高温胁迫下紫花苜蓿抑制消减文库的构建

Construction of differently expressed cDNA library of alfalfa by using suppression subtractive hybridization under the high temperature

为分离和获得紫花苜蓿高温胁迫诱导相关基因片段的EST序列。以秋眠6级的紫花苜蓿品种(ABI 700)为材料,以45℃高温胁迫下的紫花苜蓿幼嫩茎叶cDNA为试验方(tester),以正常生长(22℃)的紫花苜蓿幼嫩茎叶cDNA为驱动方(driver),利用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建了高温胁迫下紫花苜蓿幼嫩茎叶的正向抑制消减文库。从消减文库中随机挑取120个阳性克隆,进行菌液PCR鉴定。结果显示,消减文库的滴度为4 230pfu/mL,重组率为97.8%,插入片段大小主要集中在300～750bp。本研究为紫花苜蓿抗热基因克隆和系统研究高温胁迫下紫花苜蓿基因的表达奠定了一定的理论基础。

In order to isolate the high-temperature stress-induced ESTs from alfalfa,we took the alfalfa variety＇ABI 700＇as material whose fall dormancy level was 6,and 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 300-750 bp.This research of the alfalfa differential expression has laid a theoretical basis for the cloning of heat-resistance gene and the gene expression under high temperature.