干旱胁迫下斑茅消减文库的构建及分析  

Construction and Analysis of Suppression Subtractive Hybridization Library for Saccharum arundinaceum Retz.Leaves Exposed to Drought Stress

作　　者：刘文荣[1] 张积森[1] 饶进[1] 蔡秋华[1] 翁笑艳[1] 阮妙鸿[1] 阙友雄[1] 陈如凯[1] 张木清[1] 

机构地区：[1]福建农林大学农业部甘蔗生理生态与遗传改良重点实验室,福建福州350002

出　　处：《作物学报》2007年第6期961-967,共7页Acta Agronomica Sinica

基　　金：国家高技术研究发展计划(863计划)项目(2001AA241191);国家自然科学基金项目(30370901);教育部高等学校博士学科点专项科研基金项目(20040389009)

摘　　要：以干旱胁迫下斑茅（Saccharum arundinaceumRetz.）叶片组织的cDNA为Tester,正常生长条件下斑茅叶片组织cDNA为Driver,利用抑制性消减杂交技术（Suppression Subtractive Hybridization,SSH）构建斑茅干旱胁迫消减文库。文库克隆总量约为30 000个,克隆重组率为99.2%;随机从文库中挑取3500个克隆分析表明,插入片段长度主要集中在200-1 000 bp之间,500 bp以上的有463个克隆,500 bp以下的有3 037个克隆,平均长度约450 bp;通过随机对16个文库阳性克隆的测序及分析,3个克隆与ATP-NAD kinase、Aldo/keto reductase和锌指蛋白高度同源,3个克隆没有同源性,10个与逆境相关,是功能未知的EST。选择编号为EL0149和EL0145两个克隆进行Northern杂交验证,结果表明EL0149克隆为干旱胁迫上调表达的EST序列,而EL0145在正常供水和干旱条件下均无明显的的杂交信号。说明本研究克隆的SSH文库质量较高,可以进一步利用SSH文库结合芯片表达谱分析作物水分胁迫下的相关抗旱基因网络。Saccharum arundinaceum Retz., one of the most important wild relatives of Saccharum offwinarum L., is a kin species with desirable attributes including excellent vigor and ratooning, adaptability to environmental stresses such as moisture deficits or excesses, and resistance to Pachymetra root rot for sugarcane genetic improvement. Efficient construction of cDNA libraries enriched differentially expressed transcripts is the most important step in biological investigations. Suppression subtractive hybridization （SSH） is a highly effective method for the generation of subtracted cDNA libraries. It is based primarily on a described technique called suppression PCR and combines normalization and subtraction in a single procedure, in which the normalization step equalizes the abundance of cDNAs within the tester population and the subtraction step excludes the common sequences between the tester and driver populations. An SSH library was constructed from the cDNA of leaves in S. arundinaceum, subjected to water stress as Tester and from cDNA of the leaves in S. arundinaceum, planted in the normal condition as Driver. Titer results indicated that about 30 000 clones were included in the library, and the percentage of recombination was up to 99.2 %. Further, 3 500 clones were picked out randomly from the SSH library for cDNA microarray analysis. The size of inserts ranged mainly from 200 bp to 1 000 bp, of which 463 clones were larger than 500 bp and 3 037 clones smaller than 500 bp, with 450 bp on an average. After picking up randomly 16 clones for sequencing, the results by using BLAST revealed that there were three drought-stressassociated genes （ATP-NAD kinase, Aldo/keto reductase, and zinc finger protein）, three novel genes and other 10 function-unknown expressed sequence tags（EST）. Two out of 16 clones, which were termed as ELO149 and ELO145, were chosen for further analysis. It was also confirmed by Northern blot that ELO149 up-regulated in the moderate water stressed leaves, while no hybridization si

关 键 词：斑茅 干旱胁迫 抑制性消减杂交 表达序列标签 

分 类 号：S566.1[农业科学—作物学]