Construction Of RNAi Expression Vector And Transformation Of CHR1Gene Into Soybean

Soybean is one of the important food crops in our country, which has five thousand yearsof cultural history and has a plenty of physiologically active substances. As one of the moreimportant polyphenolic compounds, Soybean Isoflavone is important secondary metabolites inthe development of soybean growth and has been extensively studies because it contains avariety of biological activity. The content of Soybean Isoflavone is limited and it is specificpresent in legumes Papilionoideae plants. Because its function is similar to estrogen, it is alsocalled "phytoestrogens".Daidzein is important physiologically active substances in Soybean Isoflavone, whichpossess the highest concentration in soybean seedling hypocotyl and account for about30%oftotal Isoflavone. Daidzein can improve the resistance to microbial pathogens, modern medicalstudies dedicated that Daidzein can also regulate various physiological functions of humanbody, which can prevent and cure of cardiovascular disease and hormone-dependent tumors,breast cancer, osteoporosis and female hormone-like and so on. Integrated other related reports,Daidzein possess good physical functions in improve immunity, anti-hemolytic, protect theliver and improve liver function, migraine, diabetes, prevent skin aging, prevent skin burns thatcaused by the sun ’s ultraviolet radiation, and prevent postpartum depression and so on.Therefore, in-depth research of Daidzein, especially researches in the molecular biology fieldcan significantly enhance the value of Daidzein.Chalcone reductase (Chalcone reductase, CHR) is one of the key enzymes in the synthesisof Daidzein. CHR and chalcone synthase (Chalcone synthesis, CHS) in common to come outcatalytic synthesis of isoliquiritigenin, while isoliquiritigenin biosynthesis is the necessaryprecursor compounds of Daidzein biosynthesis. Based on the cloned analysis of GmCHR genein soybean of Liu Jiang et al, we know that CHR gene is co-existence in form of multi-gene andco-synthesis isoliquiritigenin in legume genome, and possess the maximum expression in leaf.Therefore, analysis of different kinds of CHR gene can help clarify efficiency and relationshipof regulation in the catalytic process.The principle of the test based on RNA interference, cloned known chalcone reductasegene, constructed for screening herbicide labeled pCPB-CHR1-RNAi interference carrier,through Agrobacterium infection method and pollen tube pathway method the carrier interference transferred receptor " Jinong28", through to the plants by PCR initially beenpositive transgenic plants, and then the plants were positive for southern blotting, quantitativePCR, HPLC analysis, to the adoption of RNA interference technology, without affecting theother components of soy isoflavone content of chalcone reductase specificity interference,lower levels of daidzein, found that the conversion of soybean resistance to Phytophthora rootrot significantly reduced.Impact Analysis CHR1gene synthesis of daidzein, an initialverification catalytic function and efficiency CHR1genes in order to identify changes indaidzein content of soybean resistance to Phytophthora root rot impacts, the use of transgenictechnology to improve the content of daidzein, improved disease resistance of soybean toimprove the theoretical basis, a basis for further efficient use of genetic engineering in CHR1genes.Our main results:1. Cloned RNA interference vector include the necessary sense and antisense fragments,fragments including introns. Expression vector based on pCPB through double digestionmethod, and then the positive and negative fragments were ligated into the expression vector,and finally obtained RNA interference expression vector pCPB-CHR1-RNAi with the bar geneas selectable marker.2. We applied Agrobacterium-mediated and pollen tube pathway to come out thetransform the expression vector RNA interference into soy "Jinong28" receptor, and receivedpositive transgenic plants. Thirteen T0positive plants of "Jinong28" were obtained throughAgrobacterium-mediated method, and the transformation efficient was2.21%. And then thepositive T0generation plants seeds growing, seven T1positive plants of the generation of"Jinong28" were obtained through resistance screening and preliminary PCR detection.3. The plants obtained by pollen tube pathway was carried out PCR detection, and finallyobtained T0generation of transforming seeds325of "Jinong28", and then the seeds werecultivated indoor. Eighteen T1positive plants of the generation of "Jinong28" were obtainedthrough resistance screening and preliminary PCR detection, average transformation efficientwas1.82%.4. Southern blotting results demonstrate that pCPB-CHR1-RNAi expression vectorT-DNA region has been indeed integrated into the soybean genome; the results of quantitativePCR dedicate that the RNA interference play a role in post-transcriptional level and HPLCdetection dedicate content of Daidzein precursor isoliquiritigenin is reduced.