Determination Of Amylose Content In Buckwheat Seeds And Sequence Analysis Of Waxy Genes

Buckwheat is a traditional food and medicine homologous crop in China.It is rich in nutrition and has strong health value.At the same time,it is widely planted as a crop for leisure and disaster relief.The content of amylose in buckwheat grain is related to the nutrition and processing quality of buckwheat.However,the conventional amylose determination method itself has a large demand for samples,so it is unable to determine trace samples.So far,the research on the amylose content of buckwheat grains is only limited to the analysis and comparison of the conventional differences among different varieties,and it is unable to carry out the research such as the differences of amylose content among single grains of same buckwheat Varieties harvested in the same season,the differences of amylose content in grains of the same plant spatial distribution.In addition,due to the long coding sequence of GBSSI gene,which is a key enzyme controlling the biosynthesis of buckwheat amylose,the sequence characteristic information of this gene is still lacking.Only one Tartary buckwheat Waxy(also called the Granule Bound Starch Synthase I,GBSSI)coding sequence and one Tartary buckwheat GBSSI protein sequence were also included in the NCBI database.In view of the above problems in the study of buckwheat amylose and its coding sequence,this project started with the construction of a trace high-throughput identification system for single grain amylose of buckwheat,planned to systematically analyze the distribution characteristics of grain amylose content of buckwheat germplasm which stored in Guizhou Buckwheat Engineering Technology Research Center,and tried to analyze the single grain amylose content of different branches on the same buckwheat plant.In addition,the project planned to systematically analyze the GBSSI sequences of the main cultivated buckwheat varieties by homologous cloning method.Through the above research,the following main results have been obtained:(1)Construction and application of micro identification system for single grain amylose of buckwheat.Based on iodine colorimetric method and enzyme scanning,a quantitative system of single grain amylose content in buckwheat was established.The results showed that the average amylose content in grains of 191 bitter buckwheat varieties(lines)was20.43%,and the range of variation was 11.59%~24.50%,The results showed that the average amylose content of 179 buckwheat varieties(lines)was 18.09%,and the variation range was 11.41%~22.80%.The 191 buckwheat varieties(lines)and 179 buckwheat varieties(lines)could be divided into three groups by cluster analysis,among which the first group had higher amylose content,the second group had middle amylose content,and the third group had lower amylose content.(2)Study on spatial distribution characteristics of grain amylose in buckwheat plants.The results showed that there was a significant correlation between single grain quality and single grain amylose content of Tartary buckwheat,but there was no stable relationship between single grain amylose content and single grain amylose content of common buckwheat;The amylose content of single grain of 0 branch of Tartary buckwheat was significantly higher than that of subsequent branches,but that of common buckwheat was not.(3)Homologous cloning and sequence analysis of GBSSI gene encoding key amylose synthesis enzyme in buckwheat grains.Through homologous cloning method,the GBSSI sequences of 3796 bp,3793 bp,3794 bp and 3793 bp were isolated from common buckwheat,Tartary buckwheat,golden Tartary buckwheat and rice Tartary buckwheat.They were named fe GBSSI,ft GBSSI,ftc GBSSI and ftg GBSSI,respectively.The Software deduced amino acid sequences were 603 AA,605 AA,605 AA and 605 AA,respectively,the relative theoretical molecular weights were66.71 k Da,66.60 k Da,66.72 k Da and 66.60 k Da,and the isoelectric points(p I)were 7.08,8.35,8.35 and 7.95.There is no transmembrane region and signal peptide,it is hydrophilic protein.Phylogenetic tree analysis showed that GBSSI from different sources were divided into three typical branches.Among them,GBSSI from buckwheat which used in this experiment and Tartary buckwheat GBSSI from database were clustered into cluster 1,In this branch,two kinds of Tartary buckwheat which from different sources and rice Tartary buckwheat(as a variety of Tartary buckwheat)were closely related.The golden Tartary buckwheat which breed from the the cross progeny of Tartary buckwheat and golden buckwheat closely followed the branches.While the most distant one belonged to different origin common buckwheat in pedigree.Through the above research,the trace identification system of single grain amylose content in buckwheat was established.With the help of this set of methods,the interspecific distribution of amylose content in cultivated buckwheat and the spatial distribution of amylose content in single grain in the same buckwheat plant were preliminarily revealed.In addition,through homologous cloning and a series of bioinformatics analysis,the key enzyme controlling amylose synthesis genes,sequence characteristics and evolution characteristics of cultivated buckwheat were preliminarily obtained.The above results can provide important reference for the improvement of amylose,the innovation and utilization of buckwheat germplasm resources,and the evolution of buckwheat system.