The Cloning And Analysis Of Salt-tolerant Gene From Mangrove

Cell accumulates organic osmotic regulators in the cytoplast (Mannitol, trehalose, betaine and praline, for example), while inorganic osmotic regulators in the vacuole from the cytoplast (one and most of them is K⁺), when the plant was stressed by salt or water. That will keep osmotic balance between the cytoplast and outsides (vacuole), avoiding the ion poison to the enzymes and metabolize when high concentration ion in the cytoplast. Beside keeping the cell normal swell press, the glycinebetaine(thereafter GB) can be a innocuity osmoprotectant and stablish the high structure of complex proteins. So the main enzymes in metabolize may keep activity under osmotic stress.In plants, it has reported that GB is synthesized by two steps. The second step is catalyzed by betaine aldehyde dehydrogenase (BADH). Betaine aldehyde dehydrogenase is a rate-limiting enzyme, and the effective expression of BADH gene will enhance the plant's salt-tolerance for referring to the betaine's increase. Researches about BADH gene were reported after the BADH gene isolated for the first time from high plant in 1981. cDNAs of BADH gene were cloned from E.coli, yeast and kinds of high plant. Monocotyledon's BADH genes are located on microsome and dicotyledon's BADH genes are located on choloroplast.This study aimed to explore the clone and analysis of BADH gene of mangrove. Main results are followed:1. The total RNA was extracted from Avicennia marina using CTAB, tested by 1.0% agrose gel electrophoresis and OD₂₆₀,OD₂₈₀. The strength of 28S rRNA was over twice to the 18S rRNA from the electrophoresis. The value of OD₂₆₀/OD₂₈₀ was very close to 2.0. It indicated that the total RNA we extracted had good integrality and high purity for non-degradation and low impurity.2. We got the cDNA with RNase from the extracted total RNA. A fragment about 250bp was amplified using a pair of degenerate primers by touch down PCR. It had a 80% homology to the reported BADH gene of Avicennia marina.3. Based on the fragment above, we designed primers and processed 3'RACE and 5'RACE respectively to amplified the unknown 3' terminus and 5' terminus of BADE gene. About 350bp(3'RACE) and 1.5kb(5'RACE) special fragments was amplified. Results of sequencing and analysis indicated that we obtained successfully the 3' RACE and 5' RACE of BADE gene.4. A pair of gene special primers was redesigned and a 1.5kb fragment was amplified from the cDNA reversed from the total RNA. The fragment was confirmed after sequencing and analysis. The coding section of BADE gene was 1509bp and coded a polypeptide of 502 amino acids. Analysis of BADE gene indicated high homology to other BADE gene, and over 90% homology to the reported Avicennia marina BADE gene.5. The BADE gene was ligated to pGBKT7 with T4 Ligase, and the recombined plasmid was transformed to Saccharomyces cerevisiae AH109. The tolerance to NaCl of recombined Saccharomyces cerevisiae AH109(pGBKT7-BADH) enhanced to 14% from 9%. It showed the protein transcriped and translated in recombined Saccharomyces cerevisiae AH109(pGBKT7-BADH) has biological activity.