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Construction Of Differentially Expressed Cdna Library Of Galega Orientalis Under Salt Stress By Suppression Subtractive Hybridization And The Clone Of Ca2+/H+ Antiporter Gene

Posted on:2011-02-18Degree:MasterType:Thesis
Country:ChinaCandidate:Q X SongFull Text:PDF
GTID:2143360305985452Subject:Grassland
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Galega orientalis is an important forage introduced species, in many ways to show superior performance, but in the promotion of regional impact due to soil salinization, to a large area planted under a lot of restrictions. Our study is to clone and split with the Galega orientalis salt-induced genes, in order to reveal the molecular mechanism of salt tolerance of Galega orientalis, and provide the basis for their salt tolerance breeding. The results of this study are as follows:1. Library was constructed by suppression subtractive hybridization (SSH) using cDNA from Galega orientalis. The library contained 92% recombinants with insert size ranged from 0.2 to 1 kb.2. Five hundred positive clones were randomly sequenced and analyzed, 258 cleanESTs were obtained. By the aid of cluster and phrap software, 132 Unigene were identified, consist of 32 contigs and 100 singlets. The redundancy rate of the library is 24%. Function prediction analysis classified them into several sections, such as signal transduction, transcription regulation, osmotic and metabolic regulation, host defense, and the photosynthesis.3. The subtraction efficiency of the SSH cDNA library was confirmed by randomly testing 4 independent sequences using semi-quantitative RT-PCR, of which differential expression were detected. These results suggest that SSH is an effective technique to identify novel genes in Galega orientalis.4. RACE were proceed utilized the EST sequence homologous to Medicago truncatula in library.We clone a gene of 1769bp.We name it GoCAX.Which has a ORF of 1377bp and contain 458 aa. Its homology plant is Medicago truncatula, go to 91%.5. Protein sequence analysis showed that: There is no Signal peptide,Hydrophobic amino acids is in the peptide chain, significantly more than the hydrophilic amino acids, Which may be inferred that the performance of the entire polypeptide chain is hydrophobic, which is similar to other known CAX proteins. Secondary structure analysis showed that:β-turn and random coil is the larger proportion; transmembrane fragment analysis showed that there is eleven transmembrane segments.
Keywords/Search Tags:Galega orientalis, Salt treatment, Suppression subtractive hybridization (SSH), Expressed sequence tags (ESTs), RT-PCR, Ca2+/H+ antiporter protein, RACE
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