Cloning, Expression, And Functional Analysis Of Caffeic Acid-O-methyltransferase Gene (GhCOMT) From Gossypium Hirsuturm L.

Cotton fiber is a single and highly elongated epidermal cell of the outer integument of the ovule. Lignin in cotton is synthesized via the lignin specific biosynthesis pathway which is just downstream of the common phenylpropanoid pathway, and many enzymes are involved in this process. Caffeic acid-3-O-methyltransferase (COMT) is a key enzyme of lignin specific pathway and catalyzes caffeic acid,5-Hydrocy-coniferyl aldehyde and 5-Hydrocy-coniferyl-coniferyl alcohol to produce ferulaic acid sinapyl aldehyde and sinapyl alcohol. It participates into the pathway of synthesizing S-lignin. Here we examined the role of GhCOMT in cotton development.1. In this study, full-length cDNAs of a key enzyme genes GhCOMT1/2/3(GenBank accession no.FJ479708, FJ479709, FJ848869), related to lignin metabolism in cotton (Gossypium hirsutum, cv. Xuzhou 142.) were isolated. And the GhCOMT1/2/3 genes were inserted into the pET-28a vector to construct recombinant vector pET-28a-GhCOMT1/2/3, which were then transformed into E.coli BL21(DE3) strain. After induction and expression, the target proteins were purified by using His TrapTM HP to get degenerated protein, SDS-PAGE and Western blotting detection were then employed to identify target proteins which had the molecular mass about 39.748kD,40.062 kD, and 39.119 kD respectively, the results were consistent with expectation.2. To investigate the function of GhCOMT1 gene, in the present study, we constructed the sense and anti-sense expression vectors of GhCOMT1 gene, and the coding region of the gene was placed under the E6 promoter in either sense or anti-sense orientation. In addition, a 450 bp fragment of GhCOMT1 was also cloned, and this fragment was inserted into plant vector to construct RNAi expression vector of GhCOMT1. These constructs were then transformed into Agrobacterium LBA4404. The sequencing result of different recombinant expression vectors showed that the GhCOMT1 gene has been correctly constructed.3. In this study transient expression vector of cotton GhCOMT1 gene was constructed. Firstly, the GhCOMT1 gene cDNA was obtained from pGEM-T-GhCOMT1 by PCR, then inserted into transient expression vector pRTL2-GUS/NIa. The transient expression vector pGUS-GhCOMTl is driven by 35S promoter with GUS reporter gene and the target gene, which expressed simultaneously and formed a fusion protein. Secondly, the vector pGUS-GhCOMT1 was transformed into cotton ovule by using PDS-1000/He biolistic particle delivery system. The results indicated that the transient expression vector pGUS-GhCOMT1 could be expressed efficiently in the epidermal cells of cotton fibre.Based on this data, we suggest that GhCOMT may play an important role in the morphogenesis and secondary wall thickening of cotton by positively/negatively regulating the structure of cell wall in growing course. Our study presents important experimental evidence for the function of lignin and provides gene candidates for genetic improvement of cotton fiber quality.