Construction Of BetA/BADH Plant Expression Vector And Transformation Of Tetraploid Black Locust (Robinia Pseudoacacia)

The salinization of soil is one of the most important influencing factors to limit the agricultural development. Improving the salt tolerance of the plants by genetic engineering is a focus research field in recent years. Tetraploid black locus(tRobinia pseudoacacia)is a new species introduced from Korea. It grows rapidly, has plump leaves and strong resistibility and could be used in improving ecosystem and feed. To improve the salt tolerance of the tetraploid black locust through genetic transformation is very important for expanding its cultivated area, improving ecosystem and developing animal husbandry.Glycine betaine is one of the improtant organic matters in osmoregulation. It can increase the osmotic pressure of the cytoplasm, balance the water potential, protect the membrane system and the other biological macromolecule such as protein. The choline dehydrogenase gene(betA) and the betaine aldehyde dehydrogenase gene(BADH) are the key genes of the glycine betaine's synthesis pathway. They adjust and control the two steps respectively of the glycine betaine's synthesis. Transferred the two genes into tetraploid black locust could be help for improving its salt tolerance. So, in this research, the salt tolerant genes betA and BADH were subcloned and the plant expression vectors were constructed. Then the vectors were used to tansform the Tetraploid black. The results were as follows:1. The genes betA and BADH were subcloned from pCPA-betA and pC2300-BADH respectively. The restriction enzyme cutting site SacⅠon the betA gene was changed to SmaⅠ, so as to insert into the vector pCAMBIA2300-35S-OCS and construct the binary genes plant expression vector.2. Constructed the betA plant expression vector (pCAMBIA2300-betA) and the betA/BADH binary genes plant expression vector (pCAMBIA2300-betA–BADH). pCAMBIA2300-betA contained"CaMV 35S promotor-betA gene-OCS terminator sequence"and an NPTⅡgene promoted by CaMV 35S promotor; pCAMBIA2300- betA– BADH contained"CaMV 35S promotor-betA gene-NOS terminator sequence-CaMV 35S promotor-BADH gene-OCS terminator sequence"and an NPTⅡgene promoted by CaMV 35S promotor. The sequences of the vectors were conformed by enzyme cutting and order-checking, and the results showed that the sequences were correct.3. Optimization of the genetic transformation system. Three influencing factors of the transformation efficiency were studied, such as the use of Acetosyringone (As), activated ways of Agrobacterium rhizogenes and low-temperature pregrowth. The results showed that steeping scratched explants by As solution and activing the Agrobacterium rhizogenes on the LB solid culture medium could increase the survival percent of resistence buds greatly; low-temperature pregrowthing the explants could increase the survival percent and the growing development of resistence buds in a short time(30 days).4. Took the tetraploid black locust stems as explat, transformed the genes betA and betA-BADH via the Agrobactrium rhizogenes. The transgene plants with betA and betA-BADH were got and conformed by PCR.