Cloning And Characterization Of Five Genes Related With Fiber Development In Gossypium Hirsutum L.

Cotton fibers are important textile materials that play important role in nationaleconomy and people's lives. Each cotton fiber is a single cell of ovule epidermis thatelongates to 2.5-3.0 cm within approximately 16 days post anthesis (DPA). There are agreat deal of cellulosic and noncellulosic polysacchafide been synthesized during cottonfiber development to participate in the formation of fiber configuration, and thisdevelopmental process consists of myriads of saccharide progressing complicatedbiochemical reactions. So cloning the developmentally regulated genes from cotton fibersmay play an important role not only in improving the quality of cotton fiber, but also instudying plant cell development and cellulose biosynthesis.Five cDNA clones were separated from developmentally different cotton fiber pool ofpremium material 7235 library:(1)cotton fiber expressed protein (GhCFE, GenBankAccession numbe:DQ073045), the insert fragment of the cDNA clone was 1274bp,its openreading frame was 996bp, and encoded a polypeptide containing 331 amino acids. Blastxanalysis indicated GhCFE had 95%,98% and 96% homologies with CFE1,CFE2 and CFE3respectively;(2)homogentisate 1,2-dioxygenase (HGD), the insert fragment of the cDNAclone was 1697bp,its open reading frame was 1422bp,and encoded a polypeptidecontaining 473 amino acids. Blastx analysis indicated this gene had a 84% identity withhomogentisate 1,2-dioxygenase of Lycopersicon esculentum and 81% with homogentisate1,2-dioxygenase of Arabidopsis thaliana(At5g54080);(3)two peroxidases (POD), one wasa 1489bp cDNA sequence obtained via 5'RACE technique, its open reading frame was816bp,and encoded a polypeptide containing 271 amino acids. Blastx analysis indicatedthis gene had 70% homologies with a peroxidase of Arabidopsis thaliana, so this gene wasdesignated as GhPOD1; the insert fragment of the other cDNA clone was 1355bp,its openreading frame was 999bp,and encoded a polypeptide containing 332 amino acids. Blastxanalysis indicated this gene had a 99% identity with peroxidase of Gossypium hirsutum Lreported (pod8, L08199) and their protein sequences were almost similar. We designatedthis gene as GhPOD2;(4)pectate lyase (PL), the insert fragment of the eDNA clone was1539bp,its open reading frame was 1236bp,and encoded a polypeptide containing 411 amino acids. Blastx analysis indicated this gene had an 85% identity with pectate lyase ofMalus×doraestica.So this gene was designated as GhPL (GenBank Accession number:DQ073046).We predicted their signal peptide via signalP process.They all had typical signalpeptide except GhHGD.We also analyzed their conserved domain and phylogenetic tree.Judging from their expression characters,GhCFE was preferential expressed in fibercells and did not express in root or stem but only had a weak expression in leaf, while itcould be detected in ovules and fiber cells of different developing period, especially inelongating fibers;GhHGD could be detected in every tissue,but in leaf and ovules itsexpression was weak and it was preferential expressed in fiber cells from 5DPA to 14 DPA;GhPOD2 just did not express in root and its expression was decreased after 14DPA in fiberdevelopmental stages;GhPL was specifically expressed in fiber cells, while it could not bedetected in root, stem or leaf;GhPOD1 was constitutive expression and detected in everytissue.Southern blotting analysis shows that there are two copies of these five genes in thegenome of upland cotton; sub-genome A and sub-genome D contained each.Sense expression vector containing 35S promoter and both sense and antisenseexpression vectors containing E6 promoter using pBI121 plasmid were constructed forGhCFE and GhPL; both sense and antisense expression vectors containing 35S promoterusing pBI121 plasmid were constructed for GhPOD2 and RNAi vector was constructed forGhPL.The work of transferring these recombined vectors into Gossypium hirsutum L. byAgrobactedum tumefaciens is ongoing.GhCFE, GhHGD and GhPL were integrated into yeast expression vector (pREP5N),and electro-transformed into fission yeast S. pombe Q-01. The results showed that GhHGDleaded a significant change of length and length/width ratio; GhPL leaded a significantchange of width and length/width ratio; GhCFE had no significant effect on elongatingcells or thickening cell wall in the transformed yeast.We have used the BC₁ mapping population derived from the hybridization between theupland cultivar TM-1 and the island cultivar Hai7124, further TM-1 as recurrent parent.GhCFE and GhPL were localized on the chromosome 18 and chromosome 3 respectively.