干旱胁迫下甘蔗苗期根系全长cDNA文库构建及EST序列分析  

Construction of full-length cDNA library and its EST sequence analysis in sugarcane root at seedling stage under drought stress

作　　者：罗海斌[1] 曹辉庆[1] 魏源文[1] 邓智年[1] 潘有强[1] 郭元元[1] 何海波[1] 李杨瑞[1,2] 

机构地区：[1]广西作物遗传改良生物技术重点开放实验室 [2]农业部广西甘蔗生物技术与遗传改良重点实验室,南宁530007

出　　处：《南方农业学报》2012年第6期733-737,共5页Journal of Southern Agriculture

基　　金：广西自然科学基金创新研究团队项目(2011GXNSFF018002);广西科学研究与技术开发计划项目(桂科攻10100005-9);广西科学基金项目(桂科基0778006-6;桂科基0832026;2010GXNSFA013102);广西科学基金项目应用基础研究专项项目(桂科基0991021);广西农业科学院基本科研业务专项项目[200803Z(基);200902Z(基)];广西农业科学院科技发展基金项目(2007023);广西重点实验室建设项目(12-071-09)

摘　　要：【目的】构建甘蔗根系全长cDNA文库,并进行测序,为分离克隆甘蔗抗旱候选基因奠定基础。【方法】在干旱胁迫条件下,以新台糖22号甘蔗根系为材料,采用SMART技术构建cDNA文库,并进行EST序列分析。【结果】文库构建质量鉴定结果显示,文库库容量为1.0×106PFU/mL,重组率约95%,文库插入片段长度在500～2000bp,平均长度约1102.1bp。挑选526个阳性克隆进行单向测序后,共获得523个ESTs序列,去除载体、接头序列以及长度低于100bp的序列后,进行聚类分析并组装,共得到468个Uni-ESTs,其中congtig29个,singlets439个,分别占总数的6.5%和93.5%。通过与NCBI非冗余蛋白库进行BLASTX比对,发现444个Uni-ESTs在GenBank有同源序列,占94.87%;获得24个未知蛋白功能基因,占5.13%。【结论】构建了干旱胁迫下甘蔗苗期根系全长cDNA文库,获得523个ESTs序列和468个Uni-ESTs,并获得一批新的未知蛋白功能基因。[Objective ]The present experiment was conducted to construct cDNA library of sugarcane root length and sequencing to provide referrence on isolation and clone of expressed drought genes. [Method]Under drought stress conditions, cDNA library of ROC22 sugarcane root was constructed using SMART technology and its sequence was analyzed. [Result]The identification results of library construction quality showed that the library storage capacity was 1.0xl0^6 PFU/ mL and recombination rate was about 95%, the inserts length was about 500 to 2000 bp and the average length was approximately 1102.1 bp. After unidirectional sequencing of 526 positive clones, a total of 523 ESTs sequences was obtained. Among them, a total of 468 Uni-ESTs was obtained by cluster analysis and assembling after removing vector, adapter sequence and the sequence with length less than I00 bp, in which included 29 congtigs and 439 singlets, accounting for 6.5% and 93.5% of the total, respectively. With the NCBI non-redundant protein database BLASTX comparasion, 444 UniESTs had homologous sequences in the GenBank, which accounted for 94.87%; 24 unknown protein functional genes was also obtained, which accounted for 5.13%. [Conclusion]The present research contructed cDNA library of sugarcane root length under drought stress,and obtained 523 ESTs sequences,444 Uni-ESTs and a batch of unknown protein functional genes.

关 键 词：cDNA文库 甘蔗 根系 表达序列标签 测序 

分 类 号：S566.1[农业科学—作物学]