Characterization of Some Genes Related to the Cotton (Gossypium hirsutum L.) Fiber Quality

Abstract

EXPANSINs are the intrinsic proteins in a plant cell and are involved in disentangling the cellulosic microfibrils of the cell wall. The mechanism of EXPANSIN action is generally concerned with cellular expansion. This family of proteins has also been documented to have tissue specific members. Multiple variants of this protein have been identified in specific tissues, which are temporally regulated and functionally specific. The fiber specific EXPANSINs play a key role in the development of cotton fibers. Various isoforms of EXPANSINs were isolated by screening the cDNA libraries constructed at different developmental stages of cotton (Gossypium hirsutum) and Calotropis procera fibers. Nucleotide sequence analysis of the screened clones helped to identify two major variants in cotton (GhEXPA8 and GhEXPA15) and four EXPANSIN isoforms in fast elongating C. procera fibers. The C. procera CpEXPA3 was selected for further analysis on the basis of its close relatedness with cotton fiber EXPANSINs. The comparative analysis of these EXPANSINs with existing database of the gene family revealed that they belong to the third clade of EXPANSIN A family having two characteristic domains. Eight conserved cysteine residues were found in the N-terminal of the deduced amino acid sequence of GhEXPA8, GhEXPA15 and CpEXPA3, while one was in the signal peptide region. Five tryptophan residues were conserved in C-terminal region of these EXPANSINs. The Amino Acid sequences of GhEXPA8 and GhEXPA15 have 98 % identity, while they have 69.8 % and 69.6 % identity with CpEXPA3 respectively. The presence of the signal anchor sites, the hydrophobic regions and the transmembrane regions at the N-terminus suggested that these proteins are targeted to the cellulosic microfibrils through the secretory pathway. The expression of these variants in different tissues was quantified by real time PCR. The transcripts of GhEXPA8 and GhEXPA15 were observed only in fibers, while CpEXPA3 was found to be transcribed nonspecifically in all tissues of the respective plant. The different transcription patterns of GhEXPA8 and GhEXPA15 at various stages of fiber development indicated that they are functionally different genes. The real time PCR analysis indicated the presence of EXPANSIN variants in developing cotton fibers from 0-15 DPA. The RT-PCR demonstrated that transcripts of LTP3 gene could be detected in developing fibers from 0-20 DPA. A plant expression vector was constructed by fusing LTP3 promoter with a reporter gene (GUS with intron) for in 20Generated by Foxit PDF Creator © Foxit Software http://www.foxitsoftware.com For evaluation only. Masooma Thesis vitro expression assay of the promoter strength and specificity in comparison with 2X35S. Transient expression studies on cultured cotton ovules, sepals, petals and stem revealed that the LTP3 promoter activity was confined only to the trichomes. The GUS gene in the expression cassettes was replaced with GhEXPA8, GhEXPA15 and CpEXPA3 generating six EXPANSIN genes constructs under the two promoters (2X35S and LTP3) with an aim to prolong the EXPANSIN gene expression in developing fibers. The construction of the expression cassettes was verified by DNA sequencing and the constructs were handed over to cotton transformation group at NIBGE for their utilization in fiber modification program.