Genes Cloning Of Salt Tolerance On Algae And Its Preliminary Research Of Transformation On Cotton

With the more and more arable land becoming saline worldwide, crop genetic improvement of salinitytolerance has become a mainstream research direction to effectively develop and utilize saline-alkali soil.As the pioneer crop on saline land, cotton （G.hirsutum L.） was particularly studied worldwide to cultivatesalt-tolerant varieties by molecular means and genetic engineering techniques to improve the salt tolerance.Two salt-tolerance-related genes, carbonic anhydrase gene （CA） and glyceraldehyde-3-phosphatedehydrogenase gene （GAPDH）, screened from Laminaria japonica and Dunaliella salina respectively, withcomplete sequences of genes information from NCBI library, were cloned by RT-PCR technology withbioinformatics analysis and sub-cellular localization by GFP.26accessions of cotton varieties were used asthe materials to study to detect the spontaneous phenomenon of green fluorescent on cotton pollen. Geneexpression analysis was conducted by gene gun to transform cotton in vivo on farm. Cotton seed of T₀generation was harvested to identify salt tolerance and molecular detection. The main research results wereas follows:1Complete ORFs of two salt-tolerance-related genesThe complete sequences of two salt-tolerance-related genes were screened from NCBI library ofLaminaria japonica and Dunaliella salina respectively, with the ORFs of the two salt-tolerant gene clonedby RT-PCR technology. The complete open reading frame of CA gene from Laminaria japonica was873bp,encoding polypeptide of290amino acid. The complete open reading frame of GAPDH gene fromDunaliella salina was1131bp, encoding376amino acids.2Bioinformatics analysisBioinformatics analysis showed that CA protein had the similarity of the89%,79%,78%and70%toDunaliella salina, Oryza stiva, Sinorhizobium meliloti and Oncorhynchus mykiss, The phylogeneticanalysis showed CA was the closest to Dunaliella salina, The secondary structure prediction showed thatthe protein was composed of α-helix, β-sheet, turn and random coil. Bioinformatics analysis showed thatGAPDH protein had the similarity of the90%,92%,96%and97%to Arabidopsis thaliana, Medicagotruncatula, Zea mays, Vitis vinifera respectively. The phylogenetic studies showed that GAPDH was the closest to Volvox carteri.3Sub-cellular localization for protein encoded by genepBI121-CA::GFP and pBI121-GAPDH::GFP,the fluorescence vectors of plant expression,wereconstructed and transformed into onion endepidermis cells by gene gun. The result showed that the CAprotein was expressed and located at the cell nucleus, GAPDH protein was located at the cell membrane.4Instantaneous expression analysis of the cotton pollenThe spontaneous phenomenon level of the was fluorescencewere was detected by the pollen of26accessions of cotton, among which23accessions of cotton were eliminated and ruled out because of thestronger level of the spontaneous green fluorescencewere, and3accessions of cotton with the weakspontaneous green fluorescent were changed by CA gene transformed by gene gun than untransformed, Thetransient expression analysis showed that the green fluorescence of3accessions of cotton were enhancedby CA gene, which proved the feasibility of transgenic plants in vivo transforming pollen by gene gun andlaied the foundation for the next step experiment.5Gene transformation of cotton by gene gunEukaryotic expression vectors, pBI121-CA and pBI121-GAPDH, were constructed and transformatedinto the cotton materials by gene gun.389bolls of T₀transgenic generation were harvested.6Salt-resistant screening of T₀generation of the seedsSalt-resistant screening was conducted with0.8%NaCl solution by the double the sandwich filterpapers method. The germination rate of CA transgenic cotton seeds was22.2%of162seeds, and thegermination rate of GAPDH transgenic cotton seeds was16.3%of490seeds, which were all higher thanthe control germination rate of0.08%of120seeds.7Molecular detection on DNA levelAfter the detection of salt-resistant, the germination and non-germination of seeds were nurseried andtransplanted to the greenhouse for the further molecular detection. The CA transgenic seedlings weredetected that12plants had specific bands, with the conversion efficiency of7.4%, the GAPDH transgenicseedlings were detected that26plants had specific bands, with the conversion efficiency of5.3%; The tworelatively bright strips of the GAPDH transgenic were selected, and the PCR product of the two strips weresequenced directly, Comparative analysis of sequencing results showed that the two cotton individuals were positive and were transformed successively.