Molecular Cloning And Functional Analysis Of GbWRKY32?GbWRKY40 And GbHCT Genes In Cotton(Gossypium Barbadence L.)

The total output of cotton in China occupies first place in the world but the inferior fiber quality caanot meet domestic requirements.The development of cotton fiber is controlled by various transcription factors and genes jointly. The related genes separated now cannot systematically present the developmental mechanism of cotton fiber, so we still need to identify more transcription factors and genes involved in the development of cotton fiber. WRKY transcription factor is an important transcription factor involved in various physiological responses of plants.AtWRKY44 gene has been reported to regulate and control the development of the WRKY transcription factor in the development of the plant cuticle, which has attracted the attention of researchers. HCT gene is a family member of the formyl transferase and HCT is a key enzyme in the lignin specific synthesis pathway. Studies have shown that the lignin content in cotton seeds with good fiber quality is less than that in ordinary cotton seeds. Therefore, it is of important significance to study genes related with the lignin synthesis related genes.The conclusions are presented as follows:1.The primers were designed cotton fiber development transcriptome and sea Island Xin hai 21 was choseen as experimental materials to cloning 2 members of the WRKY transcription factor and lignin biosynthesis related Gb HCT gene. The open reading frame of GbWRKY32 gene has 1077 bp, encoding 358 amino acids,and the open reading frame of Gb WRKY40 gene has 942 bp, encoding 313 amino acids.GbWRKY32/ GbWRKY40 has a conserved and specific sequence of WRKY transcription factors.HCT gene has 2 unique and conserved sequence called HTLGD and DFGWG. The open reading frame of the gene has1311 bp, encoding 436 amino acids. Phylogenetic tree analysis showed that the three genes were closely related with the upland cotton, Asian cotton and Raymonds cotton.2.The yeast expression vector of WRKY protein was constructed and was used to transform yeast strain to detect the activating effect of GbWRKY protein in the amino acid-defect medium. The results showed that Gb WRKY32/GbWRKY40 had no transcriptional activation effect.3.Total RNA was extracted from different tissues and developmental periods of Sea Island Cotton Xinhai 21, and real-time PCR was used to detect the expressing quantity of Gb WRKY gene and GbHCT gene. The results showed that The expression amounts of the GbWRKY32/GbWRKY40 genes are respectively the highest in the stems and roots among different tissues. In different development periods of the cotton fiber, the two genes are in dominant expressions in the beginning of the fiber development and elongation period. The expression amount of GbHCT gene is almost the same in the flowers and bracts of different tissues and higher than the other three tissues. The largest GbHCT gene expression amount is in the two periods of fiber elongation and secondary wall thickening periods.4.Plant expression vector pCAMBIA1304-Gb WRKY32 was constructed and used to transform model plants. The transgenic tobacco was detected by PCR and fluorescence quantitative PCR,Over-expression plants of Gb WRKY32 gene was obtained.observe overexpressing plants find that: GbWRKY32 transgenic tobacco plants showed rapid growth, well-developed lateral roots, and increased epidermal hairs. It could be initially speculated that the Gb WRKY32 gene may be involved in the development of epidermal hairs.