Cloning Of Upstream Regulatory Sequences Of Sugar Beet Bvlhvb-1 Gene And Verification Of Its Promoter Activit

Promoters play a very important role in regulator gene expression.Cloning promoters and analyzing their functions have important application research value for regulating exogenous gene expression in genetic engineering breeding,and also have considerable theoretical research value for studying the expression law of genes in plants.There is still a significant gap in the promoter research of sugarbeet related genes in China compared to major crops,and information on the molecular mechanisms of gene expression regulation is also very limited.Therefore,for sugar beets,starting from differentially expressed genes and obtaining relevant promoter information through mining and analyzing their upstream sequences is a more suitable solution for studying sugar beet promoter resources.It can not only enrich sugar beet promoter resources,but also lay a good foundation for further studying the function and mechanism of sugar beet genes.In the early stage,the research team cloned the gene BvLhcb-1 with different expression levels from two representative germplasms with color difference of beet leaves,J9451 and DZ-6.The difference in the expression of this gene in the two germplasms may be related to the difference in promoter.Therefore,this study cloned the upstream sequences of this gene from the genomes of the two materials mentioned above,named JBvLhcb-1 and DBvLhcb-1 respectively;Using bioinformatics methods to analyze the types and distribution of JBvLhcb-1 and DBvLhcb-1 sequence elements,respectively,for vector design and construction of plant expression vectors;Then perform tobacco transformation and transient expression on it to verify the promoter function of the upstream sequence;At the same time,by analyzing the regulatory elements closely related to leaf color and their distribution characteristics,a subsequent validation analysis plan and a plant expression vector with missing mutant fragments are designed for further research.This study is expected to obtain new sugarbeet promoters and their structural information,and lay a foundation for further research on the expression regulation mechanism of BvLhcb-1 gene,as well as the specific function and mechanism of action of its promoters.The main research findings include:Two upstream sequences JBvLhcb-1 and DBvLhcb-1 were cloned and analyzed.The upstream sequences of the BvLhcb-1 gene were cloned from two germplasm samples,with promoter characteristics of 1828 bp and 1854 bp in length,named JBvLhcb-1 and DBvLhcb-1,respectively.There are many core promoter elements and several functional annotations related to signaling and regulation in both sequences,including regulatory elements ACE,G-box,G-BOX,I-box,GATA-motif,GT1-motif,and TCCC-motif that participate in photoresponse;Elements such as ABRE involved in abscisic acid induction;TGACG-motif and other elements that respond to methyl jasmonic acid induction.The number and distribution of similar elements in the two sequences are different,and DBvLhcb-1 lacks one WUN-motif reaction element related to wound response compared to JBvLhcb-1.We have designed and constructed plant expression vectors for the target fragment(p CAMBIA1381xb-Dlhcb-1,p CAMBIA1381xb-Jlhcb-1).Using the commercial plant expression vector p CAMBIA1381 xb as the base vector,insert the target fragment into the upstream of the GUS reporter gene and retain the plant screening marker gene expression box on the vector,so that both vectors are suitable for both transient and stable expression.At the same time,the first stage deletion mutation scheme was designed and corresponding plant expression vectors were constructed for subsequent research by the research group.Verified the activation activity of JBvLhcb-1 and DBvLhcb-1.Plant expression vectors p CAMBIA1381xb-Dlhcb-1 and p CAMBIA1381xb-Jlhcb-1 were transformed into tobacco leaves for transient expression.After GUS staining,it was found that the full length fragments of JBvLhcb-1 and DBvLhcb-1 can drive the expression of GUS gene in tobacco leaves,indicating that these two upstream sequences have promoter activity and can be considered as promoters.