The Expression Analyses Of Transcriptional Factors And Molecular Cloning And Characterization Of Two Genes In Gossypium

Cotton{Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world. Cotton fibers used in textiles are derived from the pubescence of maturing seeds. The high fiber quality is one of the major goals in the cotton breeding all over the world. So, it is important to elucidate fiber development process and major factors related to fiber quality by molecular biology. The expression pattern of transcriptional factors were detected in fiber mutants and pubescent lines. Then, the molecular cloning of GhCPC was processed and predicted as a trans-element for fiber development in cotton. The other gene, GhDIS2, was isolated and detected in fission yeast.1. The expression pattern of transcriptional factors in cotton fiber and leafTranscriptional factors, trans-acting factors, are DNA binding proteins, which can promot or repress gene transcription, In this study, several transcriptional factors coding GL1, GL2, GL3, WD40 proteins were analyzed on transcriptional level in cotton. These transcriptional factors included GhMYB2, GhMYB25, GhMYB109 coding GL1, GhHOX1, GhHOX2, GhHOX3 coding GL2, GhDEL61, GhDEL65 coding GL3, GhTTG1, GhTTG2, GhTTG3, GhTTG4 coding WD40, and the other MYB genes.Cotton fibers are classified into two types, lint and fuzz. Fuzz fiber development usually occurs after flowering but this varies among genotypes. As many homologous genes have been isolated from cotton and shown the similar role to trichome initiation in Arabidopsis, the GL1-GL3/EGL3-TTG1 protein complex may control the cotton fiber formation (Guan et al.,2007). The results gained from the transcriptional level showed that GhTTGl, GhTTG3、GhDEL61、GhDEL65, GhMYB25 were lowly expressed in fiberless ovules, and GhTTG3. GhTTG4, GhDEL6、GhMYB38 were lowly expressed in naked-seed ovules.The Ligon-lintless mutant is a monogenic, dominant mutant characterized by short fibers and distorted plant growth in the leaves, stems and flowers. The results gained from the transcriptional level showed that GhTTGl, GhDEL61, GhDEL65, GhMYB25, GhMYB38, G042C02A were lowly expressed in Ligon-lintless ovules. And G042C02A was a novel transcriptional factor cloned from the 7235 cDNA library in our lab.The Gossypium homologues to MYB, WD40, GL2 and GL3 genes in Arabidopsis were employed to detect the expression patterns of leaf trichomes in leaf pubescent line T586 and glabrous line TM-1. GhMYB25 (AF336283) and GhDEL61 (AF336279) expression level of the glabrous line were significantly lower than the pubescent line.2. Molecular cloning and characterization of CAPRICE gene in GossypiumMYB transcriptional factors are the largest gene family of transcriptional factor families in plant. Many MYB genes from Arabidopsis have been identified to control the initiation and morphogenesis of the trichomes. TRY, ETC1 and CPC are the trans-elements for the trichome development. TRY and CPC are single-repeat MYB proteins showing no DNA binding activity, but can interact with N-terminal domain of bHLH protein of GL3 and EGL3. CPC disrupts MYB-bHLH-TTG protein complex by competitive binding with WER, leading to repression GL2 expression. CPC inhibits trichome initial in cells surrounding trichome precursors in leaves, and move form root hair cells to non-root-hair cells in root.CAPRICE (CPC) is a trans-element in trichome development in Arabidopsis thaliana. Here, we isolate the CPC gene from Gossypium TM-1 ovules using candidate homological genes on the day of flowering. qPCR showed that the expression levels of lintless-fuzzless and naked-seed mutants were significantly higher than that of the wild line in ovules 0,1,3 days post anthesis (DPA). This gene was expressed at peak values in 3 DPA ovules, with the absence of transcriptional expression in stems. Genomic sequence alignment found fewer differences in exons relative to intron regions between wild-type and lintless-fuzzless mutants. The CPC gene ORF maintains sequence conservation among allotetraploid and diploid cotton species. A GARE-motif was detected in the CPC gene promoter in G. hirsutum and G. herbaceum, but presumably lost in G. ramondii. In in vitro cultured ovules, fiber development was inhibited by the CPC gene and promoted by gibberellins, while this hormone inhibited fiber growth under high CPC transcribe accumulation. These results suggest that the CPC gene may be a key inhibitor of cotton fiber development and used for the further genetic enhancement of the fiber quality and quantity.3. Molecular cloning and characterization of GhDIS2 in cottonTranscriptional factors are transported to the regulation sites along the cytoskeleton pathway. So, the cytoskeleton genes are very important in the functional exertion of transcription factors. DISTORTED2 encodes the Arabidopsis homolog of the ARPC2 subunit of the ARP2/3 complex, which binds to the sides of existing actin filaments and efficiently nucleates new filaments.The Gossypium homology of the ARPC2 subunit named as GhDIS2 was isolated from the TM-1 ovule on the day of flowering. GhDIS2 contains 975 bp ORF, encoding a polypeptide containing 324 amino acids. GhDIS2 was expressed constitutively in every tissue with different expression levels, with the peak in 12 DPA fiber cells and low expression levels in 18,24 DPA fibers, roots and stems. Southern blotting showed GhDIS2 had two copies in allotetraploid cotton genome. A shuttle vector contained GhDIS2 cDNA sequence was transformed into fission yeast. GhDIS2 led to a significant increase in cell length and width. The results indicated GhDIS2 had the same function to DIS in other plants.