Cloning And Functional Identification Of Chalcone Reductase CHR Gene

Original distribution area of soybean (Glycine Max) is in our country that has a5000yearshistory of planting soybean. Soybean nutritional value is high and soybean is the oil-bearingcrops and high protein food crops, which is recognized in the world. Soybean contains avariety of secondary metabolites such as flavonoids, isoflavone compounds are a kind offlavonoids. It not only can regulate physiological function, but also is of great significance forhuman health. Daidzein is one of the key components of the soybean isoflavones. Daidzein isimportant physiological active substances and also has important influence on soybean diseaseresistance. Therefore, the study of synthesis and regulation pathway of daidzein has importanttheoretical and application value.Chalcone reductase is a kind of reductase protein relying on NADPH in the biosynthesisof daidzein. The studies of T. L. Graham have demonstrated that when the synthesis ofchalcone reductase is interfered by using RNAi technology, which directly leads to reduce thecontent of catalytic product isoliquiritigenin that is necessary precursor synthesized material ofdaidzein. Therefore, for enriching all the efficacy of isoflavone product, chalcone reductase, asa key enzyme for the synthesis of daidzein, inevitably are studied. But, type, function andmechanism research of chalcone reductase are weak, and related reports of the research is less.The effective regulation of daidzein can not be achieved. This test cloned chalcone reductasegenes, and pBI121-CHR-GUS, pBI121-CHR, two expression vectors are respectively built andexpressed in the tobaccos, in order to lay the foundation for further studying the synthesismechanism of daidzein. The test results are as follows:1. Soybean varieties "JiNong28" as material, the RNA is extracted and reverse transcribedinto cDNA, about1300bp chalcone reductase gene is isolated by using RT-PCR technologyand connected to cloning vector, then its open reading frame is selected according to the genesequence.2. Primers with enzyme sites are designed according to sequence of the open reading frame,the open reading frame of the gene is amplificated from cloning vector with CHR gene by PCRand recombinant plant expression vector pBI121-CHR-GUS, pBI12-CHR are constructed.After PCR, enzyme digestion and sequencing identification respectively, expression vectors areproved to build successfully and transferred into agrobacterium EHA105for preparation ofengineering strains. 3. The above recombinant expression vectors and pBI121vector were transfered into themodel plant tobaccos through the method of agrobacterium mediated,30strains of kanamycinresistance of the transgenic tobaccos were identificated by using PCR technique,14positivestrains of their tobaccos were preliminary obtained, Each part of the positive transgenictobaccos with pBI121-CHR-GUS and the positive transgenic tobaccos with pBI121wasdetected by GUS staining, and the wild type tobacco was as a negative control, each place oftransgenic tobaccos was blue, it is proved that the pBI121-CHR-GUS vector was transferedinto tobaccos successfully, and expression elements can be normally express in the tobaccos.the part of positive plants were identificated through the method of southern blot, it is provedthat the CHR gene has been integrated into tobacco genome.4. The positive transgenic tobaccos were analysised by real-time fluorescent quantitativePCR and HPLC, it is proved that the gene was expressed at the transcriptional level andexpression quantity of the gene were different among different plants, the average contents ofisoliquiritigenin what was catalytic product by CHR respectively were7.5280.018umol/g,10.6240.134umol/g in leaf tissues of the number12tobacco with pBI121-CHR-GUS and thenumber21tobacco with pBI121-CHR, but isoliquiritigenin can not be detected in the wild typetobacco. Therefore, CHR gene plays an important role in the synthesis of isoliquiritigenin whatis precursor substance of daidzein.