【作者】 陈昕；

【导师】 王保莉；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 干旱是限制农业生产的重要因素之一,解决干旱问题的重要途径之一就是提高作物自身的抗旱性。硫作为作物所必需的营养元素,在作物的生长代谢过程中起着许多重要的作用。已有研究表明,硫素不仅对植物有直接的营养作用,还可以提高植物的水分利用率,有利于植物对干旱逆境的适应。然而,人们目前对硫营养与提高作物生理抗旱性的分子机理的认识还十分有限,有待开展系统深入的研究。硫在被植物吸收同化利用的途径中,硫转运蛋白表达的调节是最初也是最基础的一个调节部位,研究其在干旱胁迫条件下的表达模式是了解硫营养对作物生理抗旱性调节分子机理的基础。本实验选用水肥敏感型小麦郑引一号作为实验材料,采用室内水培的方法,通过控制不同的硫水平,研究了在干旱胁迫条件下硫素对小麦硫转运蛋白基因表达的调节,取得了以下结果: 提取小麦根系总RNA,自行设计引物,应用RT-PCR 的方法克隆小麦硫转运蛋白基因片段,将其连入克隆载体pGEM-Teasy 后测序,测序结果比对发现该片段属于StA1.1a基因部分片段;用地高辛随机引物标记试剂盒对该片段进行标记后,对不同水分和硫营养条件下提取的小麦根系总RNA 进行斑点杂交。结果显示,该片段的表达受到硫营养的负调控,同时,硫养分胁迫下正常供水和干旱处理之间有一定的差异,证明水分胁迫对硫营养的负调控很可能有一定的抑制作用。选用跨内含子的持家基因α-Tubulin 片段作为内参基因对小麦硫转运蛋白(ST)基因的表达进行半定量RT-PCR 检测,通过对试验参数的优化,建立了检测硫转运蛋白基因表达两步法RT-PCR 技术,结果同样显示,该片段的表达受到硫营养的负调控,同时,硫养分充足情况下,正常供水和干旱处理间也可检测到微弱差异,但该影响远远小于硫营养胁迫带来的负调控作用。本文对作物利用硫养分生理抗旱的分子机理做了初步探讨,同时建立起来的稳定扩增小麦α-Tubulin 基因RT-PCR 体系可直接用于实验室检测小麦反转录体系是否成功,另外,根据序列分析结果和表达检测结果对该片段的一些性质测定也为硫转运蛋白的进一步研究提供了一定依据。更多还原

【Abstract】 Drought is one of the severe constraints on farming. One of the important pathway in the resolution to drought is to increasing the drought tolerance of crops. Sulfur is an essential element in crops nutrition, which plays a vital role in crop growth and metabolism. It has been reported that Sulfur nutrition regulates the functions of some channel proteins and has a crucial role in the trans-membrane transport of water and inorganic nutrient elements. Whereas on the whole regulation of sulfur assimilation, sulfate transporter, the first key protein, plays a central role. Therefore, to study the expression of sulphate transporter under different water and sulfur condition would provided a preliminary research of the function of sulfur in helping crops fighting against drought on a molecular level. Total RNA was extracted from the roots of a type of high-sensitive-to-drought wheat and RT-PCR was proceeded for cloning a piece of sulphate transporter gene. Sequencing was done after it linked to cloning vector pGEM-Teasy. Sequence alignments showed that this partial piece belongs to wheat sulphate transporter gene StA1.1a. After random labeling with DIG, RNA dot blot was proceed to test the expression of this gene under different growth condition. Results showed that the expression of this gene was positive regulated by sulphate starvation, and a weak inhibition was found when drought condition was involved at the same time. A straightforward semi-quantitative RT-PCR method was developed to study the expression of the same sulphate transporter gene piece, using a cross-intron α-Tubulin partial sequence to be the inner control. A stable two-step reverse transcription of a specific transcript within total RNA was involved and product amount determination by densitometric analysis of ethidium bromide fluorescence upon gel electrophoresis. Results also showed that the expression of this piece was positively regulated by sulphate starvation; Minor variance was also detected between samples with different water growth condition under sufficient sulphate supply, but this effect was far less than the effect of sulphate starvation. All these results provided a preliminary discussion of the function of sulfur in helping crops fighting against drought on a molecular level, and the stable two-step RT-PCR system of the cross-intron RT-PCR product of housekeeping gene α-Tubulin could also provided a good method to test the reverse process of wheat mRNAs.更多还原