Preliminary Study On The Tissue Culture And The Na～+/H～+ Antiporter Gene Of Zostera Marina L.

Zostera marina L .is a marine higher angiosperm, which lives in the sea water with the salinity of 600mmol/L. Its salt-tolerance is obvious and therefore Zostera marina L . is one of the absolutely good objects for the research on the salt-tolerance mechanisms of the higher plants. Na~+/H~+ antiporter is the antiport of Na~+, which distributes in both the plasma membrance and the vacuole membrane. It plays an important role in plant salt tolerance. In this paper, Zostera marina L. is utilized as the research object for the preliminary study on its tissue culture and Na~+/H~+ antiporter gene. Firstly, the limited factors about Zostera marina L.' life in the sea water, the optimal tissue or organ for the tissue culture , the optimal hormone combination and concentration is determined by means of the transplantation of Zostera marina L. and its tissue culture. The experimental result shows that the main determinative factor is the temperature of the sea water for the growth after the transplantation; the optimal temperature for the growth of Zostera marina L. is 10~20℃; it is strongly salt-tolerant and it can grow at the salinity range of 20‰~48‰ normally; its rhizome is the optimal organ for culturing and the optimal hormone concentration for the calluses induction is 2mg/L or so. Secondly, the genomic DNA is extracted from Zostera marina L. and then amplified by PCR with two oligonucleotide primers synthesized to obtain the partial nucleotide sequences of the Na~+/H~+ antiporter gene. The Na~+/H~+ antiporter gene clone was obtained after a series of manipulation: purificatioin of PCR product, ligation and transformation of recombinant plasmids, blue-white selection and so on. DNA sequence analysis shows that its DNA sequence is highly homologous with the genomic DNA sequence of E. coli. It is probably due to the mistake that happens when the product is ligated to the vector for sequencing or the mistake that happens when the product is sequenced. Perhaps the result of the sequence is the sequence of the bacterium. Then in order to obtain the genomic DNAclone of the Na+/H+ antiporter gene, some measures should be taken, for example that the sample product to be sequenced should be purified or the primers for the cloning should be redesigned. At the same time, based on the extracted total RNA from the leaves of Zostera marina L., the cDNA clone could be obtained and the Na+/H+ antiporter gene could be studied by applying the techniques of RT-PCR and RACE. Its expression profile could be studied when applying the Northern technique. The research on the Na+/H+ antiporter gene from Zostera marina L. can help to find the structural and functional difference of the different plant Na+/H+ antiporter gene and help to mastering the salt-tolerance mechanisms of the Na+/H+ antiporter gene. In addition, the Na+/H+ antiporter gene from Zostera marina L. could have the strong ability for the Na+ anti-transporting. It will hold an application meaning when the crop salt-tolerance is enhanced after they are transgened with the Na+/H+ antiporter gene from Zostera marina L. Moreover, the research on the conditions for the transplanting and culturing of the Zostera marina L. will make the other researches on Zostera marina L. more convenient, which include the breeding > the gene screening of Zostera marina L and so on.