Screening And Cloning Of Alfalfa Genes Induced By Salt Stress

Soil salination is one of the main restriction factors to crop yield and quality. In plants, salt tolerance is controlled by quantitative traits with polygene. Recently, though a large development in salt-tolerance research in plants, the mechanism is still not known clearly. Through screening and cloning genes related to salt stress, this assay laid a foundation for description of salt-resistance manchanism in alfalfa (Medicago sativa) and alfalfa salt-tolerance breeding. The main results obtained are listed bellow:1. Salt-stressed and control alfalfa cDNA were amplified by SMART cDNA PCR. Product with special bands and smear mostly distributed among 0.7-2.0kb; it was matched with most species′mRNA. After amplification, suppression subtractive hybridization was followed and a differentially expressed alfalfa cDNA library containing 810 clones was constructed. The length of insert fragments in library was between 250bp and 750bp which is expected.2. 119 positive clones were acquired in library by reverse Northern dot-blotting. After sequencing, 82 uniESTs containing 16 contigs and 66 singlets were acquired in 110 available ESTs. 82 uniESTs were submitted to GeneBank and corresponding accession numbers from FE896855 to FE896936 were obtained.3. The result of BLASTn showed that 45 ESTs had homologous genes with function known clearly, 37 ESTs′homologous gene with function described unclearly, 4 ESTs had no homologous sequence. Statistic analysis showed that myo-inositol-1-phosphate synthase, ribulose-1,5-bisphosphate carboxylase, lipid transfer protein, aquaporin, galactinol synthase, cold-induced special protein and dehydrin had the highest frequency. These ESTs can be classified into 9 groups by their founction through the result of BLASTx. The 9 groups contained material and energy matebolism, transcription, protein fate, binding protein, signal transduction, organism defence and self-rescue, development, transport, unclassified, function-unknown.4. 3′-RACE and 5′-RACE were proceed utilized the EST sequence homologous to soybean pGmPM10 (accession No.: AAA91965.1) in library. And then, a gene with a 1728bp cDNA was cloned and initially termed as MsLEA3-1 (genebank accession No. EU665182). Its maximum ORF code a protein containing 436 aa, with a 5.18 pI and a 47.0 KD MW. BLASTp showed that the amino acid sequence of MsLEA3-1 protein has the most homologous to soybean pGmPM10 (E value=1e-108).5. Bioinformatics analysis of amino acid sequence reviewed that: 1). Amino acid composition and content of MsLEA3-1 was similar to other LEA proteins. The very high contents of small and hydrophilic residues result in high hydrophilia, while the contents of cysteine and tryptophane were low; 2). There were 31 11-mer repeats in aa sequence, which was the common characteristic of group 3 LEA protein. The consensus sequence of the 11-mer repeats is TKDYAGDAAEK; 3). The aa composition and distribution of the 11-mer repeat was extremely similar to soybean pGmPM10 & pGmPM8, but different from type I and Type II of group 3 LEA protein. 4). There was an evidence signal peptide in the N-end of aa sequence and a ER-residue signal liked sequence in the C-end. It was proposed that the MsLEA3-1 functioned in endoplasmic reticulum. 5). Secondary structural prediction reviewed that theα-helix was the main structural. The amphiphilicityα-helix formed by 11-mer repeats may be important to cells under dehydration.