Molecular Cloning And Expression Analysis Of The Genes Specifically Expressed In Fibers Of Gossypium Hirsutum

Cotton is one of the most important economic crops in the world, and cotton fiber is the major natural fiber material for textile industry. Apart from the economic importance of fiber, developing cotton fiber is a perfect experimental model for studing the mechanism of cell elongation, wall development, cellulose biosynthesis in plants. Fibers are the seed hairs of cotton, and they originate from the epidermal cells of the ovular surface. Fiber development is composed of four distinct and overlapping stages. There are lots of genes actively expressed during fiber development. Isolation and analysis of genes expressed preferentially in cotton fiber will be helpful in further understanding the complicated process of fiber cell elongation and cellulose biosynthesis and ultimately does good to improve cotton fiber quality and yields.The fibers elongate rapidly in 10DPA and undergo the developmental switch from elongation to secondary cell wall deposition in 20DPA. To gain insight into the molecular mechanisms associated with the elongation of cotton fibers and the biosynthesis of cellulose, two cDNA libraries were constructed from upland cotton genetic standard line 'TM-1' by suppression subtractive hybridization(SSH). The cDNA from 10 and 20DPA fibers were respectively used as testers of the two SSH libraries, and cDNA reverse transcribed from a RNA pool of nonfiber tissues(root, hypocotyl, leaves, petal, and ovules) as the same driver. Through differential screening, 292 clones in both libraries were identified as preferentially expressed genes during fiber development. Sequence analysis showed that 66 unique sequences were found in two SSH libraries. Many previously reported cotton fiber related genes were included in both libraries. Some genes, such as encoding malate dehydrogenase, putative gibberellin regulated protein, arabinogalactan proteins(AGPs), auxin responsive protein, were reported to relate with fiber development for the first time. Northern hybridization revealed genes from 10DPA fiber subtracted library were expressed a lot during early fiber development. On the other hand, those genes screened out of the 20DPA fiber subtracted library were found to be highly expressed after 15DPA. Additionally the expression differences of 17 genes among G. hirsutum, C. barbadense, and G. arboreum were analyzed by RT-PCR. It was resulted that the G. hirsutum fiber specifically expressed genes greatly accumulated in fiber cells of G. barbadense and G. arboreum. However, the gene expression in G. arboreum 'Meiguozhongrnian 971' was some weaker than that in other genotypes. The deficient expression of fiber related genes maybe result in the defective phenotype of fiber in 'Meiguozhongrnian 971', which has not fuzz but fewer lint fibers. The results of the present study have enriched the understanding of how the genes potentially control single-cell elongation, cell wall development, and cellulose biosynthesis.Two cDNA sequences encoding plant aquaporins(AQPs) were screened out of the 10DPA fiber SSH library, and their full-length cDNA sequences were obtained by 5' RACE. The two genes were named GhPIPI-2 and GhγTIP1 respectively. GhPIP1-2 is 995bp in length, and has an open reading frame(ORF) of 861bp which encodes a predicted polypeptide of 287 amino acids(aa) with a calculated molecular weight(MW) of 30.9 KDa and pI of 8.6. GhγTIP1 is 923bp in length, and contains an ORF of 756bp which encodes a predicted polypeptide of 252 aa with a calculated MW of 25.8 KDa and pI of 4.80. Searching against protein database using BLASTx in GenBank, the deduced protein sequences of GhPIP1-2 and GhγTIP1 were found to share the highest protein homology with G. hirsutum GhAQP1(ABD639041) and soybean (Glycine max) nodulin-26(AAA02947) respectively. The bioinformatics analysis of the two deduced proteins revealed they own the characteristic feature of AQPs: small and highly hydrophobic proteins with six transmembrane helices connected by five loops(A～E). B loop and E loop respectively comprise a short a-helical domain on the opposite sides. Each a-helical domain contains an NPA(Asn-Pro-Ala) motif, which is conserved for all aquaporins with rare exceptions. Phylogenetic tree analysis of GhPIP1-2, GhγTIP1 and other plant AQPs showed GhPIP1-2 belongs to PIP1 group of PIP subfamily