Cloning And Functional Analysis Of Salt-stress Related Genes In Hordeum Brevisubulatum (Trin.) Link

Salt-stress caused by the salinization of soil is a main environmental factor restraining plant growth and yield, which severely restricts the development of modern agriculture. Studies on the molecular mechanism of plant salt- tolerant and cultivating new variety of plant resisting salt-stress are of great significance for developing and utilizing saline-alkali soil. Hordeum brevisubulatum (Trin) Link, an excellent forage with high nutritional value and yield, is of high performance great economic value. On the basis of previous researches on physiological and biochemical features of Hordeum brevisubulatum (Trin) Link and its salt-resistant mutant in our Lab, we probe into the salt-tolerant molecular mechanism of Hordeum brevisubulatum (Trin) Link, which is important for the further development of saline-alkali soil.cDNA library is a foundation for researching the structure, expression and function of genes. In this study, in order to study the expression of salt-resistance related genes in Hordeum brevisubulatum (Trin) Link, a cDNA library of Hordeum brevisubulatum (Trin) Link under salt-stress was constructed using ZAP expression vector. The recombinant rate of the cDNA library was 96%, the liter was 2× 106pfu/ml and the amplification titer was 1.8×10 pfu/ml, the range of insert fragment was 0.5kb~3kb.It was found that betaine aldehyde dehydrogenase (BADH) gene is one of the stress-tolerant genes. Therefore, primers were designed according to homogeneity of BADH. The probe HDBL of 613bp was obtained by RT-PCR. The probe was DIG labeled after homologous comparison. The cDNA library of Hordeum brevisubulatum (Trin) Link under salt-stress screened by the probe and a positive cDNA clone obtained. The full-length of the cDNA was 1776bp with the open reading frame lacated from 85bp to 1602bp, which encoding 505 amino acid residues. Its 5' UTR is 85bp and 3' UTR is 174bp which contains polyA signal (AATAA) and polyA tail. Its GenBank Accession Number is AY 188952. Analysis on amino acid homogeneity revealed that the homology was 95.4% with Hordeum vulgare. A typical aldedh structure domain was found in the protein analyzed with SMART. Southern hybridization revealed two copies in Hordeum brevisubulatum (Trin) Link genome with HDBL probe. Northern hybridization showed that the expression of HDBADH is salt-induced. The expression of BADH increases 3-4 folds under the stress of 2% NaCl. With a binary expressing vector pBin438, a plant-expressing vector PHDBL was constructed. And then HDBADH gene was converted into tomato via a leafregeneration system. It was demonstrated by PCR that five transgenic tomato strains were obtained and the transgenic tomato can radicate and grow in medium with 100mmol/L NaCl.mRNA differential-display reverse-transcription polymerase chain reaction (DDRT-PCR) is an effective and quick method to study gene different expression in the same cell under different physiological status and different stages of growth and development. In this study, fluorescence DDRT-PCR was used to compare mRNA from leaves under salt-stress and non-salt-stress. 96 different fragments were obtained altogether, of which the re-amplified fragments were found in 91 different cDNAs and the re-amplification rate was 94.79%. Cloning, sequencing and reverse Northern hybridization showed that 75 fragments were positive and the positive rate was 82.43%. Analysis with BLAST and DNAsis software revealed 32 cDNAs sequences related with salt-stress, which were all present to GenBank, and accession Number was obtained. Those cDNAs were of three types: 22 of the induced expression; 7 of the enhanced expression and 3 of the depressed expression. Analysis on homogeneity revealed that 13 cDNAs were homologous with the reported genes and the other 19 cDNAs were new ESTs.On the basis of HDL2 cDNA sequence of the induced expression type from DDRT-PCR, 852bp of full-length cDNA was successfully obtained by RACE technique with the open reading frame (ORF) is located from 67bp to 570bp, which encodes 167 amino...