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摘要：本试验旨在探究高温胁迫对菊芋sRNA通路上的几个关键酶基因的影响,尝试研究高温胁迫对菊芋的影响过程及其机理，并为筛选或培育耐高温的菊芋提供一些理论基础。实验采用电子克隆的方法成功克隆出基因AGO1、AGO5、DCL1、RDR1；采用高温胁迫（25℃和30℃共2个温度梯度，分别处理10天、15天、20天）下的菊芋作为实验材料，提取其RNA，经过反转录、RT-PCR，以及转化、测序等过程来得出结论。荧光定量结果表明经高温胁迫后的样品的4种基因的表达量比起未经高温胁迫处理的样品的表达量要高，基因AGO5、DCL1、RDR1的表达量随高温处理的时间增加有增加的趋势，而AGO1的表达量则随处理时间的增加出现了先增加后减少的趋势，这四种基因可能在对高温胁迫的应答中起着一定作用，同时这四种基因的表达在应答高温胁迫的过程中可能会有时间的先后与作用机理的不同。35713
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毕业论文关键词：菊芋；高温胁迫；AGO；DCL；RDR
Expression Analysis of Several Key Enzyme Genes of Jerusalem artichoke sine pathway under high temperature stress
Abstract：The aim of this study was to explore the possible effects of high temperature stress on several key enzyme genes and their expression analysis in sargus sRNA pathway, and to study the effects of high temperature stress on Jerusalem artichoke and its mechanism, and to provide some theoretical basis for screening or cultivating high temperature resistant Jerusalem artichoke. We were successfully cloned genes AGO1, AGO5, DCL1, RDR1 by electronic cloning, the results showed that the cDNA of Jerusalem artichoke was extracted by high temperature stress (25 ℃ and 30 ℃ a total of 2 temperature gradient, respectively, for 10 days, 15 days, 20 days) as the experimental material. The RNA was extracted and analyzed by reverse transcription, RT-PCR, as well as transformation and sequencing. Finally, the gene was quantitatively analyzed by fluorescence quantitative PCR. The gene was then quantitatively analyzed by fluorescence quantitative PCR. The results showed that the length of gene AGO1 in Jerusalem artichoke is 149bp, the length of AGO5 is 153bp, the length of DCL1 is 99bp, the length of RDR1 is 81bp, and the expression of four kinds of genes after high temperature stress was higher than those of samples without high temperature stress. The expression of AGO5, DCL1 and RDR1 increased with the increase of the time of high temperature treatment, while the expression of AGO1 increased first and then decreased with the increase of treatment time. Therefore, the four genes could have some effects on the response of temperature, while the four genes in the high temperature stress response might have different mechanism.
Key words: Jerusalem artichoke; high temperature stress; AGO; DCL; RDR
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1材料与方法3
1.1实验材料3
1.2实验试剂3
1.3仪器与设备3
1.4实验方法4
1.4.1电子克隆4
1.4.2菊芋的处理4
1.4.3引物合成以及总RNA的提取5
1.4.4 RT-PCR5
1.4.5序列测定与分析 5
1.4.6 实时荧光定量PCR6
1.4.7 数据处理6
2实验结果6
2.1基因拼接结果6
2.2总RNA的提取6
2.3引物检测6
2.4实时荧光定量PCR7
3讨论8
4结论9
致谢9
参考文献9
高温胁迫下菊芋sRNA通路几个关键酶基因的克隆表达
引言：菊芋，菊科，向日葵属，多年宿根性草本植物。高1-3米，有块状的地下茎及纤文状根。茎直立，有分枝，被白色短糙毛或刚毛。叶通常对生，有叶柄，但上部叶互生；下部叶卵圆形或卵状椭圆形。头状花序较大，少数或多数，单生于枝端，有1-2个线状披针形的苞叶，直立，舌状花通常12-20个，舌片黄色，开展，长椭圆形，管状花花冠黄色，长6毫米。瘦果小，楔形，上端有2-4个有毛的锥状扁芒。花期8-9月。具有耐寒耐旱、抗风沙、耐盐碱等抗性，此外一些品种还具有耐受和富集重金属的能力[1]。