本试验对构建的辣椒育性恢复相关基因的消减cDNA文库进行质量分析.通过对文库构建过程中的各个环节,包括总RNA的提取,mRNA的分离纯化,RasⅠ酶切,接头连接,消减效率检测以及菌落PCR等,进行分析和评估,证明利用抑制消减杂交技术(SSH)所构建的消减文库是准确可靠的,富集了大量与辣椒育性恢复相关的基因,为进一步精确筛选、克隆、分析辣椒育性恢复相关基因奠定了基础.
To evaluate the subtracted library of fertility restoration2related genes in pepper,a quality analysis was conducted,including total RNA extraction,mRNA purification,Ras Ⅰdigestion,adaptor ligation efficiency analysis,PCR analysis of subtraction efficiency,and colony PCR.The result was that the subtracted library constructed using suppression subtractive hybridization(SSH)was accurate and reliable,and had the large number of pepper fertility restoration2related genes.
[1] 王述彬,刘金兵,潘宝贵.辣(甜)椒细胞质雄性不育恢复基因的遗传与分布[J].华北农学报,2007,22(1):86-89.
[2] 沈火林,蒋健箴,王志源,等.辣椒雄性不育系选育及遗传研究[J].北京农业大学学报,1994,20(1):25-30.
[3] Diatchenko L,Lauau Y F C,Campbell A P,et al.Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes and libraries[J].Proceedings of the Natlional Academy of Sciences of the United States of America,1996,93(12):6025-6030.
[4] 郭爽,沈火林,杨文才,等.利用抑制消减杂交技术分离辣椒细胞质雄性不育育性恢复相关EST[J].园艺学报,2009,36(10):1443-1449.
[5] 董霞,李文正,刘世贵.抑制消减杂交技术及其在植物基因表达研究中的应用[J].植物生理学通讯,2006,42(3):515-522.
[6] Zheng J,Zhao J F,Tao Y Z,et al.Isolation and analysis of water stress induced genes in maize seedlings by subtractive PCR and cDNA macroarray[J].Plant Molecular Biology,2004,55:807-823.
[7] Zhao Y H,Wang G Y,Zhang J P,et al.Expressed sequence tags(ESTs)and phylogenetic analysis of floral genes from a paleoherb species,Asarum caudigerum[J].Annals of Botany,2006,89(1):157-163.
[8] Wang M L,Hsu C M,Chang L C,et al.Gene expression profiles of cold-stored and fresh pollen to investigate pollen germination and growth[J].Plant Cell Physiology,2004,45(10):1519-1528.
[9] Mi G Q,Liu L Y,Dang J W,et al.Identification of low-light induced genes in cucumber(Cucumis sativus)seedlings by suppression subtractive hybridization[J].Scientia Horticulturae,2008,117:89-94.
[10] Terefe D,Tatlioglu T.Isolation of a partial sequence of a putative nucleotide sugar epimerase,which may involve in stamen development in cucumber(Cucumis sativus L.)[J].Thero Appl Genet,2005,111(7):1300-1307.
[11] 宋国琦,胡银岗,林凡云,等.YS型小麦温敏雄性不育系A3017育性相关基因的SSH分析[J].西北植物学报,2006,26(7):1301-1308.
[12] 常青山,周荣华,余增亮,等.用差减抑制杂交方法鉴定太谷核不育小麦花药败育过程中的特异表达基因[J].高技术通讯,2003,3:30-33.
