Gene Expression Profiling During Fiber Secondary Cell Wall Development And Functional Verification Of GhKAR, GhHAD And GhENR In Cotton

Cotton is not only an important raw material in textile industry, but also one of the five major oil crops in China. Oil metabolism is an important basic metabolism, the development of seeds directly influences the accumulation of oil content, and it is related to many important metabolism channels; the cotton fiber is polarized from the epidermal hairs of seeds, and the gene in seeds influencing oil content metabolism might has a certain influence during the development of cotton fiber. According to the existing researches, the oil content of cotton seeds has no significant negative correlation with the cotton yield traits, and it has positive correlation with fiber quality indicators, which indicated that improving the oil content of cotton seeds or changing structures of their fatty acids could also achieve fibers with high quality and yield. In order to further understand the molecular mechanism of the cotton fiber development, sea island cotton (Xin Hai 32, HL) and upland cotton (17-24, L and 62-33, S) were taken as research materials. The RNA-seq technology was adopted, and the expression profiling of fiber development at secondary wall composition and thickening stage (20 DPA,25 DPA and 30 DPA) were analyzed. The differentially expressed genes were screened and GO function significance enrichment analyses were performed. To seek the critical metabolic pathway and important genes related to the thickening of cotton fiber secondary wall, the expression character of fatty acid synthetase GhKAR, GhHAD and GhENR were analyzed, and also their functions were verified. The major research results are listed as below:1) From the different genotypes were obtained large amount of differentially expressed genes at different stages of secondary wall development. After comparing the differentially expressed genes of different genotypes in the same fiber development period, we found more differentially expressed genes when pairwise comparing different cotton varieties, such as 20 DPA,6215 differentially expressed genes are obtained between island cotton (HL) and upland cotton (L); 5364 differentially expressed genes are obtained between island cotton (HL) and upland cotton (S); while only 1236 differentially expressed genes are obtained when comparing the two upland cotton (L and S). The GO functions and pathway enrichment analysis indicates that SNARE pathway is highly enriched, and the expression of these genes in the island cotton (HL) is lower than the short fiber material (S), which implies that the island cotton is still in a fiber elongation phase at 20 DPA. In addition, the fatty acid elongation pathway is also highly enriched in different cotton varieties in the same period, indicating the genes related to lipid synthesis are playing certain roles in fiber elongation and thickening of secondary cell wall.2) The differentially expressed genes at the different development stages of the fiber secondary walls in the same genotype might be closely related to the fiber quality. The GO function and pathway enrichment analysis of the differentially expressed genes shows that the propanoate metabolism and the biosynthesis of unsaturated fatty acids pathway are only enriched in long fibers materials (HL and L), and these two pathways might have promotive functions to the fiber elongation and secondary cell wall synthesis; while the pathways of fatty acid elongation and the valine, leucine and isoleucine degradation are highly enriched in different materials at different time, which implies that the two pathways playing important roles in the period of secondary cell wall synthesis and thickening.3) 46 common differentially expressed genes are obtained after analyzing different genotypes and development stages of the cotton fiber with four major expression modes:a. gradually rising, and then gradually falling, totally 30 genes; b. gradually rising, totally 7 genes; c. gradually falling, totally 6 genes; d. first gradually falling, and then gradually rising, totally 3 genes. It inferred that 37 rising expression genes might have important roles in the period of the fiber development secondary wall synthesis and thickening period, and they have positive regulating role on the quality of fiber. These genes were involved in the P450 enzyme protein gene of cytochrome, the glycosyl hydrolase, glycosyl transferase, and blinding protein gene, etc; performing RT-PCR verification of 11 differentially expressed genes has proven the reliability of RNA-seq data.4) The organization expression analysis results indicated that GhKAR, GhHAD and GhENR all have highly expression in fibers, the 20 DPA is a critical period of fiber development and objective genes expression; and the objective genes could generate response to reaction of ABA, low temperature and MeJA; the excessive expression GhKAR and GhENR genes are helpful to increase the oil content and to improve the cotton fiber length and quality as well. It is proven these genes really participates in the development of fiber. Preliminary exploration is conducted on transformation GhKAR and GhENR gene cotton plants, and the results indicated that the transformation GhKAR and GhENR gene T4 lines and contrastive (WT) have significant or extremely significant differences to the content of REC, MDA and Pro stressed by different degrees of low temperatures, which indicated the excessive expression fatty acid genes GhKAR and GhENR has a certain improvement on cold resistance in plants.