Cloning,Expression Vector Construction And Genetic Transformation Of SPW5 Gene From Sweet Potato

In recent years,due to the improvement of production level and the optimization of agricultural industrial structure,as well as the adjustment of people's dietary structure,as an important food,feed,industrial raw materials and new energy,the growing area and people's demand or sweet potato is increasing,However,sweet potato is an asexually propagated crop,which is easy to accumulate viruses in the next generation,and resulting in a large decrease in yield and a deterioration in quality.The research on the disease resistance mechanism of sweet potato,and the study on the genes related to sweet potato disease resistance will provide a basis for breeding of sweet potato varieties.In the perivious study of the laboratory,several genes related to sweet potato virus resistance,including the SPW5 gene,were screened by transcriptome analysis.In this study,the SPW5 gene was cloned from the sweet potato variety Xushu22,and its nucleotide sequence was analyzed.The gene overexpression vector SPW5-p101-GV3101 was successfully constructed.The regeneration system of sweet potato was explored.Stem tips,leaves,petioles and lateral buds of four varieties of sweet potato were used as explants to establish regeneration system,and the regeneration medium was optimized.Callus was successfully induced and suspension culture was carried out,somatic embryos were successfully induced.The genetic transformation system of SPW5 gene expression vector mediated by Agrobacterium tumefaciens was explored.The main findings are as follows:1.The SPW5 gene primers were designed according to the sequence obtained by transcriptome sequencing,and the SPW5 gene CDS sequence was cloned from the sweet potato variety Xushu22.The overexpression vector SPW5-p101-GV3101 was successfully constructed using the vector pEarleyGate101.2.The SPW5 gene was predicted and analyzed by bioinformatics analysis.The coding region was 1182 bp and encoded 393 amino acids.The protein encoded by this gene is a non-membrane protein located in the nucleus.The molecular weight of the protein is 43301.21 Da,and the isoelectric point pI is 6.36.The NCBI website predicts that the encoded protein has the WRKY region of the WRKY family and is highly conserved.It is concluded that the sweet potato SPW5 gene encode a member of the WRKY family.3.The test results of the sweet potato regeneration system show that:(1)The best explants for sweet potato-induced callus are leaves;(2)MS+0.5mg/L 6-BA+30g/L sucrose had the best effect in sweet potato callus culture;(3)The optimal conditions for suspension culture are: initial seeding density of 1.0:25 mL,inoculum size of 1.5 mL,and shaker speed of 100 r/min.4.The genetic transformation system of sweet potato was explored.The results showed that when the sweet potato variety Xushu 22 was used as the genetically transformed explant,(1)the non-transgenic positive plants could still be normal selected under the condition of 50mg/L kanamycin(Kan).The concentration of resistance screening should be increased during genetic transformation.(2)Stem segments without lateral buds could not differentiate adventitious buds,but stem segments with lateral buds could differentiate 45 pseudo-transgenic plants.The results of PCR showed that there no positive plant in 45 transformed plants.