Cloning Of Chalcone Reductase1(CHR1) Gene Of Soybean And Its Transforming Into Soybean

Daidzein had wide physiological functions as a major component ofisoflavone.Such functions as anti-tumor, prevention and therapy ofcardio-cerebrovascular disease, prevention and therapy of coronary atherosclerosis，enhancing the immunity. The research showed that chalcone reductas（eCHR）existedonly in biosynthetic pathway of daidzein through comparing with the biosyntheticpathway of daidzein and genistein,genistin was another component of isoflavone.Therefore chalcone reductase played an important role in the synthesis of daidzein.pCAMBIA3301was a common plant expression vector, and it had been widelyapplied in plant genetic engineering. pCAMBIA3301had the advantages of stable、mature and high efficient screening system. So plant expression vectorpCAMBIA3301was selected as the basic vector in this experiment.In this experiment the CHR1gene was cloned,and the plant expression vector ofCHR1gene was constructed.Then the plant expression vector was transformed intosoybean of variety of Jinong28by agrobacterium mediation in order to CHR1genemight over express in soybean. Study on how over expression of CHR1gene affectsynthesis of daidzein. The test results were as follows：1、Chalcone reductase CHR1gene was cloned: The CHR1gene was amplified byPCR with soybean JN28genomic DNA as template and specific primers ofCHR1.The CHR1gene as PCR fragment was inserted into PMD18-T Vector,Thecloning vector PMD18-T-CHR1was constructed. The experimential results showedthat chalcone reductase1（CHR1) gene had been cloned successfully from thesoybean.2、The plant expression vector pCAMBIA3301－CHR1had been constructed.：So as to realiz the construction of plant expression vector pCAMBIA3301throughsubcloning of pBI121－CHR1vector. The35S＋CHR1gene was amplified by PCRwith the template of plasmid pBI121－CHR1vector as and specific primers of35S＋CHR1. The35S＋CHR1gene was inserted into modified pCAMBIA3301vector.theplant expression vector of pCAMBIA3301－CHR1was constructe.Finally the plantexpression vector of pCAMBIA3301-CHR1was constructed by the identification ofPCR and restriction enzyme.3、The plant expression vector of pCAMBIA3301－CHR1was transformed into soybean of variety of Jinong28by agrobacterium mediation and pollen-tube pathwaymethod.（1）Agrobacterium-mediated：18T0generation herbicide-resistance positivetransgenic plants were obtained by screening of medium with herbicide;（2） Pollen-tube pathway method:The plant expression vector ofpCAMBIA3301－CHR1was transformed into soybean by pollen-tube pathwaymethod.Finally350seeds were obtained and planted.4、T1generation transgenic plants were obtained by different detection:（1） PCR detection:①A grobacterium-mediated:12T1generation positivetransgenic plants were obtained through the PCR detection of5T0generation PCRpositive transgenic plants；②p ollen-tube pathway method: Seeds which weretransformed by pollen-tube pathway method were planted,and then T1generationtransgenic plants were obtained.Finally6T1PCR generation positive transgenicplants were obtained through the PCR detection.（2）Southern blot detection:Because CHR1gene was endogenous gene ofsoybean.So the probe was prepared by35S promoter, and then the experimentimplemented Southern blot detection. The result showed that the PCR positivetransgenic plants produced single hybridization signal by Southern blot,and The plantexpression vector of pCAMBIA3301－CHR1had been integrated into soybeangenome by single copy.（3）Realtime fluorescence quantitative PCR detection：The positive transgenicplants which were detected by PCR and Southern blot implemented realtimefluorescence quantitative PCR detection.The result showed that mRNA expression ofCHR1gene in transgenic plants were significantly higher than which innon-transgenic plants.（4）High performance liquid chromatography（HPLC）: The average content ofisoliquiritigenin （precursor of daidzein） which in transgenic plants andnon-transgenic plants was detected by HPLC.The result showed that the averagecontent of isoliquiritigenin in transgenic plants were higher than which innon-transgenic plants. This result implied that the content of daidzein in transgenicplants were somewhat increased.Conclusion: The plant expression vector of pCAMBIA3301－CHR1wassuccessfully constructed, and transgenic soybeans in which had gained increase ofexpression of CHR1gene were obtained.Which lies a foundation for studying how CHR1gene affect content of daidzein.