Molecular Cloning Of ADP-ribosylation Factor 1 Gene(arf1) From Gossypium Hirsutum And Function Analysis Of Promoter

The genes diaganostic expression to cotton reproductive organ showed a significant role in the economic yield of cotton, especially to the construction of cotton quality characters in fiber, therefore, studies on these genes and isolation of the gene promoters and gene expression proper elements had the important significant for understanding fertility of cotton, fiber development and molecular mechanism of forming economic yield of cotton. The full-length cDNA, DNA and promoter of ADP-ribosylation factor 1(arf1) was isolated from Gossypium hirsutum Y18 by means of isocaudarner inverse PCR(II-PCR) and rapid isolating cDNA 5' unknown sequence and promoter(RICUP) was established in our lab(unreported home and abroad). The results by Southern blotting analysis showed the gene had two copies in cotton genome, and results by Northern blotting indicated the predominant expression of arf1 in reproductive organs of cotton , including bud, flower, fiber and boll.Alternative splicing played an important role in the eukarotic growing, developing and genetic expression and control, whereas it was not reported in the process of cotton fiber developing to present. The results by RT-PCR and Northern blotting analysis in this paper conformed that there existed alternative splicing in cotton arf1 gene, and formed three types of mRNA. The region of this gene promoter had some typical promoter sequence such as Initiator, TATA box, CCAAT box and GC box and some other proper reverse repeative structure and palindrome which indicated this promoter had strong promoter activity and characteristic of tissues and organs.Now promoter used in transgenetic anti-insect cotton was CAMV 35 which had a low expression in productive organs of cotton. To resolve the problem, we applied predominant expression promoter to drive killing-insect gene to express in productive organs of cotton. Using genes of cotton line Y18 as template and PCR technique got promoter fragment, then the fragment synthesized with gus reporting gene on vector pBI121 to construct plant expression vector named Parf-p-pBI121. The wild type cotton was transformed by pollen tube pathway mediated method and 13 positive lines were screened by Kan resistance, PCR identification showed gus gene and arf1 promoter fragments had been intergrated into cotton genome. The results from chemical analysis of gus gene indicated arf1 had productive organ expression character and provided an ideal promoter for the expression in cotton bud, flower and boll on killing-insect genes.