植物衰老控制基因克隆分析及叶片衰老遗传调控

发布时间：2017-12-28 20:23

  本文关键词：植物衰老控制基因克隆分析及叶片衰老遗传调控　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

  更多相关文章： 叶片衰老 拟南芥 LRR-RLKs基因 烟草 乙醇 NAP基因 诱导基因表达

【摘要】：衰老作为叶片发育的最后一个时期,其过程高度有序,同时又可被大量衰老相关基因(SAGs)严格调控。研究表明,衰老过程中会发生细胞程序性凋亡及营养物质的再分配,因此,叶片衰老对作物产量、品质具有重要影响,因此,叶片衰老的研究具有重要理论意义及应用价值。本研究以模式植物拟南芥和烟草为研究对象,初步探讨了叶片衰老分子及遗传调控机理,主要研究内容包括通过筛选LRR-RLK(富含亮氨酸类受体激酶)基因T-DNA插入突变体,明确了LRR-RLK基因在叶片衰老过程中的重要作用,同时对两个LRR-RLK基因SRK1,SRK2进行了功能验证,另外,本研究利用乙醇诱导型启动子诱导NAP基因表达,初步探索生产上实现人工调控衰老的可行技术。主要结果包括：(1)筛选参与叶片衰老的拟南芥LRR-RLK基因,并对SRK1及SRK2进行功能验证已有报道,类受体激酶在植物发育及抗性反应中具有重要作用。LRR-RLK是其中一类最大的亚家族。然而,LRR-RLK是否参与了叶片衰老过程鲜有报道。本研究通过PCR方法鉴定到了26个LRR-RLK基因纯合T-DNA插入突变体,进一步通过衰老相关表型分析,发现其中6个LRR-RLK突变体能够影响叶片衰老进程,我们对其中2个基因SRK1(At4g08850),SRK2(At2g37050)进行了较深入的功能验证。研究表明,SRKl及SRK2基因缺失突变体都表现为延迟衰老,叶绿素含量及FV/Fm比值等生理指标进一步验证了该结论；同时,我们构建了SRK1及SRK2的过量表达载体,并转化野生型拟南芥;另外,我们分别提取野生型及srk1,srk2突变体的不同时期叶片(幼叶、完全伸展、衰老早起、衰老晚期)的RNA,反转录获得cDNA,对SRK1,SRK2进行表达模式分析。(2)在烟草中构建诱导表达模式的叶片衰老可控体系诱导系统可使目的基因在合适的时空实现表达。转录因子NAP基因已被报道可在多种植物/作物中调控叶片衰老,因此,本研究探索了通过诱导表达NAP基因实现衰老的人工调控。利用PCR方法分别从拟南芥和烟草中克隆到NAP基因的CDS序列,通过酶切连接的方式构建了AlcA启动子驱动的NAP基因的诱导表达载体；通过农杆菌介导的遗传转化方法获得T0代转基因烟草；对转基因烟草喷洒03%乙醇,10天后发现与对照相比,转基因植株有明显的早衰表型；另外,对分离的叶片进行相同处理,两周后乙醇处理的材料较对照有明显早衰表型；本研究结果为生产上实现人工可控的衰老模式提供了理论依据。本研究通过筛选拟南芥LRR-RLK突变体,明确了LRR-RLK基因在叶片衰老过程中具有重要作用,并对SRKl和SRK2进行了功能验证；另外,我们构建乙醇诱导NAP基因表达的转基因烟草,并对转基因植株进行了乙醇诱导表型观察,初步明确了生产上实现人工可控衰老调控模式的可行性。本文研究结果对深入理解叶片衰老调控机理具有重要意义,同时也为生产上应用衰老可控技术实现作物高产提供一定的理论依据。
[Abstract]:As the last period of leaf development, senescence is highly orderly and can be strictly regulated by a large number of aging related genes (SAGs). Studies show that programmed cell apoptosis and redistribution of nutrients can occur during aging. Therefore, leaf senescence has an important impact on crop yield and quality. Therefore, leaf senescence research is of great theoretical significance and application value. In this study, Arabidopsis and tobacco as the research object, to explore the molecular and genetic mechanism of leaf senescence, the main research contents include the selection of LRR-RLK (leucine rich receptor kinase) gene T-DNA insertion mutants, clear LRR-RLK gene plays an important role in leaf senescence, while the two LRR-RLK gene SRK1. The SRK2 function has been verified, in addition, this study uses ethanol inducible promoter induced NAP gene expression and explore feasible implementation of artificial aging on production control. The main results are as follows: (1) screening Arabidopsis thaliana LRR-RLK genes involved in leaf senescence, and functional verification of SRK1 and SRK2 have been reported. Receptor like kinases play an important role in plant development and resistance response. LRR-RLK is one of the largest subfamilies. However, it is rarely reported whether LRR-RLK participates in the process of leaf senescence. This study through the PCR method identified 26 LRR-RLK gene homozygous T-DNA insertion mutants, through further analysis of aging related phenotypes, found that 6 LRR-RLK mutant can affect leaf senescence, we identified 2 genes SRK1 (At4g08850), SRK2 (At2g37050) for a more in-depth functional verification. Research shows that SRKl and SRK2 gene deletion mutants showed delayed senescence, chlorophyll content and the ratio of FV/Fm and other physiological indicators to further validate the conclusion; at the same time, we constructed the overexpression vector of SRK1 and SRK2, and transformed into wild type Arabidopsis; in addition, we do not extract wild type and srk1 mutant srk2 leaves in different periods (the young leaves, fully extended, early aging, aging late) RNA, reverse transcription cDNA, SRK1, SRK2 expression pattern analysis. (2) the induction system of leaf senescence controlled by the induction of expression pattern in tobacco can make the target gene express in the appropriate time and space. Transcription factor NAP gene has been reported to regulate leaf senescence in many plants / crops. Therefore, this study explored artificial control of senescence by inducing expression of NAP gene. By the method of PCR were cloned from Arabidopsis and tobacco to the CDS sequence of NAP gene, by enzyme connection way to construct the AlcA expression vector of NAP gene promoter induced promoter driven; generation of T0 transgenic tobacco by Agrobacterium mediated method; transgenic tobacco sprayed with 03% ethanol, and found 10 days later compared to the control, the transgenic plants have obvious senescence phenotype; in addition, the same treatment leaves on separation, two weeks after ethanol treatment materials have obvious premature aging phenotype compared with the control; the results of this study provides a theoretical basis for the production of artificial aging controlled mode. This study through the screening of the Arabidopsis LRR-RLK mutant, the LRR-RLK gene plays an important role in the senescence of leaves, and the SRKl and SRK2 to verify the function; in addition, we constructed transgenic tobacco ethanol induced expression of NAP gene, and the transgenic plants were observed phenotype induced by ethanol, initially identified the production feasibility of artificial control aging regulation mode. The results of this study are important for understanding the mechanism of leaf senescence control, and also provide a theoretical basis for the application of aging control technology to achieve high yield.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2
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本文编号：1347285