Isolation And Characterization Of GhHOX2 Promoter From Gossypium Hirsutum L.

Cotton is one of the most important fiber crops and plays an important role in our economy as a major raw material for textile industry.Cotton fibers are unicellular and derived from the epidermal layer of ovule;therefore cell elongation can be evaluated independently from cell division.During the primary cell wall formation stage,fiber cells elongation is rabidly above that of common plant cells.The fiber cell could reach to 3-5 cm in final length and was 1000-3000 time longer than its diameter.Furthermore,the abundance of cellulose was synthesized during secondary cell wall formation and accounted for more than 95%of the dry weight of mature cotton fiber.Those characteristic given,the fiber cell provides a useful model for the study of cell division,cell elongation and cellulose synthesis of plant.Transcription factors which regulated gene expression may play very important roles in cotton fiber development.However,little was known about the molecular mechanism regulating the fiber initiation.GhHOX2,a putative homeobox gene which was cloned from the developing cotton fiber cell,is still poorly known about its function.In order to investigate the function of GhHOX2,the 5' upstream region of GhHOX2 were isolated from genomic DNA by the improved Y-shaped adaptor dependent extension(YADE)method.The GhHOX2 promoter was fused with GUS reporter gene and transferred into tobacco genome by Agrobacterium-mediated transformation.The main results are as follows:1.Characterization of genomic structure and expression profile of GhHOX2Southern blotting analysis showed that there should be two homologous GhHOX2 gene copies in the Gossypium hirsutum genome.RT-PCR showed that GhHOX2 was expressed in root, stem,leaves,cotyledon,bud,petal,fiber,and ovule.Real-time PCR revealed that expression in cotyledon and petal relative to GhHIS gene was the highest,but expression in roots,bud and fiber were lower than in other tissues.These results indicated that GhHOX2 was a constitutive expression transcription factor gene.2.Isolation of 5' -upstream sequence of GhHOX2 The full sequence of GhHOX2 was 4.29-kb in length containing 10 introns and 11 extrons,in which two Hindâ…¢sites were observed.According to the genomic sequence,a 1218 bp GhHOX2 promoter fragment was obtained by the modified YADE method.However,two fragments of GhHOX2 promoter were amplified and the substaintial difference between them was validated.So the two promoter of GhHOX2 were designated as pHOX2-1 and pHOX2-2 by a sequence of 1218 bp and 1195 bp,respectively.The pHOX2-1 sequence showed 85%identity with pHOX2-2 promoter and the main difference between them was the base bias located in the regions from -1140 to -483 bp, where only a Hindâ…¢site was observed in pHOX2-1 while two in pHOX2-2.Furthermore,two fragments,which contained the promoter and partial coding region of GhHOX2,were cloned by specific PCR amplification using cotton genomic DNA as template.Sequence analysis approved the promoter sequence in the two cloned fragment were identity with that obtained by YADE extending, which supported our conclusion that the two promoter fragments of GhHOX2 were really existed in cotton genome.We also characterized the promoter sequences of HOX2 from Gossypium raimondii Ulbr.,Gossypium herbaceum L.and Gossypium arboretum L..The result indicated that the pHOX2-1 might inherited from D₅ genome of Gossypium raimondii Ulbr., and the pHOX2-2 might inherited from A₁ genome of Gossypium herbaceum L..3.The function of GhHOX2 promoter in transgenic tobaccoTo investigate the function of GhHOX2 promoter,two expression vectors,pGhHOX2-1-GUS and pGhHOX2-2-GUS,were constructed by fusing the two GhHOX2 promoters with GUS and introduced into tobacco respectively via Agrobacterium-mediated transformation.GUS activity was detected strongly in stem,receptacles and ovary at Odpa,and also detected in sepal weakly by histochemical assays in pHOX2-2::GUS transformants.However,GUS activity was detected only in stem and ovary in pHOX2-1::GUS transgenic tobaccos.Quantitative GUS assays in various tissues from individual lines of transgenic tobaccos holding pHOX2-1::GUS,pHOX2-2::GUS and CAMV35S::GUS show that the strength of different promoters expression is variable.The mean GUS activity in ovsry at Odpa,receptacles and stem was 3445.81, 3192.98 and 2178.36 pmol/mg/min respectively from pHOX2-2::GUS transgenic tobaccos,however it was only 3%,2.2%and 1%than that from CaMV35S::GUS transformants.Moreover,in pHOX2-1::GUS transgenic tobaccos,the average GUS activity was measured in ovary at Odpa and receptacles with a value of 875.61 pmol/mg/min and 501.63 pmol/mg/min,however it was only 25% and 15.7%than that of in pHOX2-2::GUS transgenic tobaccos,respectively.These results suggested that the difference of expression pattern in transgenic tobaccos between pHOX2-1 and pHOX2-2 may be resulted from the difference sequence of two promoters.