Identification Of Granule-bound Starch Synthase GBSS Genes And Functional Analysis Of FtGBSS1-2 In Tartary Buckwheat（Fagopyrum Tataricum）

Tartary buckwheat（Fagopyrum tataricum）is a medicinal and food homologous crop,with high development and utilization value and good development prospects.Seeds are the main edible part of buckwheat,and the main nutrient of seeds is starch.The content and properties of starch directly affect the processing quality and taste of buckwheat products.However,the current determination method of Tartary buckwheat starch content is not accurate enough.Meanwhile,the synthesis mechanism of Tartary buckwheat starch,especially the synthesis mechanism of amylose that determines the quality and taste of Tartary buckwheat,is still unclear,which makes the research on Tartary buckwheat starch greatly lagging behind.The granule-bound starch synthase GBSS gene is a key gene for amylose synthesis.The study of Tartary buckwheat GBSS gene is of great significance to improve the quality and properties of Tartary buckwheat starch.In this study,a more accurate method for the determination of plant amylose and amylopectin content was first compared and determined,and then the genome-wide identification and analysis of Tartary buckwheat GBSS was performed using the Tartary buckwheat genome,and real-time quantitative PCR（q RT-PCR）analysis was performed.The expression of GBSS gene in different tissues and seed development of Tartary buckwheat was screened out,and a GBSS gene with extremely high expression in seeds was screened,and its function was cloned and transgenic.Below are key research findings:（1）For the four commonly used methods for the determination of linear and amylopectin content（single-label single-wavelength colorimetry,single-label dual-wavelength colorimetry,double-label single-wavelength colorimetry,double-label dual-wavelength colorimetry）were compared in terms of accuracy,precision and sample determination,and it was found that the double-standard double-wavelength colorimetric method could more accurately determine the content of amylose and amylopectin.The amylose determination wavelength was 597 nm,the reference wavelength was 480 nm,and the regression coefficient was0.997;the amylopectin determination wavelength was 541 nm,the reference wavelength was 700 nm,and the regression coefficient was0.9964.The amylose and amylopectin content of different concentrations of amylose/amylopectin standard solution and three Tartary buckwheat varieties（Xiaomiqiao,Jinqiao No.2,and Guimiku No.11）were determined by double-standard dual-wavelength colorimetry.The results are within the expected range,indicating that this method can accurately measure the content of amylose and amylopectin.The amylose content of Xiaomiqiao was determined to be 22.30%,the amylose content of Guimiku No.11 was 24.42%,the amylose content of Jinqiao No.2 was31.88%,and the amylose content of Xiaomiqiao and Guimiku No.11 was31.88%.There was little difference in the content of chain starch,and compared with Jinqiao No.2,the difference was larger.（2）Five buckwheat GBSS genes were identified by genome-wide analysis,named as Ft GBSS1-1,Ft GBSS1-2,Ft GBSS1-3,Ft GBSS1-4 and Ft GBSS1-5,respectively.The full lengths of these five genes ranged from1803 bp to 1824 bp,and the coding amino acid lengths ranged from 600 to 607.The identity of the five sequences was 79.07% at the nucleotide level and 78.54% at the amino acid level.The structure of the five Tartary buckwheat GBSS genes and the physicochemical properties and evolutionary relationship of the encoded amino acids were analyzed.The gene structure analysis showed that the Ft GBSS1-1 gene contains 12 exons and 11 introns,Ft GBSS1-2 gene contains 13 exons and 12 introns,Ft GBSS1-3 gene contains 12 exons and 11 introns,Ft GBSS1-4 gene contains 13 exons and 12 introns,Ft GBSS1-5 gene contains 10 exons and9 introns.The analysis of physical and chemical properties showed that the hydrophobicity values of the five Tartary buckwheat GBSS proteins were all negative,indicating that the Tartary buckwheat GBSS proteins were hydrophilic proteins;Evolutionary analysis shows that Ft GBSS1-1,Ft GBSS1-2,Os GBSS1,Ta GBSS1,Sb GBSS1 and Zm GBSS1 cluster on the same branch,Ft GBSS1-3,Ft GBSS1-4,Ft GBSS1-5,At GBSS1,Cp GBSS1,Sl GBSS1,St GBSS1,Nn GBSS1 and Ad GBSS1 are clustered on the same branch.Among them,Ft GBSS1-1 and Ft GBSS1-2 have the closest genetic relationship with Os GBSS1,which mainly controls the synthesis of seed amylose,indicating that Ft GBSS1-1 and Ft GBSS1-2may be involved in the synthesis of seed amylose.（3）In order to clarify the tissue specificity of the five Tartary buckwheat GBSS genes,Using Guimiku No.11 and Jinqiao No.2 with large differences in amylose content as materials.Detection of the expression of five Tartary buckwheat GBSS genes in roots,stems,leaves,flowers and seeds of Guimiku No.11 and Jinqiao No.2.Among the different tissues of Guimiku No.11,the expression levels of Ft GBSS1-1and Ft GBSS1-2 were the highest in seeds,Ft GBSS1-3 in leaves,Ft GBSS1-4 in stems,and Ft GBSS1-5 in Relatively high expression levels in roots and flowers;Among the different tissues of Jinqiao No.2,Ft GBSS1-1 and Ft GBSS1-2 had the highest expression levels in seeds,Ft GBSS1-4 and Ft GBSS1-5 had the highest expression levels in flowers,and Ft GBSS1-3 had the highest expression levels in leaves,indicating that The highest expression levels of Ft GBSS1-1 and Ft GBSS1-2 were found in Tartary buckwheat seeds.During the development of Tartary buckwheat seeds,the expression levels of Ft GBSS1-1 and Ft GBSS1-2were higher in the late-filling stage of Guimiku No.11 than in the mid-filling stage,and those of Jinqiao No.2 in the late-filling stage were lower than those in the mid-filling stage.The high expression of Ft GBSS1-1 and Ft GBSS1-2 in the middle and late grain filling stages may be involved in the synthesis of amylose in Tartary buckwheat,which is the main factor determining the amylose content of mature seeds of Tartary buckwheat.（4）Since there is no stable genetic transformation system for Tartary buckwheat,the Ft GBSS1-2 plant overexpression vector was constructed and transferred into Arabidopsis and rice to verify its function.In transgenic Arabidopsis,a total of 8 positive lines were identified.The expression levels of Ft GBSS1-2 genes in transgenic Arabidopsis were significantly higher than those in wild-type Arabidopsis.However,we found that starch in Arabidopsis was mainly in the form of the form of amylopectin（content about 45～48%）and the content of amylose are extremely low（content about 0.8～1.4%）,and the transformation of Ft GBSS1-2 gene in Arabidopsis cannot explain the function of this gene well.In Ft GBSS1-2 transgenic rice,a total of 27 positive lines were identified,and the starch content of 5 transgenic lines was determined.It was found that the amylose content of 5 transgenic rice was significantly higher than that of wild type rice,and amylopectin content was significantly higher than that of wild-type rice.The content was significantly lower than that of wild-type rice,and the total starch content was lower than that of wild-type rice,indicating that the transferred Ft GBSS1-2 gene could promote the synthesis of amylose in rice,thereby increasing the amylose content of transgenic rice.