Screening Tartarian Buckwheat Varieties For Chongqing Low-altitude Areas And Cloning Of Chalcone Synthease

Tartarian buckwheat(Fagopyrum tataricum(Linn)Gaertn)is one of the main minor grain crops and important medicinal crops with rich flavonoid.Buckwheat has a great efficacy to reduce blood sugar and blood lipids,and even can be used to prevent and cure diabetes,high blood fat,hypertension and other diseases as adjuvar because of rich nutrient and high flavonoid.With the development of modern biotechnology and genetic engineering,it is possible to get new Tartarian buckwheat varieties with high flavonoid,the premise of which is the in-depth study on flavonoid biosynthesis.Most researches were done on Tartarian buckwheat of high-altitude areas,but research of low-altitude area Tartarian buckwheat is comparatively backward.Given this situation,it is a very meaningful and important research topic for us to study Buckwheat in low-altitude areas.Researches involve introduction and screen,selection of high yield and high flavonoid varieties,flavonoid metabolic engineering of Buckwheat and so on.In present paper we chose 17 new varieties of Tartarian Buckwheat coming from different regions to identify their major economic traits in areas of 248 m under sea level. The result showed that the productivity of 17 new Tartarian buckwheat varieties were generally low except Jiujiang Tartarian buckwheat,Jin No.2 Tartarian buckwheat and Xi No.7-3 Tartarian buckwheat,which had higher yield of grains among all varieties participated in the experiment.According to result of determination,total flavonoid content of Jin No.2 Tartarian buckwheat is the highest.Charles ketone synthase(chalcone synthease,CHS,EC 2.3.1.47)is a poly-ketone-synthase,which catalyse the first rate-limiting step of flavonoid biosynthesis.In order to reveal the function of CHS in the flavonoid biosynthesis and provide candidate gene to metabolic engineering of Buckwheat,we cloned full-length of CHS cDNA,designated as FtCHS(GenBank Accession Number:EF562533),via RACE technology from Jin No.2 Tartarian buckwheat of high yield and high flavonoid content. And detailed analysis of bio-informatics was conducted on both gene and its protein.The results were as follows:based on conservative sequence of known Charles ketone synthase genes from Medicago truncatula,Petunia x hybrida and Rheum palmatum,primers were designed to successfully amplify core fragment(868 bp);BLAST analysis revealed homology between core fragment and CHSs from other plants and firstly identified this core fragment is the fragment from gene CHS of Buckwheat;according to the particular sequence,3'-RACE and 5'-RACE gene-specific primers were designed to amplify the 3'-end(517 bp)and 5'-end(359 bp);sequences of core fragment,3'-terminal and 5'-end were spliced to get electronics full-length of CHS cDNA of Tartarian buckwheat and then primers were designed to amplify physical length.FtCHS with a full-length of 1463 bp, bioinformatics prediction of which indicated that the coding region is 1179 bp,codes a protein with 393 amino acid.FtCHS molecular weight is 43.156 kDa and isoelectric point is 5.86;BLAST analysis indicated that sequence similarity between FtCHS and CHS of palm leaf rhubarb(Rheum pahlmatum L.)is up to 83%and FtCHS was successfully cloned; Molecular Evolution Tree of FtCHS and CHSs from bacteria,fungi and other plants reveals that FtCHS has closer evolutionary relationship with Rheum palmatum and Persicaria hydropiper,and FtCHS has a common origin with bacteria,fungi and other plants, matching with results of BLAST analysis;It is predicted that secondary structure of FtCHS containsα-helix(41.73%),layers(16.28%)and random coils(41.98%);further research on three-dimensional modeling of FtCHS contains 11α-helixes and 15β-layers,which showed that protein was able to fold to a typical CHS protein with three-dimensional structure highly similar to that of MsCHS2.The results of present study theoretically explain the reason why Buckwheat yield low in low-altitude areas and show light on function and mechanism of CHS in flavonoid biological synthesis of Buckwheat in molecular genetics level.Based on this,further research on Buckwheat genetic transformation could be conducted,which pave the way for realization of Buckwheat flavonoid metabolic engineering and eventually obtainment of new gene-modified materials with high flavonoid or new varieties.