玉米ZmPIS启动子的胁迫响应特性分析及核心功能区段的鉴定

发布时间：2018-04-24 19:45

本文选题：转基因烟草 + 玉米ZmPIS启动子　；参考：《山东大学》2014年硕士论文

【摘要】：土壤干旱和盐渍化等严重限制了植物的生长发育,已成为影响作物品质和产量的重要因素。作物应对干旱和土壤盐渍化不良环境,促进产量的生物学过程受到包含多个关键基因在内的协同调控过程。发掘并克隆耐逆关键功能基因及调节元件,采用转基因技术培育抗旱、耐盐作物新品种,是解决上述问题的有效途径。目前,利用植物基因工程手段改良作物相关性状时,转化的目标基因在植物宿主细胞中的高效表达和组织或时空特异性等受到越来越多的关注。基因的表达调控是在DNA水平、转录水平、翻译及翻译后修饰水平等多层次协同调控的过程,其中转录水平的调控主要是通过启动子上的顺式作用元件与反式作用因子的相互作用来实现功能基因在特定的时间、组织中表达。分离克隆可用于作物抗逆遗传改良的启动子并获得自主知识产权,了解各启动子中的核心功能区段及调控元件的功能基础具有重要的理论意义和应用价值。在实验室前期工作中,翟淑梅等发现玉米ZmPIS基因具有明显的盐和干旱等胁迫诱导特性。官赞赞等克隆了ZmPIS基因的启动子序列并初步确定了该启动子启动目的基因表达的能力。本工作在上述工作基础上对ZmPIS基因的启动子的功能及胁迫响应特性进行了系统地分析。ZmPIS基因启动子的生物信息学分析表明该启动子含有多个胁迫响应元件。我们构建了该启动子的5’端系列缺失突变植物表达载体PZ1-PZ8,启动Gus报告基因的表达,转化烟草。对含有启动子不同长度片段的转基因烟草叶片进行了GUS荧光活性测定,发现PZ7在PZ1-PZ8转基因烟草叶片材料中表现出了最高的启动目的基因表达的能力。以PZ1、PZ7和35S启动子转基因烟草2周龄植株及花、果、种子进行GUS组织化学染色,发现PZ7相对于PZ1全长启动子在上述不同组织器官中均具有更高的GUS表达强度。为进一步研究ZmPIS启动子胁迫响应特性,我们对PZ1-PZ8转基因烟草进行了NaCl和18% PEG等胁迫处理分析。转基因烟草离体叶片在200mMNaCl、18% PEG处理下的GUS组织化学染色结果表明,盐和渗透胁迫下PZ1-PZ7有相似的诱导表达模式,即处理1 h、3 h、6 h时与未处理的对照相比无显著差异,12 h时表现出一定诱导趋势,处理至24 h、48 h和72 h时GUS染色强度显著上调,且PZ7在胁迫诱导前后均具有最高GUS表达强度。相对而言PZ8和35S启动子在上述两种胁迫环境下均未表现出明显的诱导表达变化。为进一步验证上述离体实验的结果,我们以PZ1、PZ2、PZ6、PZ7、PZ8、35S转基因烟草活体植株为材料开展了相应的NaCl和PEG胁迫处理实验。GUS组织化学染色及荧光活性测定结果表明,处理24 h时PZ1、PZ2、PZ6和PZ7转基因烟草中的GUS酶活性达到最高(均达到处理前的2倍以上),且在处理48 h和72 h时一致保持较高的诱导水平,也就是说PZ1-PZ7均具有明显的高盐和渗透胁迫诱导活性,而PZ8的高盐和渗透胁迫诱导活性却明显丧失。值得注意的是PZ7在胁迫前后与其它突变体材料相比均体现出了最高启动活性,可达到PZ1(全长启动子)的20倍以上,PZ6的3倍以上。正常条件下PZ7的启动表达活性为PZ8的1.1倍左右和35S启动子的20%左右；而盐和渗透胁迫后可达到PZ8的2倍以上和35S启动子50%左右。上述结果意味着PZ7-PZ8缺失的110 bp片段(-466到-357 bp)中可能含有高盐和渗透胁迫响应元件,且PZ7(-466 bp片段)为ZmPIS启动子具有盐和渗透胁迫响应特性的高效启动基因表达的关键序列区段,体现出了一定的应用潜质。本工作通过对玉米ZmPIS启动子的功能及胁迫响应特性分析,发现了PZ7(-466 bp)片段为该启动子具有盐和渗透胁迫响应活性的高效启动基因表达的核心功能区段,而-466 bp到-357 bp之间的110 bp序列可能存在盐和渗透胁迫响应顺式作用元件。这些实验结果加深了我们对玉米ZmPIS基因启动子上核心功能区段及胁迫响应特性的了解,为作物抗逆基因工程育种提供了一定的启动子资源和参考资料,有望为我国作物抗逆遗传改良贡献自己的绵薄之力。
[Abstract]:Soil drought and salinization have seriously restricted the growth and development of plants. It has become an important factor affecting the quality and yield of crops. The biological process of crop response to drought and soil salinization and the biological process of promoting yield are coordinated by several key genes. The use of transgenic technology to cultivate drought resistant and salt tolerant crop varieties is an effective way to solve the above problems. At present, more and more attention has been paid to the efficient expression and tissue or time space specificity of the target gene in plant host cells by using plant genetic engineering methods to improve crop correlation. Regulation is a multi level coordination process, such as DNA level, transcriptional level, translation and post-translational modification. The regulation of transcriptional level is mainly through the interaction of the cis acting element and the trans acting factor on the promoter to realize the functional genes in a specific time and tissue. The isolated clones can be used for crop resistance. In the early work of the laboratory, Zhai Shumei found that the maize ZmPIS gene had obvious salt and drought stress induction characteristics. The promoter sequence of the ZmPIS gene was augmentation and the ability of the promoter to start the gene expression was preliminarily determined. On the basis of the above work, a systematic analysis of the biological information of the promoter of the.ZmPIS gene was carried out on the basis of the function of the promoter of the ZmPIS gene and the response characteristics of the promoter, and it was shown that the promoter contains multiple stress responses. Components. We constructed