Cloning And Expression Analysis Of Cotton ABHcad Gene A Key Enzyme In The Biosynthesis Of Gossypol

Cadinene synthase is a key enzyme of gossypol biosynthesis. Gossypol is a kind of terpene compound limiting the utilization of cotton seed protein. Therefore, it would be helpful to understand the relationship between pigment gland formation and gossypol biosynthesis and provide theoretical basis for low phenol cotton breeding through cloning cadinene synthase genes, studying the expression patterns of these genes in different cotton varieties with different pigment gland and analyzing the relationship between pigment gland formation and gossypol content.In this study, three nearly isogenic lines (G11, G12 and G13) bred from (G arboretum×G. bickii)×G. hirsutum was selected as the research materials. At the three development stages (seedling stage, bud stage, the blooming stage) of GI1 and GI2, we observed the distribution of pigment glands, measured the density of glands and compared the accumulation contents of gossypol in different tissues (root, stem, leaf, bud, flower and fluid).We cloned the cDNA sequence of cadinene synthase gene from G11, analyzed the expression patterns of cadinene synthase gene in different tissues of three materials and made a correlation analysis for gossypol content and relative expression level of ABHcad in roots at different development stages. The results were as follows:1. There was no pigment gland in root epidermis of three nearly isogenic lines GI1, GI2 and GI3, as well in other tissues of GL3, but during the whole development period, there existed pigment glands in other tissue epidermis of G11 and G12. We found the formation of GI2 seed pigment gland was delaying.2. Gossypol contents of different tissues of GI1 and GI3 at three development stages were measured by the method of national standard, the results showed that:the gossypol content was highest in the roots of GI1 and GI3, which indicated the root was the place for gossypol biosynthesis. There was gossypol in the stem, leave, bud and bell of GI1, but the contents were significantly lower than that in root tissue; There was without the presence of pigment glands in the whole plant of GI3, but also had the existence of gossypol; Compared with G13, the gossypol content in all tissues of G11 during whole development period was higher, which indicated that the existence of pigment glands was helpful for the accumulation of gossypol.3. Applying RACE, we had cloned a cadinene synthase gene, namely ABHcad, with the length of 1956 bp and encoding 366 amino acid residues predicted from NCBI databases. Phylogenetic tree analysis showed that the target gene was most close to the gene family of cadinene synthetase C. The genome sequence of ABHcad indicated that three cotton materials had compound chromosomes including A、G and D, so there might be multiple cadinene synthase gene allelic loci.4. Compared with the root tissue of cotton lines, the expression levels of cadinene synthase gene in the tissues of stem, leaf, bud and flower were lower and did not show specific patterns. The correlation analysis results showed, within a certain range, it did not show a linear relationship between gossypol content and the expression level of ABHcad in root tissue at different development stages, but presented "U" type or inverted "U" type.