【作者】 夏建冰；

【导师】 齐桂年；

【作者基本信息】 四川农业大学 ， 茶学， 2010， 硕士

【摘要】 茶树(Camellia sinensis(L.) O. Kuntze)是我国重要的经济作物之一,不仅栽培历史悠久,而且分布广泛。已有研究表明,我国西南部是茶树的起源中心,孕育了丰富的茶树种质资源,为优良品种的选育奠定了优良的基础。干旱是限制世界作物生产的主要环境因子之一,全球干旱、半干旱地区约占土地总面积的36%,占耕地面积的43%。我国干旱和半干旱地区面积约占全国土地面积的1/2,同时,随着全球温室效应的增强,对抗旱品种的选育将是未来品种的选育的一个重要方面。传统的茶树品种选育主要是通过系统选育和单株选育,但是有遗传背景复杂、遗传不稳定、选育时间较长、选育盲目性较大等缺陷,同时抗旱指标的选择多是以环境性状为主。分子育种直接在分子水平选育,通过对遗传物质DNA的筛选进行品种选育,能有效避免上述缺点。DD-PCR(差异显示PCR技术,或DDRT-PCR)有操作简便、灵敏性良好和效率较高的优点,本研究首次将技术成熟的差异显示PCR技术应用于茶树干旱环境下的差异表达基因的筛选。本实验意图将在干旱环境下有表达变化的基因片段作为以后抗旱品种研究和选育的一个筛选指标,对干旱品种的选育做出一定的贡献。通过实验得到了以下实验结果：1、幼龄茶苗利用试剂盒提取RNA的最佳部位是嫩叶和幼根,因为积累的次生产物少,对提取到的RNA污染少,但根部RNA保存较困难,降解较快,说明应用该试剂盒提取的RNA中有其他杂质,影响根部RNA的保存。2、PCR反应时用Mixmixture的扩增效果要好于传统的Taq酶体系；电泳条件是120mv,电泳1h左右；电泳所用凝胶浓度约为1.5%分离效果较好；取材部位为嫩叶时,退火温度为45℃时,扩增条带的假阳性较低,条带的重复性较好,取材部位为幼根时,根据处理时间,应适当降低退火温度；PCR反应参数以普通PCR反应参数为对照即可。3、经过PCR反应后,发现条带有上调表达、下调表达、常量表达三种表达量的变化；发现部分条带对锚定引物有一定的兼并性：通过引物筛选,确定了进行不同研究所需要的引物组合,以幼根为材料进行试验,需要重新确定胁迫剂浓度和胁迫处理时间。4、根据测序结果可以看出,茶苗在干旱胁迫处理下,有很多基因条带表达产生变化,部分条带的变化趋势和有研究者报道的酶活变化趋势相近。5、确定应用DDRT-PCR技术在茶树抗旱分子机制的探讨中是可行的,在所选择的茶树品种福鼎大白茶幼苗上取得的较好的实验效果,可以应用该方法对其它茶树品种及不同发育期茶树品种进行相关研究。更多还原

【Abstract】 Tea plant (Camellia sinensis (L.) O. Kuntze) is one of the important economic plants in China, which had long planting history and distributes widely. It has been proved that Southwest of China is the origin of tea plant, having rich of tea resources and good foundation for breeding. Drought is the main environmental limit factors in the world’s crop production, one of the world’s arid and semi-arid regions account for about 36% of the total land area, accounting for 43% of arable land. China’s arid and semi-arid land area accounting for about 1/2, while, with the enhancement of Greenhouse effect, the of drought-resistant varieties breeding will be an important aspect in the future. The traditiong way of tea breeding is maine system breeding and plant breeding,but there are some drawback like genetic backgroud complex,breeding instability,needing long time and other defects,while the drought indicators are mostly based on environmental characteristics. Molecular breeding can effectively avoid these problems for it directly screening genetic material DNA. DD-PCR (differential display PCR, or DDRT-PCR) have advantages of simple,high sensitivity and efficiency.It is first time that study gene differentially expressed under arid environment.This experiment try to screen different exprement gene to drought-resistant index,intended to make some contribution to drought variety, and got results as followed:1. The best part extracting RNA from young tea using kit is leaves and new roots,because its have less secondary products to pollution of the extracted RNA.But RNA from roots difficult to preservation as the same environment,rapid degradation,illustrating the exracting RNA with kit had other impurities affect the preservation of root RNA.2.Amplification reaction with Mixmixture better than conventional Taq enzyme system in PCR; gel electrophoresis is 120mv;electrophoresis time about 1h;separation gel concentra-tion about1.5%;experimentparts part is leaves;the annealing temperature 45℃,when meeting these codition getting better result. When experiments parts is roots,we need put down.Annealing temperature.PCR reaction parameters is like ordinary PCR reaction.3.There are three changes afer PCR:up expression,down expression and constant expression. Some bands have mergers on anchor primers.Identified anchor primers and random premers combinations after screening.Need to determine the stress time when experiment part is young roots.4.There are many gene bands expression changed after drought stress from sequencing results,and some bands changed trend similar other researchers reported.5.Making sure this method and technique is feasible in studying anti-drought molecular nechanism on the tea,for example,we achieved good experiments results in FuDing seeding,so the method can be applied to research other tea varieties and different breeding statement of tea.更多还原