Electronic Cloning And Structural Analysis Of Coding Region Of Genes Related To Sugar Beet Marker Locus BvRE049

Sugar beet(Beta vulgaris L.)is an important sugar crop in China.The genetic resources of sugar beet are the basis of all the research on any molecular mechanism,and also the prerequisite of various applied researches such as the genetic improvement of sugar beet traits via biotechnology strategy.The genetic resources of sugar beet in China are scarce at present,and gene isolation remains the primary stage.Therefore,is is a key task of sugar beet biotechnology research at present that sufficient gene sources be obtained.In this paper,a SSR molecular marker site Bv RE049 related to biennial traits of sugar beet obtained by our research group was used as the starting material,and the marker site and related genes were obtained by in silico isolation.Firstly,the self-built transcript database combined with the public database was used to conduct in silico extension for the marker sites.Then,through biological experiments,the obtained fragments were verified and cloned to obtain the exact sequence of the target fragments.The above process is repeated until the complete locus in the region of the marker locus is obtained.Finally,the structure,sequence characteristics and coding product characteristics of the gene locus were analyzed and predicted.Through this study,we will deepen the understanding of beet marker site Bv RE049 and its related traits,provide new gene resources and annotation information for the research of beet biotechnology,and lay a foundation for the further study of this marker,gene and trait..The main research results are as follows:(1)Through the in silico extension of the marker site,a sequence of about 3000 bp was obtained,located on chromosome 2 of the beet genome,possibly located in the gene coding region.(2)The results of the in silico extension were isolated,cloned and verified by experiments.After PCR amplification,purification,linking,transformation and sequencing,the full length of the gene locus was obtained,with a total length of 3005 bp.The sequence of this gene was similar to the PCNA-like gene in spinach and quinoa,so it was named as Bv PCNA-like.(3)Bioinformatics analysis on this gene locus had two aspects.At the nucleotide analysis aspect,the gene locus was predicted to be on chromosome 2 and contain a1218-basepair-long open reading frame(ORF)encoding 405 amino acids,and the gene structure was predicted to consist of four exons.At the amino acid aspect,it was inferred that the protein encoded by this gene has no obvious transmembrane region(not a membrane binding protein),and that it may be a hydrophilic protein,which is more likely to be located in the nucleus.The function of this protein is related to proliferating cell nuclear antigen(PCNA).(4)The structure of the gene locus was analyzed,the gene structure was predicted,and it was inferred that the gene locus might be composed of four exons.The validation scheme of gene splicing site and corresponding primers were designed according to the structure,and the regulatory elements upstream of the gene were analyzed,and a variety of cis-action elements were predicted.