Cloning And Analysis Of GaMYB7 And GbGLU Gene Promoter From Cotton

Promoter can control foreign gene's expression feature:where,when or how much.Constitutive promoter,which drives the foreign gene expressed in all plant organs and throughout the plant developmental period,is widely used in plant genetic engineering studies now.This may squander the plant nutrient and energy and results in increased metabolism burden of the plant.We can control foreign gene's expression if we instead the constitutive promoter by a fiber-specific promoter or a disease inducible promoter in cotton genetic engineering.In this study,we try to isolate a cotton fiber-specific gene(GaMYB7) promoter and a Verticillium dahliea induced up-regulation expression gene(GbGLU) promoter.The mainly result includes two sections:The ordered array of the pooled BAC clones was screened and the BAC clones containing the GaMYB7 gene were identified.With the BAC-DNA serving as template for the first round of TAIL-PCR,we successfully obtained a sequence 1566 base pairs in length upstream of the ATG start codon of the GaMYB7 gene.Cis-acting element prediction results showed the basic sequence structure was consisted of identified putative core promoter elements and other upstream promoter elements, including light response elements,auxin and gibberellin response elements.The effects of GA₃,IAA and light on GaMYB7 expression in fiber cells were studied by in vitro cotton ovule culture and real-time fluorescent quantitative PCR.The results showed that GaMYB7 expression could be significantly induced by 10uM IAA,5uM GA₃ and 10d light treatment,respectively.In transient assays with cotton ovules cultured in vitro,both this promoter sequence and a series of tnmcations could drive theβ-glucuronidase reporter gene(GUS) specifically in ovules and fibers.Therefore, the promoter is considered to be a useful expression element for deep researches on molecular mechanisms of cotton fiber development,modification of fiber quality and expression of foreign genes in fiber. We also identified the BAC clones containing the GbGLU gene from upland cotton genetic standard line TM-1 BAC library screening and isolated a sequence 1357 base pairs in length upstream of the ATG start codon of the GbGLU gene, successfully.Several cis-acting elements,including W-box,GT-1 box,ARE,BOX4, as-2-box,3-AF1 binding site,Root-motif elements,were recognized with the aid of PLACE program.Four different regions of the promoter sequence of the GbGLU gene were fused to theβ-glucuronidase(GUS) coding region.In Agrobacteriummediated transient expression assay,the transcriptional activations of the promoter deletions were examined in cotton embryogenic callus.