【作者】 张国青；

【导师】 翁跃进；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2002， 硕士

【摘要】 本实验以野生二棱短芒大麦(Hordeum brevisubulatum(Trink．)Link．)为试材，运用mRNA差异显示方法比较了正常培养(CK)和430mM NaCl盐溶液胁迫24hrs(T)基因转录水平上的差异，并对差异表达的cDNA片段进行了克隆分析。3种3’锚定引物和18种5’随机引物构成的54个引物组合中，有41个引物组合可以扩增出差异条带，其中以G和A结尾的锚定引物扩增效果较好。 对回收差异片段进行二次扩增，并从琼脂糖上回收，共得到41条分子量介于200-1000bp之间的差异片段。将这些差异片段转入pGEM-T easy载体，获得了41个克隆。 用反向Northern杂交技术证实7个阳性差异片段，其中，片段14和片段31在盐处理材料中特异表达，而在对照中不表达；片段1、片段2、片段13、片段15和片段19共5个cDNA片段在处理材料中的表达量高于对照材料。 对以上经反向Northern验证的7个差异片段进行测序，在GenBank中进行同源性比较。结果发现只在盐处理材料中表达的cDNA片段14与大麦未知功能cDNA克隆具有95％同源性，并与冷、热及盐胁迫处理植物的cDNA克隆具有83％以上的同源性，推测此片段与植物逆境胁迫下的抗性基因相关；另一个只在盐处理材料中表达的cDNA片段31与大麦和小麦功能未知的cDNA克隆具有91％以上的同源性。在盐处理材料中表达量高于对照的cDNA片段1和cDNA片段13，同大麦牙口小麦的一段未知功能的cDNA克隆具有91％以上的同源性；其它3个在盐处理材料中表达量高于对照的cDNA片段与盐胁迫和病原感染植物cDNA克隆具有85％以上同源性，推测这3个片段与植物的非生物逆境和生物逆境抗性基因相关；其中，cDNA片段19与植物叶绿体基因组编码ATP酶亚基的基因具有88％以上的同源性，此cDNA序列代表的是与叶绿体基因组编码ATP酶亚基有关的基因。 此外，利用pTriplEx2为克隆载体，构建了430mM NaGl胁迫处理24hrs野生二棱短芒大麦叶片的cDNA文库，获得的文库滴度为2.16×10~6，插入片段大小的范围为0.5～4.0kb，平均为1.5kb。更多还原

【Abstract】 Total RNAs from the leaves of wild ba.r\ey(Hordeum brevisubulatum(Trink.) Link.) treated 24hrs with 430mM NaCl(T) and unstressed control(CK) were extracted. The difference in their mRNA transcriptions was analyzed by using mRNA differential display. Differentially expressed cDNA fragments were cloned and sequenced. Forty-one of the 54 primer sets displayed differentially expressed cDNA fragments. The anchored primers with G or A residues at the 3 ’ end were most efficient.Forty-one cDNA fragments from 200 to lOOObp expressed differentially were isolated by polyacrylamide gel and cloned in pGEM-T easy vector.After identifying by reverse Northern blot analysis, fragments of 14 and 31 were expressed only in wild barley treated with 430mM NaCl, and fragments of 1, 2, 13, 15 and 19 achieved expression enhanced in wild barley treated with 430mM NaCl compared to that in control plants.These seven cDNA fragments were sequenced. Results of BLAST analysis in GenBank showed that fragment 14, one of two cDNA fragments which expressed only in wild barley treated with 430mM NaCl had 95% identity with cDNA clone of unknown function isolated from barley, and over 83% identity with cDNA clones isolated from cold, heat and salt-stressed plants. The fragment may be related to plant stress responding genes. Fragment 31 had over 91% identity with cDNA clones of unknown function isolated from barley and wheat. Fragments of 1 and 13, two of the five cDNA fragments showed over 91% homology to barley and wheat cDNA clones of unknown function. Fragments of 13, 15 and 19, exhibited over 85% identity with plant stress responding cDNA clones. These fragments may be related to plant stress responding genes. Fragment 19 which expressed at higher level in wild barley treated with 430mM NaCl than unstressed control showed over 88% homology to chloroplast ATPase subunit gene. It represented encoding chloroplast ATPase subunit gene.cDNA library from wild barley leaves treated with 430mM NaCl was successfully-constructed by using pTriplEx2 vector. It contained 2.16X106 pfu/ml. The size of insert fragments ranged from 0.5~4kb, with an average length of 1.5kb.更多还原