistance of transgenic tobacco by reducing the extent of damage and reducing the damage of ROS oxidative stress in transgenic tobacco cell membrane.(5)Yeast two-hybrid system analysis preliminary obtained cotton antioxidant enzyme APX has an interaction with GhHSP70-26 protein in the yeast body.Function analysis of the GhHSP70-26 upstream promoter revealed that the promoter that regulates the GhHSP70-26 gene have ABA response element and high temperature response element.Yeast hybrid experiments showed that the transcription factors GhbZIP and GhHSF specifically bind to the ABRE and HSE elements in the GhHSP70-26 promoter,indicating that GhHSP70-26 may be regulated by the above two transcription factors.(6)The V-H~+-ATPase A gene was cloned by RT-PCR and named as GhVHA-A.The complete open reading frame of this gene is 1872 bp,which contains 623 amino acid residues.The predicted molecular weight is 68.41 kDa and the theoretical isoelectric point is 5.17.It was found that the V-ATPase A subunit of cotton has high similarity and homology with soybean,Arabidopsis thaliana,wheat and cocoa,and was closely related to the VHA-A in cocoa.At the same time,the V-ATPase A subunit is a highly conserved protein subunit with two conserved domains:GAFGCGKT and PSVNWLISY.(7)The expression of GhVHA-A in different tissues of cotton was analyzed.The results showed that the expression of GhVHA-A was highest in stem.And the GhVHA-A had different degrees of response in the leaves when the cotton was subjected to high salt,dehydration(PEG),low temperature and exogenous ABA stress.The expression of GhVHA-A gene in leaves of different cotton materials was positively correlated with cotton drought resistance.(8)Study of VIGS technology silencing GhVHA-A shows that the silent plant water loss fast in vitro leaves,leaf MDA content and conductivity increased,while the chlorophyll content and total antioxidant enzyme activity reduced,which indicating that silencing GhVHA-A gene makes cotton sensitive to drought stress and increased the damage degree.(9)Overexpression of GhVHA-A can reduce the water loss in transgenic tobacco leaves.And increase the survival rate of transgenic tobacco under drought stress,indicating that overexpression of GhVHA-A can enhance the tolerance of transgenic tobacco.The overexpression of GhVHA-A can also increase the drought resistance of transgenic plants by increasing the accumulation of osmotic regulators in transgenic tobacco vacuoles and promoting the growth of transgenic tobacco roots.(10)The phenotype observation of transgenic cotton with overexpressing GhVHA-A showed that the transgenic cotton had well developed root system and more lateral roots,which indicated that GhVHA-A gene might play an important role in the growth and development of root. |
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