【作者】 万佳；

【导师】 徐正君；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 干旱、低温和土壤盐渍化等逆境胁迫将严重影响作物的生长和发育,降低产量。为了保障粮食安全,改善和提高作物对环境胁迫的抗性一直是农业生产的主要目标之一。随着植物分子生物学的发展,发掘与抗逆相关的新基因,将对耐逆作物品种的选育开辟新的途径。水稻作为最主要的作物之一,提高其抗逆能力将对粮食稳产具有重要的意义。在改善水稻的抗逆方面,定向的基因调控技术具有非常广阔的发展前景。近十年来已经有许多抗逆相关基因被转入水稻中,并获得了一些提高抗逆性的转基因植株。但是,水稻中仍然有许多未知抗逆基因没有被发现和利用。因此,本文将运用分子生物学技术,期望从水稻中找到在抗逆过程中具有重要意义的共调节因子,拟通过基因调节技术最终提高水稻的抗逆能力。为此,本文主要进行了以下三个方面的研究:1.利用荧光差异显示技术克隆逆境（低温）相关基因本实验采用mRNA荧光差异显示（Fluorescence Differential Display,FDD）技术,结合大矩阵筛选,对水稻抗低温的差异片段进行了大规模的分离筛选。获得了954个低温条件下的差异表达片段。其中,受低温特异诱导表达的片段200余个,在低温下表达增加的片段300余个,表达降低的片段400余个。通过亚克隆技术及测序分析,我们从中选取了2个在逆境条件下发生选择性剪接现象的基因进行了分析。通过半定量RT-PCR技术、蛋白质功能分析、蛋白质相互作用等技术,进一步探讨了这两个基因在水稻抗逆中的功能。2.水稻选择性剪接基因OsHSP81的功能研究（1）利用RT-PCR技术,我们克隆到了一个具有HSP90家族典型结构的新基因。该基因cDNA全长3395bp,包含919bp的5′端非编码区,376bp的3′端非编码区和2100bp的开放阅读框,推测编码一个699个氨基酸的蛋白质,其预测分子量是80.2kD,等电点为4.85,命名为OsHSP81。在克隆OsHSP81基因时还发现了它在转录过程中出现选择性剪接现象,产生了另一个转录文本OsHSP81-S。OsHSP81-S与OsHSP81位于同一基因座位上,只是ATPase功能域完全缺失。（2）利用半定量RT-PCR方法对OsHSP81和OsHSP81-S在低温、高温、干旱、高盐和激素等胁迫下进行表达分析,发现OsHSP81和OsHSP81-S在未经逆境胁迫处理时,均出现不同程度的表达;在低温、干旱、GA₃和ABA胁迫下,OsHSP81和OsHSP81-S的表达量都是由降低到升高;高温、高盐和IAA胁迫下,OsHSP81表达量由升高到降低,而OsHSP81-S在这三种胁迫下,其表达量变化不显著,基本维持在一个相对稳定的表达水平下。此外,研究还发现OsHSP81和OsHSP81-S在相同逆境胁迫不同时间段存在着表达量差异。（3）使用GST融合蛋白技术,在大肠杆菌中诱导出OsHSP81和OsHSP81-S两种融合蛋白。融合蛋白经过纯化定量后进行ATPase酶活性分析,结果表明OsHSP81具有比较高的ATPase酶活性,而OsHSP81-S蛋白由于ATPase功能域的缺失不具有ATPase酶活性。同时,HSP90特异抑制物格尔德霉素（geldanamycin）的加入明显抑制了OsHSP81的ATPase酶活性,进一步证实了OsHSP81具有ATPase酶活性。（4）从酵母双杂交（Y2H）实验中筛选出与OsHSP81相互作用的蛋白OsPRP1、RBB13-3、P450,以及含ZIM domain的未知蛋白和一些水稻未知基因。其中OsHSP81和OsHSP81-S均与OsPRP1相互作用,可以确定OsPRP1与HSP81结合的部位不在ATPase酶活性区。另外,在AD和BD载体互换验证实验中,分别把OsPRP1的部分和全长序列构建到BD载体中,结果显示OsPRP1的PRR中间区域与OsHSP81和OsHSP 81-S的C端相结合。3.水稻选择性剪接基因OsCaMBP在不同逆境下的表达分析（1）经RT-PCR扩增后测序得到2094 bp的cDNA序列。该序列的编码区位于115 bp到1824 bp,编码569个氨基酸残基,预测分子量为63.2 kD。第133～162氨基酸构成IQ基序,140～150氨基酸为典型的IQ钙调素结合基序,属于钙不依赖型钙调素结合蛋白。因此,将此基因编码的氨基酸命名为水稻钙调素结合蛋白OsCaMBP。同时,在热激和低温处理时,OsCaMBP还发现了选择性剪接现象。（2）通过半定量RT-PCR方法对OsCaMBP在低温和高温胁迫下表达进行分析,OsCaMBP均不同程度地受到低温和高温诱导。在低温胁迫处理时,30 min-4 h期间OsCaMBP开始表达并逐步上升,在8 h出现表达高峰后表达量相对稳定;而在高温胁迫处理时,叶鞘在15 min即出现表达高峰,之后随着时间的推移表达量逐渐下降,而在根和叶中均为30 min～2 h表达量不断增加,并在2 h出现表达高峰,随后逐渐下降。OsCaMBP在逆境胁迫下具有器官表达差异。通过FDD技术,我们从水稻中筛选出了多个可能与逆境相关的基因。本文中发现的选择性剪接基因OsHSP81和OsCaMBP均已初步证实在不同逆境胁迫下其表达量发生改变。根据它们在逆境胁迫下的表现表达特性,可以推测它们在水稻抗/耐逆过程起着重要作用。在今后的工作中,我们将从多方面对它们在抗逆方面的功能进行分析,以及更加深入地研究它们在基因网路上的作用机制。更多还原

【Abstract】 Some abiotic stress such as drought, cold and salination can influence the crop’s normal growth and development, and reduce its yield. So improve crop’s abiotic stress tolerance can play a major role in the increase of yield to ensure food security. With the development of plant molecular biology, finding the new gene associate with abiotic stress has opened up a new era for crop breeding.As rice is considered one of the major crops, the development of new cultivars with enhanced abiotic stress-tolerance will undoubtedly have an important effect on global food production. The transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of rice cultivars. In recent years, an array of stress-related genes has already been transferred to rice to improve its resistance against abiotic stresses. Many transgenic rice plants with enhanced abiotic stress-tolerance have been obtained. But there still have many genes we do not find and isolate which can improve the rice’s tolerance yet. Therefore, we focused on isolating some co-regulation factor of rice which can improve rice stress tolerance by molecular biology technology. The main results are provided as follows:1. Isolate the rice genes which induced by abiotic stress with fluorescent differential display.In order to discover a gene associated with abiotic stress, we utilize the fluorescent differential display （FDD） to screen the differential expressed gene between cold and drought stress in rice.We had obtained 954 differential expression gene fragment which induced by abiotic stress by subcloning and sequencing. About 200 gene fragment were induced by cold specific,～300 gene fragment over-expressed and～400 under-expressed in cold and drought stress. We choose two interesting gene fragment of them for further analysis because of the common character: alternative splicing. 