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Construction and Primary Analysis of Differentially Expressed cDNA Library of Alfalfa by Using Suppression Subtractive Hybridization under the High Temperature

Author: HanMingPeng
Tutor: GaoYongGe;WangChengZhang
School: Henan Agricultural University
Course: Grassland
Keywords: Alfalfa Suppression subtractive hybridization High temperature stress Differentially Expressed Genes cDNA library
CLC: S541.9
Type: Master's thesis
Year: 2011
Downloads: 5
Quote: 0
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Alfalfa(Medicago sativa L.), known as the King of Grass, has the longest cultivating history and the largest planting area in the world among leguminous forage kinds. Alfalfa, with a high yield, rich in protein, nitrogen-fixing ability of biological characteristics and wide adaptability plays a significant role in the development of animal husbandry and food safety in China. High temperature damage, becoming more serious with the growing temperature caused by human’s increasing activities. To alfalfa can not only decrease its rate of growth and make it blasted or even dead, but can also increase the plant diseases and insect pests which directly limits its growth and seriously affect the production and quality.Researches recently mainly focus on high temperature affection to alfalfa, mechanism of adaptation to high temperature and methods to choose evaluation index of heat resistance and to evaluate reliability of the index. However, study on searching heat resistance gen of alfalfa are rare. Basing on mRNA differential display technique, suppression subtractive hybridization (SSH) technology was created by Diatchenko etc in 1996 to screen unknown differences gene. SSH can be used to obtain cDNA fragments of differentially expressed genes in a relatively short period of time and to enrich rare gene in more than one thousand times which will make it possible detect some certain expression of mRNA in low abundance.In order to isolate the high-temperature stress-induced ESTs from alfalfa, we took the alfalfa cultivar ABI 700 as material whose fall dormancy level was 6, taking the cDNA from alfalfa tender stems and leaves under 45℃as tester, while taking their cDNA under 22℃(normal growth ) as driver. The forward suppression subtractive library of alfalfa under high temperature stress was constructed by using suppression subtractive hybridization. Choosing 120 positive clones stochastically from the subtractive library to take bacterial PCR, it displayed the recombination rate of library clones was 97.8%. The size of inserted fragment was mainly between 300-750bp.Randomly picking out 120 white spots from the subtractive cDNA library, 117 differentially expressed clones were screened by colony PCR. 48 EST sequences of effective clones from the 111 acceptable quality EST sequences retained after being sequenced and being merged between the same fragments. According to the intercomparison results between GenBank from non-redundant nucleic acid database (nr) and BLASTn sequence from EST database, these EST were divided into three kinds: the first kind represented a total of 19 known genes, the second represented a total of eight known ESTs and the third kind represented a total of the 21 new ESTs. Representative of known genes, ESTs, according to gene function, can be divided into seven categories: signal transduction, cell growth / apoptosis, resistance / organism defense, physical transport, transcription regulation, metabolism and function to be determined and so on.The basic theory for researching cloning of heat-resistance gene and the gene expression under high temperature has been offered in this paper.

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