Cloning And Expression Study Of CHI Gene And CHS Gene In Vigna Radiate L

Flavonoids are the important medicinal ingredients. They not only take part in UV protection and flower coloration but also have many functions, such as anti-tumor and anti-cancer effects. Chalcone isomerase (CHI) and chalcone synthase (CHS) are the rate-limiting enzymes in the flavonoids biosynthetic pathway. Their expression or not will act as a key regulating effect in flavonoids synthesis. So, to clone these two enzyme genes and analyze their functions are valuable for improving the content of flavonoids in plants and changing flower colour by genetic engineering technology as well as could lay the foundation for genetic regulation of flavonoids biosynthetic pathway in plants.CHI and CHS, which exist in plants widely, have been isolated from many plants. However, the CHI and CHS genes of mung bean(Vigna radiate) which is rich in flavonoids have not been cloned. So, in this study, these two genes in mung bean were cloned, and then CHI gene was transferred into tobacco and soybean in order to study its functions preliminarily. The main results were as follows:1、4 primers were designed based on the conserved sequences of CHI genes in other higher plants. The fragments of CHI gene from mung bean were isolated by homologous cloning and RT-PCR. Then the fragments were spliced into a full length cDNA sequence and verified with the specific primers. The CHI gene from mung bean was 669 bp encoding 222 amino acids. The deduced amino acid sequence shared over 80% amino acid homology compared with CHI from Glycine max and Medicago satrva. Besides, the sequence contained some conserved domains, such as Chalcone superfamily, PLN02559 and Chalcone domain.2、The full length cDNA sequences of CHS gene from mung bean were obtained by homologous cloning and RT-PCR. And then the sequences were verified with the specific primers and nested PCR.3 full length cDNA sequences and 1 fragment of CHS gene family were obtained. The full lengths were all 1170 bp encoding 389 amino acids and the fragment was 713 bp. The 3 full length cDNA sequences shared over 90% nucleotide homology each other, and all contained CHS-like conserved domains. With BLAST analysis, the 3 cDNA sequences shared over 89% nucleotide homology compared with CHS from Phaseolus vulgaris and Vigna unguiculata.3、PCR detection, restriction enzyme digestion and DNA sequencing were testified that the recombinant vector named pFGC5941-CHI was constructed successfully.4、With agrobacterium-mediated method, the vector pFGC5941-CHI was transferred into tobacco and soybean. There are 13 positive tobacco and 10 positive soybeans by PCR detection.5、RT-PCR and BASTA test all showed that BAR gene in vector was insert to the genome of tobacco and soybean and expressed in the two plants. Smeared the leaves of genetically modified and non-genetically modified plants with basta, the non-genetically modified leaves were withered after 1 week while genetically modified leaves were growing normally. In addition, RT-PCR with CHI pecific primers indicated that CHI gene from mung bean had been expressed in partial tobacoo and soybeans.6-. Extracted and detected the total flavonoids content in genetically modified and non-genetically modified tobacco and soybean leaves. Compared with the non-genetically modified plants, the total flavonoids content of genetically modified plants were higher significantly. The content of genetically modified tobacco and soybean were 3.3 and 2.3 times at most to non-genetically modified plants, respectively. They all showed that the expression of exogenous CHI gene play an accelerative role in genetic regulation of flavonoids biosynthetic pathway in plants.