Analysis On Genetic Diversity Of Chinese Buckwheat Cultivars

Buckwheats are important medical fresh homologous crops.As people's living standards continue to improve,buckwheats' nutritional value and medicinal value are arousing more people's attention.The study found that the buckwheat's roots,shoots,seedlings have good medicinal value,its seeds are the best food for people who are suffering from diabetes,coronary heart disease,and so on,so the economic value of buckwheat is also gradually improving.Not only nutrient-rich,buckwheat also contains large quantity of flavonoids.Modern medical research shows that buckwheat has several pharmacology functions,such as anti-oxidation,antidiabetics,and antitumor.The main substance that plays these pharmacological activities in the buckwheat is just the flavonoids. With the discovery of the buckwheat's medicinal value on a deeper level,buckwheat,especially tartary buckwheat,has become the focus of attention for the public.Current study on buckwheat flavonoids mainly focuses on how to improve flavonoid content through cultivation measures,but study on the molecular mechanism using genetic engineering is less.In this study,several genes related to the synthesis of buckwheat flavonoids were cloned with genetic engineering methods,and preliminary analysis of these target genes have been conducted using bioinformatics method,intended to set up the theoretical foundation for further study of buckwheat flavonoids on functional verification of related genes and mechanisms of their regulation and expression.In this study,three genes related to anabolism of buckwheat flavonoids were cloned using PCR,RT-PCR, and were analyzed and compared with bioinformatics method. Results are as follows:1. Using PCR,RT-PCR method,CHS gene DNA,cDNA fragments from tartary buckwheat and common buckwheat were obtained,and named FtCHS,FeCHS,FtrCHS and FerCHS separately,which are all 1027 bp long,and contain 326 open reading frames encoding amino acids.Sequence analysis showed that the four genes have high homology with other plants' CHS genes.Analysis of these CHS gene fragments from tartary buckwheat and the common buckwheat found that they have no introns.Comparing FtCHS and FeCHS found that tartary buckwheat and the sweet buckwheat CHS genes have 38 single-base differences,so we speculate that this may be one of the reasons that lead to the flavonoids content differences in tartary buckwheat and common buckwheat.2. By RT-PCR method,four cDNA fragments of PAL genes in tartary buckwheat were obtained,and named FtPAL1,FtPAL2,FtPAL3,FtPAL4.FtPAL1,FtPAL2,FtPAL3,obtained using the same primer,were all 969 bp long,but their nucleotide sequences vary greatly,and the amino acid sequences they code also have many differences.Analysis online found that FtPAL1,FtPAL2,and FtPAL3 all have a high homology with PAL protein gene in other plants,so we speculate FtPAL1,FtPAL2,FtPAL3 are three members of PAL gene family in tartary buckwheat.FtPAL4, 934 bp long, amplified using primers designed based on FtPAL2 and PAL genes in other high homology plants,a length of,is the 3 ' extension of FtPAL2. One 1510 bp DNA fragment,named FtPAL,including a reading frame coding 503 amino acids was obtained by splicing FtPAL4 and FtPAL2.Sequence analysis showed that FtPAL has a high genetic identity with PAL in other plants.The protein encoded by FtPAL is speculated a stable protein based on the analysis of basic physical and chemical properties of the protein.3. By RT-PCR method,the cDNA fragments of DFR gene in tartary buckwheat was obtained.It is named FtDFR, 1120 bp long, includes a reading frame encoding 341 amino acids. Compared with the whole-length amino acid sequence encoded by DFR gene in Fagopyrum cymosum,the amino acid sequence encoded by FtDFR gene has equal length,with only three amino acid residue dififerences.Alignment analysis shows that FtDFR has a high homology with DFR genes in other plants.Analysis of DFR protein in tartary buckwheat indicates that its theoretical molecular weight is 38473.1 KDa, predicted pI value is 5.78, theoretical formula is C₁₇₂₂H₂₆₇₁N₄₅₅O₅₁₀S₂₂, and instability coefficient is 35.05.So,the protein encoded by FtDFR gene is speculated a stable protein.