Genetic Transformation And Functional Analysis Of Salt Stress-related Genes From Gossypium Ardium

Plants suffer from a variety of biological and abiotic stresses during their growth and development.High salt stress is one of the abiotic stresses that adversely affect plant growth and is an important factor that restricts crop yield.Cotton is also regarded as a pioneer crop of saline-alkali land and has a good level of salt tolerance.With the intensification of global land salinization,the salt tolerance level of existing cultivars can no longer meet the status ofagricultural production.Therefore,development of salt-tolerance cotton is necessary so that the cotton plantroduction area can be moved to coastal saline-alkali land and inland saline-alkali land It is of great significance to alleviating the reduction of the area of cultivated land and ensure food security in our country.Studies have shown that the CBL family and WRKY transcription factor family can regulate a variety of abiotic stress mechanisms in plants.The CBL family members are Ca2+-dependent CBL-CIPK regulatory system in response to external environmental stress,WRKY transcription factor family members are activated by transcription or suppress the expression of downstream genes to deal with the unfavorable environment.In the previous study,the laboratory cloned two members of the family from the wild species of dryland cotton,named GarCIPK8 and GarWRKY1 7,transformed and validated their salt-tolerance functions in model crops Arabidopsis thaliana and tobacco,respectively.Compared with the wild type,the strain showed significant salt tolerance.In the previous period,the laboratory has completed the genetic transformation of GarCIPK8 gene in cotton.Three transgenic cotton lines were obtained in the T5 generation.On this basis,the objectives of this study are:1.To investigate the salttolerance of transgenic cotton lines with GarCIPK8 gene and its possible salt-tolerance mechanism.2.To transforme the salt tolerance related gene GarWRKY17 into cotton receptor material R15 using Agrobacterium mediated transformation method.1.Genetic transformation of Gar WRKY1 7 gene in cottonAgrobacterium tumefaciens-mediated transformation was used to infect cotton hypocotyls with Agrobacterium containing the gene of interest.Plants transformed with GarWRKY17 were obtained through plant tissue culture techniques.The obtained transgenic plants were positively screened by PCR and 6 strains were finally obtained.More than 50 strains of transgenic cotton.In this study,we mastered the entire process of cotton transgenic technology during the experiment,optimized the process of hardening seedling regeneration,improved the operability of the seedlings,and improved the survival rate of transgenic plants.2.Functional verification of salt tolerance of GarCIPK8 geneThree cotton homozygous lines were selected from the homozygous lines of the GarCIPK8 transgenic cotton line obtained after multiple generations of selfing,homozygote,resistance screening,PCR,and RT-PCR for the germination period.And seedling salt tolerance verification.Under the 200 mM NaCl treatment,both R15 and transgenic cotton germination were inhibited,and both R15 and transgenic lines were inhibited.R15 and transgenic lines showed that GarCIPK8 had an effect on cotton germination under salt stress.Not obvious.The growth of R15 and transgenic lines were inhibited under 300 mM NaCl treatment at seedling stage,but compared with R15,the transgenic lines grew more vigorously and the root system was more developed,indicating that GarCIPK8 gene can enhance the salt tolerance of cotton under salt stress..To further explore the salt-tolerant pathway of GarCIPK8 gene,the physiological indices of transgene lines and R15 leaves were detected under 300 mM NaCl stress.The results showed that compared with R15,SOD and POD activities in the transgenic lines were significant.The increase in MDA content was significantly reduced,indicating that GarCIPK8 gene improves cotton's salt tolerance by increasing the antioxidant stress of cotton.The ion content of transgene lines and R15 at different stages and different tissues and organs(roots,stems,and leaves)were determined under 300 mM NaCl stress.The results showed that the sodium accumulation in roots of transgenic lines was 15 days after salt treatment Below the level of R15,the accumulation of sodium in the stem was significantly higher than that of R15;the accumulation of potassium in the roots of the transgenic lines was higher than that of the receptor,and the amount of sodium accumulated in the stem was significantly higher than that of the receptor.The results showed that under salt stress,with the R15 Compared with the transgenic strains,they are less affected by salt damage and exhibit better salt tolerance.