Studies On The High-Throughput Expression Technology Of Wild Soybean Genes In Arabidopsis

Low temperature,salinity and drought are a major issue in our country and world agricultural production.Improving the tolerance of these abiotic stress of crops,is of great strategic significance for increasing food production,developing farming resources and solving the food problem.In recent years,with the rapid development of molecular biology and the mature of transgenic technology,anti-modified crops through genetic engineering against osmotic stress.There has been a good momentum.Explaining the molecular mechanism of osmotic stress in plant and finding "key genes" from a large number of stress-related genes,is an important prerequisite for osmotic stress genetic engineering of crops.Our research aims at these problems,constructed plant expression vector generic card boxes with the matrix attachment regions,MAR.Taking the wild soybean of northeast as the material,established high-throughput gene over expression system,and the system of screening the transgenic plants against osmotic stress.Therefore,our study is of great significance for tolerance basic theoretical research,foundation of new technologies system,agriculture utilizing,and other areas.Main research results are as follows:1.Plant Expression Vector ConstructionWe had cloned P-MN and E-MN sequence including MAR sequence by overlap extension PCR method.At the same time,we constructed one plants common effective expression vector card boxes pEMT-5 with MAR and SfiA,SfiB restriction sites,promoted by E12.The vector is suitable for constructing over-expression library which is obtained by SMART technology.2.Plant Expression Vector functional analysisThe vectors pEMT-5 and pBHT-5 were transferred into tobacco by Agrobacterium Tumefaciens.Semi-quantitative RT-PCR and Real-time PCR analysis the same 005 gene expressed in pBHT-5 and pEMT-5 vectors in transgene plants.With the analysis of transcription levels,005 expression levels in pEMT-5 are more 3.89 times than in pBHT-5.3.Wild Soybean conditions of osmotic stress of a full-length cDNA cloneWe obtained the full length cDNA of wild soybean under the conditions of osmotic stress by Clontech SMART^TM technique.Then,a full-length cDNA cloned the vector pEMT-5.The result of the analysis is that the primitive titer of the library was 1.96×10⁸cfu/mL and the end titer of the library was 2.0×10¹⁰cfu/mL,the recombination efficiency of the cDNA library was 96.29%by PCR identification.The library fits gene over-expression.4.Gene library transferred into Arabidopsis and transgene plant were selected (1) Gene library transferred into Arabidopsis by Floral dip and harvested Seeds.(2) In order to select transgene seeds,Km selection pressure is 10mg/L.(3) We harvested 10000 seeds,there are 110 Strains by Km selection pressure, transformation ratio 1.1%.