【作者】 殷得所；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 化学诱导表达系统是植物基因控制表达的重要工具，在实验室和大田具有很大的应用潜力，此类系统的实用性依赖于诱导的特异性和可调控性，以及使用成本和环境相容性。本研究用一种杀菌剂(丙环唑)和两种除草剂安全剂(AD67、二氯乙酰胺)对水稻品种9311的幼苗进行了诱导处理，采用差异显示技术分离回收了诱导表达的基因片段，鉴定了10个阳性表达片段，对诱导基因的功能及转录元件进行了分析，构建了启动子功能分析载体。主要结果如下：1．差异表达基因片段的获得。采用3种诱导剂对3叶期水稻幼苗进行诱导处理，处理时间分别为0h(CK)、6h、12h、24h、48h、3d、4d、5d。通过对各处理总RNA的分离和差异显示分析，获得了67个差异表达片段。2．差异表达基因片段阳性验证。根据不同的引物组合，对差异片段进行了重扩增、电泳分离、转膜和杂交，从中筛选得到经反式Northern Blotting验证的10个阳性表达片段。3．对阳性片段的序列分析。采用PMD18-T载体对阳性片段进行克隆并测序。序列提交GeneBank进行同源序列搜索，获取基因功能的相关注释。结果表明，Y2和Y11是未知序列：其他8个片段中Y1和Y7是未知功能的基因，Y3为二磷酸鸟苷-甘露糖-焦磷酸化酶基因(GDP-mannose pyrophosphorylase，GMP)，Y5为核糖体蛋白F因子蛋白(SM-F)，Y6为叶绿体1，5二磷酸核酮糖羧化酶(RBCL)基因，Y8为YIPPEE基因，Y9为Vps55蛋白，Y10为富含甘氨酸的RNA结合蛋白GRP／A。4．对诱导基因的5’端启动子区域进行了转录元件分析。因Y2和Y11无法进行物理定位未做分析。在其他8个基因的5’端上游搜索到多种转录元件，其中除了TATA框等核心启动子序列外，还有多个CAAT增强子调节元件。比较还发现，诱导基因的上游普遍含有一些植物逆境应答元件，出现频率较高的有ABA应答元件ABRE，光应答元件ACE、BOX4、SP1和GATA，还有干旱应答元件MBS和茉莉酸甲脂的应答元件GGTCA、TGACG-motif、CGTCA-motif等。另外，还发现参与抗病和逆境胁迫应答转录元件(TC-rich repeats)，以及水杨酸应答的TCA元件。5．对GMPase和YIPPEE基因进行了表达分析。RT-PCR和NORTHERN分析表明，两个基因的表达都在诱导剂处理6小时开始出现上升，GMPase基因在处理2d时达到高峰，YIPPEE基因在诱导处理12h时达到高峰，两基因在5天内持续表达。6．构建了YIPPEE基因启动子的表达载体。将pCAMBIA1303载体GUS基因的CMV35S启动子替换为YIPPEE基因上游不同长度的启动子片段，拟用于启动子的功能验证。更多还原

【Abstract】 Chemical inducible expression system is a powerful tool with which to regulate and control the gene expression of plant potentially in both laboratory and field. Their utility is dependent on the specificity and controllability of induction ofgene expression, as well as the cost and environment profile of the inducing chemicals. Searching new regulable gene by chemical is a basic work for the research and development of inducible promoter. In this study, a fungicide and two herbicide safeners were used as the inducer to treat the rice seedlings, and ten genes were isolated and characterized by differential display as the positive ones induced by the inducers. Analysis about the function and transcript element were carried out, and constructed plant were prepared. The following are the main result:1. Rice seedlings were induced by 3 inducers (Propiconazol、AD67、dichlopronamide) and expression analysis were carried out. Treatments were conducted at 3-1eaf stage, the samples were collected at 6h, 12h, 1d, 2d, 3d, 4d and 5d after induced and compared with the CK which treated by sterile water. Total RNA were extracted and differential display analysis show that 67 fragments were increased or decreased apparently in transcription level after induced.2. Fragments on the poly-gel were recycled and verified by reverse Northern blotting,ten positive fragments were identified as induced gene.Recycled DNA respond to induced gene were linked into T-vector and transferred to complementary cell of DH5α, after resistant screening, the positive clones were prepared for sequencing.3. Sequencing and homologous analyzing were processed for the 10 fragments.The searching on GeneBank revealed that fragments Y2 and Y11 come from the unknown sequence, and 2 fragments (Y1 and Y7) represent the unknown genes, the rest fragments respond to GMPase, Sm-F, RBCL, yippee, Vps55 and GRP/A respectively. 4. The transcript elements of 8 genes were searched in the 5’ untranslation region. Multiple elements were founded in the 5’ UTR, including the TATA-boxes as well as CAAT-boxes and G-box which is reserve in plant. And serial inducible elements such as ABRE which is respond to ABA induction, and ACE, BOX4, SP1, GATA which involved in light responsiveness were found. The MBS, GGTCA, TGACG-motif, CGTCA-motif which specifically respond to MeJA were also detected. Another kind of important element TCA-box that involved in defense and stress responsiveness to salicylic acid were found.5. RT and Northern assay were carried out to ascertain the induction profile of GMPase and YIPPEE. Primers were designed according to the complete RNA s or cDNAs. Expression assay showed that the transcripts of two genes started to increased from 6 h after induction, and the level can be detected until 5 d.6. The constructed vectors for promoter characterization were prepared. Candidates of promoter regions were cloned and replaced for the 35S sequence of GUS gene in the pCAMBIA1301 for further study.更多还原