| An important process of plant physiology is plants-photosynthesis that green plants use chlorophyll under visible light irradiation, the carbon dioxide and water into organic matter, and release oxygen. Photosynthesis is a process of the energy savings and organic synthesis. This process is crucial for plant survival, and photosynthesis is essential for the whole ecosystem carbon cycle. The previous experiment make the different producing areas of Yunyan 87 tobacco leaves as experimental material, using methods of comparative proteomic to explore the differential proteome of tobacco in different ecological regions, and make a leaf proteomics research of four representative characteristic flue-cured tobacco ecological zones from Yibin, Guangyuan, Xichang and Miyi,. Tobacco leaf in Miyi ecological region indicating in protein Marker in SDS-PAGE electrophoresis a significant differences around the size of 17KDa of strip. And then identified five proteins include in a two-dimensional gel electrophoresisidentified from the differential band, they are phospholipid hydroperoxide glutathioneperoxidase, cytochrome, thioredoxin, ubiquitin and carbonic anhydrase. Carbonic anhydrase (CA) is an enzyme that is associated with photosynthesis, according to research found in the literature, it had a significant correlation between the carbonic anhydrase activity of soybean, maize and rice plant and net photosynthetic rate, and carbonic anhydrase activity have great effects on net photosynthetic rate, however, associated with carbonic anhydrase in tobacco is seldom reported. This study cloned early test from differences in the strips of carbonic anhydrase gene, and made analysis of the gene by bioinformatics and functional The main research is as follows:(1) By homology cloning method, NtCA gene was cloned from NtCA homologue, show that the length of the coding region of the gene was 525bp, encoding 174 amino acids,1 carbonic anhydrase domain, belonging to the beta -CA. Homology analysis found that, the gene sequenceand Japanese tobacco with 97% similarity; and tomato with 90% similarity; and potato with 89% similarity; and Sesame with 78% similarity.Cloning of sequences and other scholars to login to NbCA sequence in the same plant, that in the very high degree of gene conservative.(2) The physico-chemical properties of the CA protein, protein isoelectric point is 5.92, CA proteinmolecular weight is 34.51 KD, chemical formula C858H1334N222O248S9, instability index is 55.11, the half-life of 30h, aliphatic index was 95.25, the total average hydrophobicity (GRAVY) of 0.262, by GRAVY know, tobacco CA protein GRAVY values between 2 and -2, and negative, the CA protein is a hydrophilic protein.(3) Predict the secondary structure of carbonic anhydrase by using the IBCP online tools,results as follows:the enzyme protein secondary structure is mainly composed of 63 amino acid residues accounted for 32.20% of the alpha helix (alpha helix),62 amino acid residues accounted for 29.25% of random curl (random coil), extending chain (extended strand) 27.83%, is composed of a small amount of amino acid residues beta corner accounted for 13.21%. Using homology modeling forecasting the three-dimensional structure of tobacco CA protein, and it is concluded that the tobacco carbonic anhydrase type belongs to the beta carbonic anhydrase.(4) The construction of pBI121-NtCA expression vector, transferred it into Agrobacterium LBA404,through the colony PCR identifiCAtion of positive clones. Using the leaf-disc transformation method for transgenic manipulation, was obtained by screening for transgenic strains, and lay a solid foundation for the further functional research.(5) Using the PCR technology, detection of tobacco CA gene expression in different tissue is found that the plants of leaf, stem, flower is not significant difference in the expression of quantity, therefore, carbonic anhydrase in tissue expression may not specificity.and transgenic plants with CA gene expression quantity is higher than that of non-transgenic plants. |
