Cloning And Functional Analysis Of Fibre Specific Expression Promoter From GhManA2 In Cotton

By using the method of PCR-based cDNA library screening,oneβ-mannosidase clone,GhManA2,which was specifically expressed in fibre cells had been previously isolated and characterized.According to the cDNA sequence,three pairs of primers were designed for PCR-based BAC library screening.Five positive clones were screened.And using these clones as PCR amplication template,5' flanking sequences and gene sequences of GhManA2 in different cotton species were isolated via chromosome walking technology and PCR method.Through genetic engineering technique,the plant chimeric expression vectors contained different 5' truncations promoters of GhManA2 from tetraploid upland cotton A subgenome were constructed,and then were transformed into ovules by Agrobacterium-mediated transformation technology and particle bombardment.Transient GUS expressions were observed to analyze the function of these constructs.The results were as follows:(1)By using the Real-time PCR,the expression level of GhManA2 in cotton fibres(0 to 25 DPA fibres tested) was detected.The result showed that GhManA2 gene is begin to express at 5 DPA fibres which has a high expression level relatively and the level is 6/5 times higher than 10 and 15 DPA;the expression level of 10 and 15 DPA is similar;the expression level of 20 DPA is 2 times higher than 10 and 15 DPA;the expression level of 25 DPA which 6 times higher than 10 and 15 DPA,is highest in 5～25 DPA fibres.So,the whole results show that GhManA2 acted on primary cell wall synthesis and secondary cell wall deposition of fibre development.(2)According to the GhManA2 cDNA sequence,three pairs of primers were designed and were used for PCR-based BAC library screening.Five positive clones were screened:G13,B7,B14 are from upland cotton 0-613-2R BAC library;F20 and M12 are from Asian cotton cv.Jiangling BAC library.(3)A 5' flanking sequence of GhManA2 was isolated from the DNA of G13 BAC clone via chromosome walking technology at first.A 2075bp fragment was obtained.The two core promoter regions and some upstream regulatory elements were found in the fragment using the PROMOTER PREDICTION and PLANT CARE softwares.Several possibly transcription start sites(TSS) were predicted.TATA-box,CAAT-box, Skn-1-motif,GARE-motif,HSE,ERE,ABRE,G-box,I-box and other cis-elements were also found.(4)The promoter sequences of other four BAC clones of F20,M12,B7 and B14 were further obtained by PCR,The length of promoter sequences are 1795 bp,1795 bp, 1821 bp and 1821 bp,respectively,and are all different from the expected length of 1797 bp.In addition,the sequence similarity and phylogenetic relationship of five clones showed two clusters,one was G13,F20 and M12,and another was B7 and B14.Because F20 and M12 clones were from Asian cotton BAC library,we speculated that the line of G13 belonged to tetraploid A subgenome,and B7 and B14 belonged to tetraploid cotton D subgenome.(5)The genome sequences of GhManA2 which contain in G raimondii and the five clones of F20,M12,G13,B7 and B14 were also cloned.The analysis of structure of GhManA2 revealed that GhManA2 from different cotton species were all composed of 11 exons and 10 introns.The length of ORF was 2931 bp.The sequence similarity and phylogenetic relationship of ORF for the five clones showed the high consistency with the result from promoter sequences analysis.Further,sequence similarity and phylogenetic relationship of each intron for F20,M12,G13,B7,B14 and G raimondii showed the introns were not conserved in different genomes and GhManA2 gene's concerted evolution was also detected.Further study showed that cDNA sequence of GhManA2 reported previously was from tetraploid cotton D subgenome and speculated that promoter of GhManA2 in D subgenome could activate the expression of GhManA2.(6)Four plant expression vectors were constructed with different 5' truncations promoters of GhManA2.35S promoter of pBI121 were replaced and fused with the gus gene.These constructs were named as pBI121-M1(1857 bp),pBI121-M2(1468 bp), pBI121-M3(869 bp),and pBI121-M4(245 bp).Further,the four plant expression vectors were transferred into cotton ovules using the Agrobacterium-mediated transformation method and particle bombardment.Both of the Agrobacterium-mediated transformation and the particle bombardment showed the same results:except the pBI121-M4 with the shortest 5' deleted flanking region of putative GhManA2 gene promoter and the putative core promoter region deletion,other constructs all detected promoter activities.A GUS histochemical assay showed that GUS activity was only found in fibre cells on the cultured ovules and showed the 5' flanking region of GhManA2 gene could specifically expressed in fibre cell.(7)By analyzing the cis-acting elements of the GhManA2 gene upstream sequence and the transient expression of gus gene via Agrobacterium-mediated transformation and particle bombardment,it was supposed that the promoter fragment which located at -953～-309 bp have the function to promote specifically fibre expression.