Arabidopsis Thaliana Cell Wall Invertase AtCWINV4Function Research And Genetic Transformation Of Brassica Napus

Invertase is one of the key enzymes in sucrose metabolism of plants, which catalyzes hydrolysis of sucrose into glucose and fructose irreversibly. According to the different locations, it can be divided into the cell wall invertase, vacuole invertase and cytoplasmic invertase. They are involved in the regulation of plant photosynthesis, plant gowth and development, cellular response to stress.They also play an important role in signal transduction.AtCWINV4is specific expression in flowers of Arabidopsis thaliana(Coumbia). Researchers have found that cwinv4lines cannot produce nectar,mutant flowers displayed greatly reduced cell invertase activity when compared with wild-type plants, and cwinv4flowers also accumulated significantly lower levels of total soluble sugar.These results implicate CWINV4as an absolutely required factor for nectar production. We have taken AtCWINV4overexpression lines as materials and analyzed its expression,invertase enzyme activity, soluble sugar content and transformed Brassica napus"zhong shuang11" on this basis. Research results are as follows:At first,we have constructed four different overexpression vector pSN01, pSN02, pSN03, pSN04around AtCWINV4(with pZP212as a template), infected wild-type Arabidopsis thaliana and obtained transgenic plants. We have analysed the expression of AtCWINV4in transgenic plants with RT-PCR. The expression of AtCWINV4is higher than wild-type plants in several pSN01、pSN02transgenic lines, and cell wall invertase activity is increased in these lines. In terms of soluble sugar content, glucose content,fructose content is increased in three lines of pSN02transgenic plants compared with wild-type plants. These results demonstrate that improving flower nectary AtCWINV4expression to increase yield of nectar through transgenic means is possible.But the expression of AtCWINV4in pSN03, pSN04T1transgenic lines is reduced compared with wild-type plants,the two constructions may not be able to be used to add nectar production in crop improvement.Secondly, for the first part of the results,we have transformed Agrobacterium GV3101with vector pSN01, pSN02,then transformed Brassica napus"zhong shuang11",and obtained regenerated plants from the screening culture medium. We have identified six positive plants from thirty four regenerated plants through PCR. Transformation efficiency is about seventeen percent. We have harvested the seeds of T1 generation and lay the foundation for exploring to increase yield of nectar of Brassica napus through transgenic means.Thirdly, in order to clone invertase gene of Brassica napus,we have spliced cDNA sequence of AtCWINV4homologous gene in Brassica napus by using the in silico cloning technique as AtCWINV4probe sequence. We have designed specific primers according to the sequence of cDNA and obtained the cDNA sample through reverse transcription of mRNA of Brassica napus..Then we have amplified the cDNA of related gene through RT-PCR and jointed vector T for sequencing. The results show that the DNA sequence obtained by experimental means is consistent with the electronic cloning patchwork sequence completely. It reveals that EST public database and electronic cloning in plant functional genomics research has important application value.