| The content of secondary metabolite is closely related to the quantity of storage organ. Study on the molecular mechanism under the development of the storage organ can provide the theoretical direction for the effective agricultural practice. As the result, it is meaningful to study the correlation between the secondary metabolites and storage organ. As is well known, Gossypol and gland of cotton have been a good model for the research of genetic engineering.Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. More interestingly, this ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level.In this study, the material we used is Xiangmian18, a mutant which had the special property of delayed pigment gland morphogenesis, i.e., the glands in the leave appeared to be normal in the plant, but the seeds have low gossypol level. Firstly,we studyed on the characteristics of gland development and detected the changes of the gossypol content and antioxidant enzyme activity. Secondly, differentially expressed genes were screened by high throughput screening technology based on difference in gland of cotton. Furthermore,we established a normalized and full-length cDNA library of its pigment formation phase and cloned candidate genes related gland formation followed by bioinformatics analysis of the selected genes. The results may pave the way for the further investigation the molecular mechanism of gland formation and the identification of genes directly related to gland formation.The main results of this research are as follows:1.The observation to gland formation and study on the resistance-related physiological parameters in cotton.The glands were formated after 48h germination in xiangmian18. In chuanmian2802 new gland grew after 36h germination. The diameter became bigger during the gland foramtion.The content of gossypol decreased during the early germination and rised slowly after 5d gernmination in chuanmian2802. The content showed a slow body growth tend in xiangmian18, consistent with chuanmian2802.The content of soluble suger and the content of soluble proteins tended to decrease in general during early germination of cotton and rise gradually after a weekly germination.The activity of POD,CAT and SOD increased during germination of cotton. The activities of antioxidant enzyme were highest in chuanmian2802, xiangmian18 was in second place, and xianwuN5 showed low activites.2. High through-out analysis of pigment gland formation related genesTo reveal features of cotton gland development, the transcriptomes on several mutantants during the gland development have been compared. We studied gene expression profiles of Xiangmian18, chuanmian2802, XianwuN5 during cotton gland development using Affymetrix cotton GeneChip array that contains probes for about 23,977 genes. Comparative analysis among the post-gland formation, the undevelopment gland of Xiangmian 18 and chuan2802, the samples showed that 112 genes were up and 191 were down-regulated. Comparative analysis among chuanmian 2802, the post-gland formation, Xiangwu N5, the samples showed that 218 genes were up and 331 were down-regulated. These genes were classified into six major GO categories relevant to such biological processes as protein polymerization, microtubule-based, lipid biosynthetic process movement, response to oxidative stress, photosynthesis gene. Among them, 6 genes have been further confirmed with RT-PCR and the results were in accordance with the Genechip's data. These genes were regulated by each other, playing roles directly or indirectly in the gland formation of cotton.3. Establishment of a normalized and full-length cDNA library at the stage of pigment gland formationA normalized cDNA library was constructed from upland cotton during its pigments gland forming age with SMART (Switching mechanism at 5 end of RNA transcript) technique and DSN technique (Duplex-specific nuclease). mRNAs were isolated from upland cotton;Then double-stranded cDNAs were synthesized from mRNAs and processed by normalization; digested with SfiI; and then the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E.coli JM109 by electroporation. Tittering results revealed that approximately 5.86×105 independent clones were included in this library. Electrophoresis gel results fragments ranged from 800 bp to 2 kb, with an average size of 1400 bp. Random picking clones showed that the recombination rate was 94%. Our successfully constructed cDNA library is a full-length library with high efficiency, and can be used to identify the forming genes related to development of pigments gland cottons.After normalizing, abundant classes of different genes tended to equalize, which can increase the chance to discover new genes.4 Cloning and analysis on the representative pigment development related gene GAP1.Based on the result of screening cotton microarray, the primers were designed according to DW513956. Using PCR to screen the constructed library, we got full length cDNA, named GAP1 (gland related protein1, GenBank number: EU3703012) The complete nucleotide sequence of GAP1 cDNA has 1203 bp and contains a long open reading frame of 747 bp. The predicted protein sequence derived from the open reading frame produces a 248-amino acid polypeptide, with a calculated molecular mass of 33.09 KD and a theoretical iso-electric point of 8.99; Protein subcellular location was predicted in the cytoplasm; the protein is hydrophobic property with a signal peptide, a peroxidase domain. There were 75%, 75%,73% homologic comparison with Nicotiana tabacum, Vigna angularis, Euphorbia pekinensis respectively.In situ hybridization was performed to analyze the expression pattern of GAP1 gene during the xiangmian18 different organs. The results showed that at the stage of gland formation in the seed germination, GAP1 gene expression at the gland development was stronger than gland no-development. The experiment showed that this gene was tissue specific. This gene was highly expressed in the vessel of stem cotton and phylogenetic of the leaves.5. Cloning and analysis on the representative pigment development related gene GhZIF.Based on the result of screening cotton microarray, the primers were designed according to DT463838. Using PCR to screen the constructed library, we got full length cDNA, named GhZIF (gland related protein 2, GenBank number: EU3703033) respectively.The complete nucleotide sequence of GhZIF cDNA has 1989 bp and contains a long open reading frame of 1464bp.The predicted protein sequence derived from the open reading frame produces a 487-amino acid polypeptide, with a calculated molecular mass of 53.75kD and a theoretical iso-electric point of 5.79; Protein subcellular location was predicted in the plasma membrane. The protein is hydrophobic property, which anchors membrane, containing a transporter domain.In situ hybridization was performed to analyze the expression pattern of GhZIF gene during the xiangmian18 different organs. The results showed that at the stage of gland formation in the seed germination, GhZIF gene expression at the gland no-development was stronger than gland development. This gene was highly expressed in the vessel of stem cotton,phylogenetic of the leaves and parenchyma cells.Therefore, the solid foundation had been laid for finding relevant genes and investigating their functions of cotton gland formation. It is significant to control the gland and the regulation and biosysthsis of gossypol at molecular level.
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