Cloning And Functional Verification Of GhSAD2 Gene In Cotton

In this study,GhSAD2 gene was amplified by RT-PCR and GhSAD2 gene was cloned by RT-PCR.The GhSAD2 gene was analyzed by qRTPCR.And to explore the expression pattern of the target gene in the process of cotton seed development and under low temperature stress.In this study,we constructed the overexpression vector of GhSAD2 gene and mediated the transformation of Agrobacterium tumefaciens to tobacco and The results showed that the cold resistance and fatty acid composition of the seedlings were analyzed by the method of transgenic GhSAD2 interferon carrier T3 and receptor material 2074 B.The main results were as follows:1.GhSAD2 gene was cloned and the bioinformatics analysis was carried out.GhSAD2 is registered in NCBI as KX197920,its cDNA is 1188 bp in length,encoding 396 amino acids,containing two histidine clusters is a typical fatty acid desaturase family protein.The Gh SAD2 gene encodes a molecular weight of45.3 kD,a theoretical isoelectric point of 5.88,and an average hydrophilicity(GRAVY)of-0.464,belonging to a hydrophilic protein.Phylogenetic analysis showed that the gene was closely related to the evolution of the homologous gene of the cocoa tree.2.The overexpression vector of the target gene was constructed and transformed into tobacco and cotton.The plant overexpression vector p BI121-GhSAD2 was transferred into Sanxi tobacco and Xinlu early 33 through Agrobacterium tumefaciens-mediated leaf disc and callus transformation respectively.A total of 9 transgenic tobacco plants with GhSAD2 gene overexpression were obtained,The conversion rate of tobacco was 75%;cotton positive plants were not obtained,positive cotton callus was obtained and GUS was tested.3.The expression of target gene was studied by fluorescence quantitative PCR.The results showed that the expression level of GhSAD2 gene in cotton leaves was higher than that in stem and root.The expression of GhSAD2 gene in cotton seed development showed a trend of gradual decrease and then decreased in the seeds at 25 days after anthesis GhSAD2 gene was able to respond to low temperature stress,and its expression was the highest at 4 ? and 15 ? for 6 h.After 12 h of stress,the expression of GhSAD2 gene was stable in the control group,2.3 times.The conductivity of GhSAD2 overexpressing transgenic tobacco was significantly lower than that of wild type tobacco at 48 ? after 4 ? low temperature stress.It was speculated that Gh SAD2 gene might play a role in cotton cold stress regulation.4.The agronomic traits and fatty acid contents of cotton T3 line of transgenic GhSAD2 interference vector were determined.The results showed that the transgenic cotton and the recipient 2074 B compared with the clothing increased by 7%-8%,clothing content increased 0.8-1.6g,seed index decreased by1.3-2.4g.The content of total fatty acids,palmitic acid and linoleic acid in transgenic cotton seeds decreased significantly,the content of total protein,the content of stearic acid and the content of oleic acid were significantly increased,and the GhSAD2 gene was affected by the desaturation process of fatty acidsThe ultimate oil content of the child.5.The expression characteristics of the target gene in the seed development of transgenic plants were studied by fluorescence quantitative PCR.The results showed that the expression pattern of the target gene was lower than that of the recipient 2074 B at the 15 th day after anthesis,and the expression pattern was the same as that of the recipient plant.The results showed that the expression of the seeds was the highest in the seeds after 30 days.6.The conductivity,malondialdehyde content and proline content of transgenic plants were determined.The results showed that RNA tolerance of transgenic plants was stronger than that of CK,and the cold tolerance was weakened and the heat tolerance was enhanced.It was speculated that the cold tolerance of cotton seedlings could be related to the expression of GhSAD2 gene in cotton seedlings.