Cold Resistance Analysis Of Two Beta Vulgaris Var.cicla And Cloning Of Glycosyltransferase Genes BvGT708 And BvGT74

Beta vulgaris var.cicla,with a beautiful appearance and attractive color,is a potential foliage plant.It is mainly distributed in the southwest region and part of the Yangtze and Yellow River basins,and it can not be overwintered in open cultivation in the northeast region.If new varieties of landscaping adapted to the climate of Northeast China are to be cultivated,it is necessary to evaluate the cold resistance of various Beta vulgaris var.cicla and study the molecular regulation mechanism of cold tolerance.In this study,the leaf electrical conductivity,SOD,CAT,POD activity,soluble sugar,and proline content of two ornamental sugar beets under low temperature stress were determined,and the cold resistance of the two Beta vulgaris var.cicla was evaluated.Through high-throughput sequencing data analysis under low-temperature stress,the glycosyltransferase genes Bv GT708 and Bv GT74 that obtained cold stress response were screened.Glycosyltransferase?GT?is an important class of modified enzymes in the glycosylation process of betalains.It is involved in the synthesis of betalains and is an impor tant factor for beet to appear red and ornamental.At the same time,glycosyltransferase genes regulate plants in response to a variety of abiotic stresses,especially low temperature stress.This study uses bioinformatics and molecular biology techniques to predict the biochemical properties of Bv GT708 and Bv GT74 and clone two genes to construct a plant expression vector,and transfer the Bv GT708 gene into model plants tobacco and Arabidopsis in order to be in model plants The study of gene function ultimately lays the foundation for the development of new cold-resistant Beta vulgaris var.cicla varieties and the synthesis of ornamental plant pigments.The main findings are as follows:?1?At-10?,with the prolongation of low temperature stress,the relative c onductivity and proline content of Red chard and Yellow chard showed a gradual upward trend;the activities of SOD,CAT,POD showed a trend of first increasing and then decreasing;The soluble sugar content of Red chard gradually increased,while the soluble sugar content of Yellow chard gradually decreased.According to the comprehensive evaluation of the membership function method,it is concluded that the cold resistance of Red chard is stronger than that of Yellow chard.Provide reference data for the cold resistance of Beta vulgaris var.cicla.?2?By measuring the content of betalains in Red chard leaves for 6 consecutive weeks,it was found that the content of betalains increased gradually as the Red chard continued to grow and develop.The q PCR results showed that the relative expression level of Bv GT708 increased gradually with the gradual increase of the development period of Red chard,Bv GT74 showed a trend of first decreasing and then increasing,which indicated that Bv GT708 was positively correlated with betalains accumulation,but Bv GT74 had no significant correlation with betalains content..It is speculated that Bv GT708 may be involved in the synthesis of betalains.?3?By measuring the content of betalains in Beta vulgaris var.cicla at-10?,it was found that the content of betalains increased first and then decreased with the prolongation of low temperature stress,indicating that betalains may respond to low temperature stress.Fluorescence quantitative PCR found that under low temperature treatmen t,the relative expression of Red chard Bv GT708 changed similarly to betalains,and also showed a trend of increasing first and then decreasing.It is speculated that Bv GT708 may respond to low temperature stress by participating in betalains synthesis.?4?In order to further analyze the role of Bv GT708 and Bv GT74 genes in betalains synthesis,Bv GT708 and Bv GT74 genes were cloned from Red chard leaves and bioinformatics related analysis was performed.Bv GT708 has an open reading frame of 494 amino acids,an isoelectric point of 6.20,and a molecular weight of 38450.37 KD.The total average hydrophobic index?GRAVY?is-0.255,which is a hydrophilic protein.The Bv GT74 open reading frame has a total of471 amino acids,an isoelectric point of 6.21;a molecular w eight of 36.1116 KD,a fat number of72.66,a total average hydrophobic index?GRAVY?of-0.130,and a hydrophilic protein.?5?Successfully constructed Bv GT708 and Bv GT74 plant expression vectors:p BI121-Bv GT708,p BI121-Bv GT74,and Bv GT708 fluorescent protein binary expression vector p BI121-GFP-GT708.Transgenic Bv GT708 was transformed into Arabidopsis thaliana by inflorescence infestation,and transgenic Arabidopsis seedlings were screened initially through resistance screening.The four T₀ generations had a positive rate of 57.1%,which laid the foundation for the cold resistance research of this gene.Agrobacterium-mediated transformation of Bv GT708 into tobacco yielded 6 strains of T₀ transgenic tobacco with a positive rate of 75.0%,providing a basis for the gene synthesis of betalains in plants.