Construction Of Palnt Expression Vector Involving HrpZpsta Gene And Its Transformation Into Soybean

Soybean is one of main economic crops in China and always provide as an essential food in popular diets. Soybean phytophthora root rot caused by Phytophthora sojae is a destructive disease for soybeans throughout the soybean production regions of world. Depending on the siol, phytophthora root rot infect plants and insentively widespread. Therefore that is a common series of problems for every researcher on soybean study to handle with reducing the harm of phytophthora root rot and keeping normal yielding of soybean production.Tranditional and transgenic breeding both development new soybean material exhaustively. Though several deficiencies exist in tranditional way, and more and more disease-resistant material were created by transgenic way. The gene hrpZpsta that codes the protein happin belongs to Hrp gene family. The protein happin can cause the hypersibilitive reaction (HR). HR is essencial reason for plants abtain the ability of disease and insect broad resistant. It is a feasible protocol to development new material by utilization of HR.With gene manipulation, gene hrpZpsta fragment was transferred into gene, the transgenic plants possessed ability of sybean phytophthora root rot resistance.Main manipulations are following:According to sequence of gene hrpZpsta on GenBank, designed a pair of specific primers and modified the end with scorresponding recognition sites. Cloning vector pMD 18-T-hrpZpsta was employed as the templates, a 423 base pair fragment of hrpZpsta gene consisted in Pseudomonas syringae pv. Tabaci was propagated by PCR way. The propagated fragment and plant expressed vector pBI121 was dealt with double digestion, then both them were connected by T4 ligase at 22℃.The recombinant expressed vector was transformed into cells of compentence agrobacterium EHA105. Then the gene hrpZpsta was transferred into soybean plant with Agrobacterium-mediated transformation methord on soybean cotyledons and pollen tube pathway.The transgenic plants were identified using PCR, Southern blotting, RT-PCR and SDS-PAGE technology.Main results are following:The recombinant expressed vector pBI121-hrpZpsta was constructed successfully.The optimal concentration of Kanamycin for selecting explants was 100mg/LEighty-four TO and T1 generation of regenerated plants were obtained using Agrobacterium-mediated transformation methord; one hundred and seventy-eight T1 and T2 generation of regenerated plants were obtained by pollen tube pathway. The regenerated plants were identified with PCR, Southern blotting, RT-PCR and SDS-PAGE technologies. Results showed there were 14 positive regenerated plants by Agrobacterium-mediated way and 5 positive regenerated plants by pollen tube pathway. This suggested that hrpZpsta gene was confirmed onto the genome of soybean preliminarily. The transformed ratio were 15.6% and 2.8% respectively.