2. Function analysis of alternative splicing gene 0sHSP81（1） A rice HSP90 family gene, OsHSP81, was cloning from rice treated with cold-stress by the fluorescence differential display （FDD） screening method. Its cDNA sequence （3395bp） contains an ORF encoding a 699 amino acids protein （80.2 kD）. Meanwhile, we found another transcript OsHSP81-S was produced by alternative splicing. OsHSP81-S and OsHSP81 were located at the same gene locus. Without the ATPase domain, the OsHSP81-S and OsHSP81 encode the same protein sequence.（2） Analysis the OsHSP81 and OsHSP81-S’s expression under different stress （low temperature, heat shock, drought, NaCl, GA₃, IAA and ABA） by semi-quantitative RT-PCR. The OsHSP81 and OsHSP81-S mRNA was already detected in control treatment, and the decrease of OsHSP81 and OsHSP81-S mRNA were apparent when rice after being exposed to low temperature, drought, GA3 and ABA, but not sooner than 8 hour. In heat shock, NaCl and IAA treatment, the expression of OsHSP81 increase very quickly, in contrast with OsHSP81-S expression smoothly. Furthermore, the result indicated that the two genes existed different expression level at the same treatment time.（3） By GST fusion protein system, we induced and purified the OsHSP81 and OsHSP81-S fusion proteins. ATPaes assay showed that OsHSP81 possessed ATPase, but OsHSP81-S not to have because of the ATPase domain deletion. Meanwhile, Geldanamycin can prevent OsHSP81 ATPase activity.（4） Screening proteins to interact with OsHSP81 and OsHSP81-S by yeast two-hybrid system. We got three proteins which names were OsPRP1, RBBI3-3, P450, and some unknown protein in rice. The interaction between the OsHSP81, OsHSP81-S and OsPRP1 were confirmed by transfer the insert library from the AD to the DNA-BD vector and vice versa, and then repeat the two-hybrid assay. The result support that the PRR of OsPRP1 interact with OsHSP81 and OsHSP81-S C-terminal.3. Cloning and Tissue-specific expression of the OsCaMBP gene in response to various treatments（1） We isolated OsCaMBP, a rice CaM-binding protein that contains an IQ domain at the N-tenninus. Its cDNA sequence （2094 bp） contains an ORF encoding a 569 amino acids protein, the predicted molecular weight was 63.2 kD. An IQ motif was found at the position of 140-150 amino acid, and IQ calmodulin-binding motif at the position of 133-162 amino acids. These motifs were the most typical for the structures belonging to calcium-free CaM binding proteins family. In addition, we found the alternative splicing of OsCaMBP under heat shock and low temperature treatment.（2） Semi-quantitative RT-PCR analysis showed that the expression of OsCaMBP was remarkably inducible not only with cold treatment, but also with heat-shock treatment. OsCaMBP was undetectable under normal condition, and induced not only with 1h cold treatment （8℃, 1h）, but also with heat-shock treatment （42℃, 15 min）, suggesting that this gene plays important roles in the signaling pathway in rice under both cold and heat-shock stresses.Using fluorescent differential display, we isolated many rice gene fragments which associated with abiotic stress. In this article, their expression pattern of two new genes OsHSP81 and OsCaMBP from rice with an alternative splicing mechanism under abiotic stress had verified. Because of these two genes’ important character, the thorough studies of their stress-resistant and the two genes interacting mechanism will carry on.更多还原