【作者】 刘晓敏；

【导师】 张世宏；

【作者基本信息】 吉林大学 ， 植物病理学， 2011， 硕士

【摘要】 低温、干旱等逆境是影响植物生长发育的主要非生物胁迫因子,严重的影响植物的产量。每年全球主要农作物的产量损失约有50%是由非生物胁迫导致的。玉米是我国三大粮食作物之一,属于逆境敏感型植物,生育期内极易受低温、干旱、盐渍等多元逆境胁迫的干扰和伤害,解决玉米生产中严重的多元逆境胁迫问题,提高玉米产量,除了常规育种技术以外,基于基因遗传转化的生物育种技术是一个重要的选择。通过转基因技术提高玉米抗逆性是有效应对逆境挑战的根本途径,而分离、克隆抗逆关键基因是解决这一重要问题的核心与关键,克隆和组建逆境交叉诱导型启动子是进行玉米多抗基因转化实现分子育种目标的重要保障。诱导型启动子可根据需要在植物特定的发育阶段、组织器官和生长环境下,诱导基因的大量表达,应答外界环境的变化。避免了植物营养的不必要浪费。近年来,为了提高植物的抗逆性,许多启动子和抗逆基因被克隆,但是玉米的逆境诱导型启动子研究较少。本研究从玉米中分离了低温、干旱诱导型启动子(LsP),并构建了植物表达载体,转化农杆菌,并对其活性进行了初步验证,为以后开展利用转基因技术改良玉米抗逆品质研究奠定基础。本研究结果如下:(1)从玉米基因组中分离了low temperature and salt responsive protein(LS)基因ATG上游1735bp的调控序列,命名为LsP。并通过启动子分析软件,对其顺式作用元件进行分析。(2)构建了该启动子的植物双元表达载体pCAMBIA1301-LsP,用叶盘转化法转化烟草。对烟草的抗性芽进行GUS染色,初步证明该启动子具有低温诱导活性。得到了转基因的烟草,并且对转基因烟草进行200mmol/LNaCl、4℃和20%PEG诱导后进行GUS染色,发现该启动子受低温和干旱诱导。(3)为寻找该启动子的核心序列,构建了4个5′-端渐变缺失载体。通过瞬时表达进行初步研究。GUS荧光定量分析发现在-1845bp~-1173bp之间存在着一些负调控元件降低基因表达,在-543bp~-110bp之间存在该启动子的核心序列。在-1173bp~-810bp之间片段中可能存在着低温诱导响应元件,在-810bp~-543bp之间处可能存在干旱响应元件。更多还原

【Abstract】 Low temperature, drought are major abiotic factors that play a very crucial role in plant growth and cause significant reductions in plant productivity. It has been indicated that more than 50% of crop loss will be caused by the three abiotic stresses every year in the world. Maize is a stress-sensitive crop being vulnerable to disturbance and damage in growth period, particularly in northeast of China. Transformation based on the biological genetic breeding technology is an important choice besides traditional breeding techniques,it is not only to resolve serious problem of multi-stress in maize production but also to increase the output.Transgenic technology is the fundamental way to enhance the effective resistance. So it is important to isolate and clone candidate genes in related to stress tolerance for solving this problem,and cloning and the formation of cross-stress inducible promoter is a security to achieve a newer crop plants that are tolerant to abiotic stresses by molecular breeding. The trait of gene expression under the control of stress inducible promoters is specific to stress conditions, avoiding unnecessary waste of plant nutrition. In recent years, To improve the resistance of plants, a broad range of promoters have been characterized and used in plant genetic engineering.However, the stress inducible promoter from maize are too less. In this study, we cloned low temperature- and drougth- inducible promoter from Maize and tested the activity of Ls gene promoter using Agrobacterium-mediated transformation of tobacco. It lays the foundation for improving maize resistance by transgenic technology.There are three results as follows:(1) we cloned 1735bp 5’-flanking region of the low temperature and salt responsive protein(LS)gene from maize, named LsP. The cis-acting elements were analyzed by the PLACE.(2) To investigate the promoter activity, The full length of Lsp was cloned into the pCAMBIA1301 binary vector, to construct the promoter-GUS fusion vector pCAMBIA1301-LsP.The plasmids were transformed into tobacco plants mediated by Agrobacterium tumefaciens EHA105, Histochemical analysis of GUS activity of resistant shoots showed that, LsP was able to drive the expression of the reporter gene under low temperature.Then, this promoter activity were analyzed in transgenic tobacco. The driving activity of LsP was strongly induced in the leaves by PEG and 4℃treatment.(3) In order to identify promoter regions that confer the activity under stresses, the 1735bp promoter fragment and four 5’-deletions of LsP were linked to a GUS reporter gene. GUS fluorescence quantitative analysis of a series of 5-deletions of the LsP suggested that there were negatively cis-elements in region of -1845 bp ~ -1173 bp and some core elements in the region of -543 bp ~ -110 bp. It is possible that the region of -1173bp ~ -810bp possesses cold-inducible elements and the region of -810 bp ~ -543 bp possesses drought-inducible elements.更多还原