Cloning Of Genes Related To Development Of Gland Of Gossypium And Genes Of The Key Synthase Of Gossypol Biosynthesis And Gossypol Analysis

Gossypol is not only the important resistant substance of Gossypium , but also a natural product which is of great value in medicine and industry. Gossypol, especially the (-)-gossypol, has the function of male antifertility, anti-cancer and anti-virus. But gossypol is toxic to human and non-ruminant animals. People anticipate an extraordinary kind of cotton with glanded root, leaf and stem but glandless seed (gossypol free) which could be used sufficiently, and the cotton could still keep its resistance. In order to control this secondary metabolism substance effectively, People have been engaged in the studies of gossypol and its storage organ-pigment gland for a long time. Up to now some cadinene synthases and their genes of the key synthase of gossypol in Gossypium arborium have been reported. Nevertheless, The study on the gland in molecular respect has not been reported yet. The cadinene synthase and its genes of Gossypium barbadense with high (-)-gossypol also has not been reported.On the basis of the comparing studies on gossypol content of different genetic types of pigment gland in the seeds of cotton newly bred, including the special and common glandless and glanded ones conducted through high-performance liquid chromatography (HPLC), we cloned and analyzed the cadinene synthase genes and their conserved domain of Gossypium barbadense and special Gossypium hirsutum resources resistant to Verticillium dahliae. Furthermore, we cloned the genes relatedhybridization (SSH).The comparing studies on gossypol content of different genetic types of pigment gland in the seeds of cotton newly bred, including 'the special and common glandless and glanded ones, were conducted through high-performance liquid chromatography on a CIS Column with methanol-0.5% acetic acid aqueous solution, 90:10 (v/v) as mobile phase at a flow rate of 0.8 m L min-1 and UV detection at 254 nm. This method was shown to be highly reproducible, with precision (as RSD) and accuracy (as RME) < 10%, both intra-day and inter-day, recoveries 100.4-103.2%. The results -revealed remarkable differences among the seeds of the different gland genetic varieties or species of cotton. The content of gossypol of the two special germplasms of G.hirsutum and the G. barbadeses were high; that of Bt transgenic materials experimented was obviously lower than the glanded resistant G.hirsutum; that of the special Xiangmian 18 with with glandless seed, but glanded root, leaf and stem was slightly higher than the common glandless ones, but still lower than the international food standard. The content of gossypol in the leaf in flowering stage of Xiangmian 18 was between the glanded and glandless cotton. The studies displayed the important resources of different gland of extraordinary cotton.Then we cloned the DNA and cDNA sequence of cadinene synthase of G. barbadense and special G. hirsutum resources resistant to Verticillium dahliae using RT-PCR method. Compared with the data of GenBank, the sequence of cadinene synthase of G. barbadense is different one from G. hirsutum and G.arborium, with identities of 97% and 99% respectively in nucleacide and protein of G.arborium, which belongs to cadinene synthase A.cDNA sequence of cadinene synthase of special G. hirsutum resources resistant to Verticillium dahliae is different from G.arborium, G.barbadense and other Gossypium hirsutum, with identities 99% in nucleotide and protein of G.arborium, which belongs to cadinene synthase C. The result of comparing analysis indicated that there was a little difference in the cadinene synthase genes between glandless and glanded Gossypium hirsutum, with identities 99%.We obtained cDNAs differentially expressed in the time of developing of the gland (stage II) using suppression subtractive hybridization(SSH). Two differentially expressed cDNA libraries of forward and the reverse subtraction were constructed. 24 cDNAs differentially expressed or preferentially expressed were obtained throughscreening the libraries. The result of virtual northern analysis veri...