Cloning Of Gene(s) Induced By Salt Stress & Analysis Of Its Function In Wild Barley (Hordeum Brevisubulatum (Trink.) Link.)

The research aim of this study is to obtain the gene(s) relevant to salt tolerance, protect the property right for stress resistance genes resource in our country and develop salt-tolerant crop variety by gene engineering.Total RNAs from the leaves treated 24hrs with 430mM NaCl(T) and control(CK)were extracted in wild barley (Hordeum brevisubulatum (Trink.) Link.) .The difference of mRNA transcriptions was detected using suppression subtract hybridization (SSH). 449 of differentially expressed cDNA fragments were cloned for composed of cDNA library and identified by reverse Northern bloting. Compared to the control, 18 of ESTs expressed under 430mM NaCl treated in wild barley and were sequenced. Two of them were repeated to have same sequence each other. One of EST Bst 16 with poly A was selected to do RACE for obtaining the full length cDNA. So 17 of ESTs and a full length cDNA were analyzed and forecasted their function in public database of GenBank.Results of BLAST analysis in GenBank showed that the salt tolerance content of many processes of plant biochemistry and physiology and are controlled by multi different genes in plant. Among the 17 ESTs, Bst3, Bst9, Bstl 8 were found to be related with barley chloroplast genes rbcL, which induced by osmotic stress and cold stress; Bstl, Bst6, Bst17, Bst22 were relevant to the protein expressed specially in salt stress; Bstl3 and Bstl9 were relevant to molecular chaperone protein, which can help keep activity of protein under stress condition; Bst4 encoding membrane protein would be protein kinase; Bst5 was forecasted to encode partial gene nad2 for dehydrogenas(NADH); Bst8 was found to have high homologous with phosphoethanolamine methyltransferase in common wheat (Triticum aestivum L.); Bst10 was found to have high homologous with ATP-dependent Clp protease ATP-binding subunit precursor cDNA in rice (Oryza sativa) and Arabidopsis thaliana ; Bstll was found to encode cAMP&cGMP -dependent protein kinase, which was related with signal transfer in plant; Bstl2 was found to have homologous with gene encodeing isocitrate lyase; Bst20 encoding protein translation factor was found to be relevant to protein synthesize process.The full length cDNA BST16 was consisted of 601bp to have an ORF from 93 to 515including initiator codon and termination codon and to encode one 140 amino acid polypeptide. Comparing with database in GenBank and TIGR, the results showed BST16 had 90% homology to mRNA for a known photosystem II (PSII) lOkD protein in barley (H.vulgare), 65% homology to PS II polypeptide in Arabidopsis thaliana and 94% homology to wheat drought-stressed cDNA clone. BST16 was also proved to encode gene of PSII lOkD protein by protein prosite and conserve domain analysis. The protein encoded by BST16 contained a conserve domain PsbR of PSII lOkD protein from 48 to 140 and its carboxy terminal had an ADH_SHORT prosite of Short-chain dehydrogenases/reductases family signature from 94 to 122, which showed the protein encoded by BST16 would be a reductases. All above showed that the full length cDNA BST16 encoded PSII lOkD protein and could be induced by abiotic stress factors and its function was relevant closely to photosynthesis efficiency and salt tolerance.