Cloning And Activity Analysis Of Fiber-specific Expressional Promoter LTP3 And FbL2A From Cotton (Gossyium Hirhsutum)

Cotton (Gossypium hirsutum) is an economically important commodity worldwide. Cotton fibers are unicellular trichomes that terminally differentiate from epidermal cells of developing cotton ovules. Initiation of fiber development is triggered by hormones and lasts up to 45 to 50 dpa (days past anthesis). During fiber development, many genes, known to be fiber-specific or preferentially expressed in cotton fiber, are developmentally regulated. The 5' flanking regions of these genes have been isolated and shown to direct expression of heterologous proteins in a tissue-specific fashion, which provide many promoters or c/s-elements for modifying cotton fiber by genetic engineering schemes. Such as E6 gene and LTP gene, activated during the primary wall synthesis stages, and FbL2A activated during late primary and early secondary wall synthesis stages etc.We cloned LTP3 (1548bp) and FbL2A (4050bp) promoter and a 614bp LTP'3 promoter 3' fragment by PCR amplification, then fused with the E. coli. P -glucuronidase gene (GUS) as a reporter gene after sequencing analysis, and named pBl-LTP3(\), pBl-LTP3(2) and p23G-FbL2A. The expression patterns of LTP3 promoter (LTP3(l) and LTP3(2) ) were analyzed in transgenic tobacco plants. Tissues that express the GUS gene stain blue when provided with a specific substrate. Which providing a convenient means to identify the expression patterns determined by promoter.As expected, GUS expressions under control of LTP3(1) and LTP'3(2) promoters were detected only in the trichomes of tobacco leaves, which means 614 nucleotides upstream of LTP3 gene were enough to confer the expression. While the positive control, contains Cauliflower mosaic Virus (CaMV) 35S promoter and GUS gene, was constitutive expressed, and the negative control (untransformation plants) was detected no GUS expression. The results indicate that LTP3 promoter can be used to target the tissue-specific expression of heterologous genes.We also transformed pEl-LTP3(l) and pBl-LTP3(2) into cotton by the pollen tube traverse method, in order to master the transformation method and detect the expression of GUS in cotton fiber.