【作者】 郑军；

【导师】 王国英；

【作者基本信息】 中国农业大学 ， 遗传学， 2004， 博士

【摘要】 水分胁迫严重影响植物生长及作物生产，它是主要的非生物胁迫因子之一。现在认为，数以百计的基因参与到植物对非生物胁迫的响应中，然而，目前只有为数不多的基因的详尽功能得到阐明，况且，这方面的大部分工作是在模式植物拟南芥上完成的。 为了在玉米中发掘水分胁迫相关基因，通过抑制差减杂交(SSH)的方法，以20％PEG处理三叶期玉米的叶及根的cDNA为tester，对照为driver，构建了一个含有960个克隆的差减文库。为了进一步确认阳性克隆，所有克隆经过差示筛选(PCR-selected Deferential Screening)，按照试剂盒的鉴定标准，从960个克隆中挑选出533个阳性克隆，并全部进行测序，共500个克隆测序成功。对所获得的EST进行Cluster分析，发现共有190个独立的(unique)ESTs，其中包括101个contigs和89个singletons。所有unique ESTs在GenBank上BLAST分析表明，该文库包含了一些已经研究过的玉米水分胁迫相关基因，如：ZMrab17、ZMDBF1、ZMGRP、ZMSUCS1等以及一些与拟南芥、水稻或其它植物的水分胁迫相关基因具有很高同源性的EST。然而，仍有许多的EST的功能未知。根据其BLASTX的结果可以将有显著同源性的EST分为13个功能组。 为了进一步研究这些unique ESTs，利用cDNA Macroarray对其进行表达谱分析。用机械手将190个克隆的PCR产物点到尼龙膜上，每个克隆重复4次。以不同处理(PEG、干旱、ABA、高盐、冷)不同时间点的总RNA反转录标探针进行杂交。以表达量是对照的2倍或2倍以上为有意义的表达上调。在PEG胁迫下分别有67、113个EST在叶、根部表达上调；共有163个EST在干旱胁迫下表达上调。在ABA、高盐、冷处理时分别有116、55、24个EST表达上调。利用Gene Cluster软件进行表达谱的聚类分析发现，在PEG处理下玉米的根和叶有不同的表达模式；PEG诱导表达的EST绝大部分也能被干旱胁迫诱导表达。通过数据分析，发现一些EST所对应的基因可能在胁迫的信号转导中起作用。 在差减库中，发现一个全长的基因ZmGST7，进一步的Northern分析表明该基因受水分胁迫的诱导，并且在胁迫后期表达量很高。为了进一步研究它的功能，构建了两个表达载体(过量表达、诱导表达)，并将进行转化拟南芥。对转基因拟南芥T2代植株进行性状检测，初步得出结果表明，该基因能够明显促进植物生长，并在低水平的渗透胁迫下能够提高植物的抗性。 除此之外，我们已经从差减文库中鉴定到许多受PEG、干旱、ABA、盐、冷诱导的cDNA，然而，绝大多数cDNA的功能是未知的，所以，下一步的工作就是要克隆这些cDNA的全长基因，这不仅对于我们了解高等植物对胁迫反应的机制，还是对于进一步的基因操作以提高作物的抗逆性都具有非常重要的意义。更多还原

【Abstract】 Water stress is one of the major environmental stresses that adversely affect plant growth and crop yield worldwide. Hundreds of genes are thought to be involved in abiotic stress responses. However, only a few molecular components have been known precisely until recently. Most of these studies were processed in model plant Arabidopsis.To identify the genes controlling water stress response in maize (Zea mays) seedlings, suppression subtractive hybridization (SSH) was performed using mixed cDNAs prepared from maize seedlings treated with 20% PEG as testers and cDNAs from unstressed maize seedlings as drivers. A forward subtractive cDNA library was constructed, from which 960 recombinant colonies were picked and amplified. Through differential screening of the subtractive cDNA library, 533 clones were identified as water stress induced. After sequencing, 190 unique expressed sequence tags (ESTs) were obtained by clustering and blast analysis, which included transcripts that had previously been reported as responsive to stress as well as some functionally unknown transcripts. The ESTs with significant protein homology were sorted into 13 functional categories.A cDNA marcoarray containing the 190 unique ESTs was used to analyze their expression profiles in maize seedling during both PEG treatment and natural drought. The results indicated that 67 ESTs in leaves and 113 ESTs in roots were significantly up-regulated by PEG-stress. 123 ESTs were found to be up-regulated for at least one time-course point in either maize leaves or roots. Correspondingly, 163 ESTs were significantly up-regulated by drought stress. Results from the hierarchical cluster analysis suggest that the leaves and roots of maize seedlings had different expression profiles after PEG treatment and that there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts. A set of transcripts has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.Among the 190 unique cDNA clones, one clone which shows 100% identity to maize GST7 gene and is identified as ZmGST7. Northern analysis indicated that ZmGST7 is induced by water stress and had highly expression level in the late time point. In order to investigate the function of ZmGST7, two vectors were constructed and introduced to Arabidopsis thaliana by Agrobacterium tumefaciens. Some transgenic T2 lines were used for molecular and tolerance analysis. The preliminary results indicated that transgenic Arabidopsis seedlings that overexpress ZmGST7 grow faster than wild type seedlings and may plays certain roles in lower osmotic stress.In the present study, we also identified many PEG-, drought-, ABA-, salt-, cold-inducible cDNAs. However, the functions of most of them remain unknown. It is important to analyze the functions oftheir corresponding full-length genes not only for further understanding the molecular mechanisms of stress response in higher plants but also for improving the stress tolerance of crops by genetic manipulation.更多还原