a 5 'terminal series deletion mutant plant expression vector PZ1-PZ8, started the expression of Gus reporter gene and transformed tobacco. The GUS fluorescence activity of transgenic tobacco leaves with different lengths of promoter was measured. It was found that PZ7 showed the highest start in the PZ1-PZ8 transgenic tobacco leaf material. The ability of the target gene expression. The 2 weeks old plants of PZ1, PZ7 and 35S promoters, flowers, fruits and seeds were stained with GUS histochemical staining. It was found that PZ7 had a higher GUS expression intensity in the different tissues and organs of the PZ1. To further study the response characteristics of the ZmPIS promoter stress, we had the PZ1-PZ8 transgene. The NaCl and 18% PEG stress treatments of tobacco were analyzed. The GUS histochemical staining results of the leaves of transgenic tobacco under the treatment of 200mMNaCl and 18% PEG showed that the PZ1-PZ7 had similar induced expression patterns under salt and osmotic stress, that is, the treatment of 1 h, 3 h, 6 h was not significantly different from that of the untreated, while 12 h showed a certain inducement. The guiding trend, when treated to 24 h, 48 h and 72 h, the GUS staining intensity was significantly up-regulated, and PZ7 had the highest GUS expression intensity before and after stress induction. Relatively speaking, PZ8 and 35S promoter did not show obvious inducible expression changes under these two stress environments. The Z8,35S transgenic tobacco living plants carried out the corresponding NaCl and PEG stress treatment experiments. The results of histochemical staining and fluorescence activity of.GUS showed that the activity of GUS enzyme in PZ1, PZ2, PZ6 and PZ7 transgenic tobacco reached the highest when treating 24 h (all reached more than 2 times before the treatment), and remained high in the treatment of 48 h and 72 h. The induction level, that is, PZ1-PZ7 has obvious high salt and osmotic stress induction activity, while the high salt and osmotic stress induced activity of PZ8 is lost obviously. It is worth noting that PZ7 shows the highest activation activity compared with other mutant materials before and after stress, and can reach more than 20 times of PZ1 (full promoter), and 3 times of PZ6. Above. Under normal conditions, the activation of PZ7 is about 1.1 times of PZ8 and about 20% of 35S promoter, while salt and osmotic stress can reach more than 2 times of PZ8 and 50% of 35S promoter. The above results mean that the 110 BP fragments of PZ7-PZ8 deletion (-466 to -357 BP) may contain high salt and osmotic stress response elements, and PZ7 (-466) Fragment) is the key sequence section of high efficient promoter gene expression of ZmPIS promoter with salt and osmotic stress response characteristics, reflecting certain application potential. This work has found that PZ7 (-466 BP) fragment has salt and osmotic response activity through the analysis of the function and stress response characteristics of maize ZmPIS promoter. The 110 BP sequences between -466 BP and -357 BP may have salt and osmotic response cis elements. These results deepen our solution to the core functional areas and stress response characteristics of the maize ZmPIS promoter, and provide the crop resistant genetic engineering breeding. Certain promoter resources and reference materials are expected to contribute to the improvement of crop genetic resistance in China.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q943.2

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