and GhγTIP1 belongs toγTIP group of TIP subfamily. GhPIP1-2 and GhγTIP1 contain three and two introns respectively. Genomic southern blot analysis indicated both GhPIPI-2 and GhγTIP1 have several copies and homologous genes in G. hirsutum and G. barbadense. Northern blot analysis with gene-specific probes showed that both GhPIPI-2 and GhγTIP1 are specifically expressed during cotton fiber elongation. Real-time PCR analysis indicated expression patterns of them in G. hirsutum and G. barbadense are similar. However, the expression level of them in G. barbadense is lower than that in G. hirsutum. As members of plant AQPs, GhPIP1-2 and GhγTIP1 may play an important role during cotton fiber elongation with responsibility for transmembrane transport of water into the vacuole.What is more, four full-length cDNA sequences encoding AGPs were obtained by 5' RACE and 3' RACE. The four genes were named GhAGP2, GhAGP3, GhAGP4, and GhFLA1 respectively. GhAGP2 is 886bp in length, and has an ORF of 729bp which encodes a predicted polypeptide of 243 aa with a calculated MW of 25.6 KDa and pI of 9.27. GhAGP3 is 915bp in length, and contains an ORF of 792bp which encodes a predicted polypeptide of 264 aa with a calculated MW of 27.8 KD and pI of 6.82. The full-length of GhAGP4 is 1090bp with an ORF of 717bp, encoding a predicted polypeptide of 239 aa with a calculated MW of 25.4 KDa and pI of 8.67. GhFLA1 is 961bp in length, and has an ORF of 732bp which encodes a predicted polypeptide of 244 aa with a calculated MW of 25.8 KDa and pI of 7.7. The deduced protein sequences of GhAGP2, GhAGP3, and GhAGP4 were searched against protein database using BLASTx in GenBank, and all of them were found to share high protein homology with G. hirsutum GhAGP1(AA092753). As for the GhFLA1, the deduced protein is highly homologous with Arabidopsis unknown protein(AAN60339), FLA9(fasciclin-like arabinogalactan protein 9, NP₅63692), and FLA6(NP₅65475). The bioinformatics analysis of the deduced amino acid sequences of four genes indicated they are classical AGPs. They contain an N-terminal signal sequence, a central domain rich in Ala, Ser, Thr, Pro/Hyp, and C-terminal hydrophobic transmembrane domain. All of them include two AGP-like domains(rich in noncontinuous Pro residues) and a putative C-terminal GPI (glycosylphosphatidylinositol) anchor signal sequence. In addition, all of them contain one FAS1 domain. Obviously the four genes encode fasciclin-like AGPs(FLAs). Genomic sequences analysis revealed the four AGPs genes have no introns. Southern hybridization indicated all of them are low copy genes and have a few of homologous genes in G. hirsutum and G. barbadense. Northern hybridization with specific probes and real-time PCR revealed that all of them are specifically expressed during cotton fiber development. GhAGP2 and GhFLA1 are abundantly expressed from 5DPA to 20DPA. However, GhAGP3 and GhAGP4 accumulate largely from 15DPA to 25DPA. Real-time PCR analysis also indicated expression patterns of them in G. hirsutum and G. barbadense are some different, especially for GhAGP3 and GhAGP4. Otherwise the expression level of GhFLA1 in G. barbadense is much higher than that in G. hirsutum. All analysis results hint GhFLA1 and GhAGP2 may play vital roles during fiber rapid elongation and the primary cell wall development. But GhAGP3 and GhAGP4 might be involved in the secondary wall deposition.In order to isolate the fiber-specific promoters from cotton, promoter sequences of four fiber specifically expressed genes, pGhPIP1-2, pGhγTIP1, pGhAGP4, and pGhFLA1 were obtained using the method of genomic walking. The length of pGhPIP1-2, pGhγTIP1, pGhAGP4, and pGhFLA1 are 840bp, 400bp, 1210bp, and 1400bp respectively. Through cis-acting elements searching in the P1antCare web site, many regulatory elements were found in four promoters, including those relate to light response, phytohormone response, stress response, and so on.Furthermore we want to verify the precise functions of these AQP and AGP genes during cotton fiber development by RNA interference approach. The silencing vectors of GhPIP1-2, GhγTIP1, and GhAGP4 were respectively transformed into upland cotton mediated by Agrobacterium. All RNAi constructs had obtained the transgenic plants. PCR analysis showed that 91ï¼…of the RNAi transgenic plants are positive. The following work of this assay will be continued when the transgenic plants set seeds.