Construction CDNA Library And Cloning Isoflavone Synthase Gene From Astragalus Membrana Ceus

The Astragalus membrcmaceus(fisch.) Bge. is considered as one of rare endangered officinal plants in Changbai Mountain area. And as rare traditional Chinese medicinal materials, their secondary metabolite are important effective constituents in biological activity of A. membranaceus and possess important medicinal value. But the research about functional gene and secondary metabolism biosynthetic pathway is relatively less in A. membranaceus. It is meaningful for preserving A. membranaceus gene resources and accelerating to studying the gene level process. In this paper, we reported the construction of the cDNA library from wild A. membranaceus roots; cloned full-length sequences of isoflavone synthase gene by the technique of RACE; analyzed characteristic of the sequence; we collected the leaf blade and adventitious roots from A. membranaceus, checked IFS gene expression using semi-quantitative RT-PCR method; our work represents the first attempt to engineer IFS gene in lettuce.The cDNA library was constructed successfully by the technique and the library qualification evaluation showed:the titer of primary cDNA library was 4.6×105 cfu/mL, the percentage of recombination was about 100%, the fragment size of inserted was 0.5-2kb. And we have cloned full-length sequences of a novel IFS gene from A. membranaedceus by the technique of RACE, the IFS gene named AmIFS(GenBank:JN882009). The length of AmIFS cDNA is 1903 bp and the sequence in coding region of IFS is 1578 bp and the IFS include 5’untranslated region (5’UTR) of 116 bp,3’UTR of 209 bp. The putative AmIFS protein consisted of 525 amino acids. Expression analysis of IFS gene was carried out by semi-quantitative RT-PCR. The results showed that in the five developmental stages IFS had lowest expression level during full flowering stage(July); IFS has the highest expression level in October; Semi-quantitative RT-PCR of adventitious roots showed that adventitious roots could produce isoflavones in the developmental stages. We transformed pBI-AmIFS into lettuce, and PCR showed that the AmIFS gene was integrated into lettuce genome.