The Study On Transformation Of Agrobacterium Tumerfaciencs-Mediated GmPHD2into Soybean

Soybean not only is an important economic crop but also has important medical values in China. However, due to a strong importer of transgenic soybeans, internal soybean plantations was declining and soybean industry was facing a serious crisis. To solve this problem, We not only use traditional breeding to increase soybean single production level but also use plant transgenic technology to develop new resistant varieties in a more efficient and more purposeful way. The new resistant varieties can adapt different living situations, including drought, salt, frost, cryogenic, so soybean plantations would likely increase under no competition for land with major food crops circumstances, which was important to increase production and fully exploit saline-alkali soil.’Zhechun NO.3’、’Huachun NO.6’and’0428’preserved by Hangzhou sub-center of national soybean improment were studied as materials in this paper. A transcription factor GmPHD2, which was cloned into two plant expression vectors (pBA002and pES002), was respectively introduced into soybean to receive new germplasms with observably-improved salt tolerance. Both cotyledonary node and hypocotyl were severed as explants in the study by studying main factors and optimizing relevant conditions affecting soybean Agrobacterium-mediated transformation system. The main results were as follows:1. Optimization of main factors and relevant conditions affecting soybean Agrobacterium-mediated transformation system. we optimized main factors (genotypes, explants etc) affecting soybean Agrobacterium-mediated transformation system by comparing gus transient expression frequency with embryonic tip, cotyledonary node and hypocotyl of’Zhechun No.3’,’Huachun No.6’and’0428’. The results showed gus transient expression frequency of the three soybean genotypes was low in the embryonic tip transformation system and tufty shoots showed no expression. Using the hypocotyl transformation system, the maximum expression frequency of’Zhechun No.3’,’Huachun No.6’and’0428’in the site of tufty shoots was43.92%,54.45%, and58.47%, respectively after5germination days and3co-culture days. There is significant difference between the three. Using the cotyledonary node transformation system (5-day germination), the optimal co-culture time for ’Zhechun No.3’was5days and the maximum expression frequency of ’Zhechun No.3’in the site of tufty shoots was23.05%, which was significantly higher than’Huachun No.6’’0428’and the other co-culture time(3d and4d). The germination time of cotyledonary node transformation system was further optimized using’Zhechun No.3’. The results showed that both gene expression and shoot induction increased dramatically in3germination days compared to5germination days.2. Construction of a plant expression vector pBA002-GmPHD2and transformation into soybean. The PHD-type transcription factor gene GmPHD2was amplified from cloning vector pMD18T-GmPHD2using high-fidelity PCR technology. The XbaI and Xhol restriction sites were introduced to the up-and downstream of the full length CDS, respectively. The plant expression vector pBA002-GmPHD2which contained both the GmPHD2gene and the selective marker Bar gene for herbicide resistance was accurately constructed through a series of molecular means, including TA cloning, double enzyme digestion, ligation, transformation and so on. The obtained vector was further verified by PCR amplification, enzyme digestion, and sequencing. Then, the vector pBA002-GmPHD2was successfully transferred into Agrobacterium tumefaciens. The transcription factor GmPHD2was introduced into ’Zhechun NO.3’、’Huachun NO.6’ via Agrobacterium-mediated cotyledonary node transformation method and into’0428’ and ’Huachun NO.6’ via Agrobacterium-mediated hypocotyl transformation method. The transformation events were confirmed using target gene-specific, selective marker gene-specific PCR analysis.3. Construction of a plant expression vector pES002-GmPHD2and transformation into soybean. Glyphosate resistance gene epsps was cut out by restriction enzyme from pTA2-EPSPS, while pBA002-GmPHD2cut out glufosinate resistance gene bar by the same restriction enzyme to a large gene fraction. The gene epsps was cloned into the large gene fraction to get the recombination vector pES002-GmPHD2. Results of restriction enzyme analysis and DNA sequence analysis showed that pES002-GmPHD2was constructed successfully. The transcription factor GmPHD2was introduced into’Zhechun NO.3’、’Huachun NO.6’ via Agrobacterium-mediated cotyledonary node transformation method and into’0428’ and ’Huachun NO.6’ via Agrobacterium-mediated hypocotyl transformation method. The transformation events were confirmed using target gene-specific, selective marker gene-specific PCR analysis.