The Cloning And Functional Analysis Of Soybean Salicylic Acid Combined With Protein Gene(GmSABP-2like)

Soybean is one of the main sources of oil,food and feed crops.In recent years,natural disasters,especially drought and water shortage,have the relative seriously influence on the yield of soybean.Therefore,one of the important ways to increase the yield of soybean is to enhance the adaptability of soybean in drought conditions.However,traditional breeding methods have some problems such as gene limitation in the process of cultivating new drought-resistant soybean varieties,and transgenic technique,as an important breeding method,can effectively solve the above problems.Therefore,genetic engineering technology was used to cultivate new drought-resistant soybean varieties in this study.M18 genomic DNA of soybean mutant was used as template,and the cloned soybean salicylic acid binding protein gene?GmSABP-2like?was obtained by homologous polymerase chain reaction?PCR?amplification technology.The sequence of the obtained gene was analyzed by bioinformatics software and linked to pMD-18T cloning vector.Using pCAMBIA-3301 as the basic vector,plant overexpression and RNAi expression vector containing screening marker Bar were constructed by in-fusion cloning technology.Subsequently,the constructed recombinant plant expression vector was transferred into the variety‘JN38'of soybean by Agrobacterium-mediated method,and the obtained positive plant seeds were preliminarily detected by PCR for indoor passage.Routine PCR,southern hybridization,quantitative-PCR and drought resistance identification were carried out on T₂generation positive plants to analyze the drought resistance function of GmSABP-2like gene,and the relevant test results were as follows.1.Design of gene-specific primers:a 378 bp soybean GmSABP-2like gene was obtained by PCR using M18 genome of soybean drought resistant mutant as template,and linked to pMD-18T cloning vector.2.The result of sequence analysis showed that the length of GmSABP-2like gene was 376 bp,encoding 124 amino acid,the molecular weight and isoelectric point of protein were 13937.25 and 8.57,respectively.3.Interference expression vector pCAMBIA-3301-GmSABP-2like-RNAi and overexpression vector pCAMBIA-3301-GmSABP-2like of plant were successfully constructed by in-fusion cloning technique.4.The constructed recombinant plant expression vector was transferred into the variety‘JN38'of soybean by Agrobacterium-mediated method,after PCR detection,2and 3 overexpressed and interfering vector plants were obtained in T₀generation respectively;5 overexpression vector and 7 interference vector plants were obtained in T₁generation;13 overexpression vector and 10 interference vector plants were obtained in T₂generation.5.Southern hybridization analysis showed that GmSABP-2like gene was mainly integrated into soybean genome in the form of single copy,and the integration site was different from other genes.6.The results of qRT-PCR analysis showed that the overexpression of GmSABP-2like gene in T₂transfected plants was significantly increased compared with that of untransformed plants;however,the expression of GmSABP-2like gene in interference vector plants showed a downward trend.7.The results of drought resistance experiment showed that overexpression vector plants significantly improved the drought resistance of soybean JN38.