簇毛麦低拷贝cDNA序列的克隆和鉴定  

Cloning and identification of low copy cDNA sequences from Haynaldia viliosa

作　　者：柴守诚[1] 刘大钧[1] 陈佩度[1] 齐莉莉[1] 李万隆[1] 刘金元[1] 

机构地区：[1]南京农业大学细胞遗传研究所

出　　处：《西北植物学报》1999年第2期177-182,共6页Acta Botanica Boreali-Occidentalia Sinica

基　　金：高等学校博士点基金;江苏省自然科学基金

摘　　要：从簇毛麦叶片中提取总ＲＮＡ，进一步分离ｍＲＮＡ。以ｍＲＮＡ为模板反转录合成ｃＤＮＡ，两端经Ｔ４ＤＮＡ多聚酶修平后加ＥｃｏＲⅠ接头分子，连接于质粒ｐＧＥＭ－７Ｚｆ（＋）的ＥｃｏＲⅠ克隆位点，转化大肠杆菌ＪＭ１０３菌株建立了ｃＤＮＡ文库。用ＰＣＲ扩增重组质粒的ｃＤＮＡ插入序列，用３２Ｐ标记后分别与ＨｉｎｄⅢ或ＸｂａⅠ酶切的小麦－黑麦附加系ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交。根据其杂交结果，目前已鉴定出４个不同的低拷贝探针。ＣＨＶ１２１和ＣＨＶ１２９与黑麦的３Ｒ附加系有特征性杂交带，因此，属第三同源群探针。ＣＨＶ１０４和ＣＨＶ１２４与黑麦３Ｒ、７Ｒ附加系有特征性杂交带，因此属第３、第７同源群探针。另外，与簇毛麦ＲＮＡ的Ｎｏｒｔｈ－ｅｒｎ杂交分析表明，ＣＨＶ１０４和ＣＨＶ１２１对应的基因分别编码分子量约为２．３和２．８ｋｂ的ｍＲＮＡ。Total RNA and mRNA were isolated from leaves of Haynaldia villosa .First strand synthesis of cDNA was driven by avian myeloblastosis virus (AMV) reverse transcriptase and an oligo(dT) containing primer with mRNA as template,followed directly by second strand replacement synthesis using RNase H and ploymerase Ⅰ. After treatment with T 4 DNA ploymerase to flush the ends, the cDNA was ligated to EcoR Ⅰ adaptor and cloned into the EcoR Ⅰ sites of plasmid pGEM 7Zf(+). The strain JM103 of E.coli was transformed, and a cDNA library was finally constructed. The inserted cDNA sequences were amplified through PCR, labelled with 3 2P and used to probe the Southern blots of Hind Ⅲ or Xba Ⅰ digested DNAs from T. aestivum-S. cereale addition lines to select low copy cDNA probe for different homoelogous groups of Triticeae. As a beginning,we have identified 4 different low copy probes.CHV121 and CHV129 detected specific bands on 3R addition line,indicating that they may be used as probe of homoelogous group 3 chromosomes of Triticeae.CHV104 and CHV124 were specific for 3R and 7R chromosomes involved in the addition lines.The hybridization of H.villosa RNA with CHV104 and CHV121 showed that the corresponding genes of them encode 2.3 kb and 2.8 kb mRNA respectively.

关 键 词：簇毛麦 CDNA 探针 SOUTHERN杂交 

分 类 号：Q949.714.2[生物学—植物学]