【作者】 陈惠英；

【导师】 蔡新忠；

【作者基本信息】 浙江大学 ， 植物病理学， 2012， 硕士

【摘要】 DCL(Dicer-like)是一类保守的dsRNA特异性核糖核酸内切酶,其在RNA降解、转录后基因表达调控和抗病毒侵染等方面具有重要作用。目前已知的植物DCL基因信息及其抗病调控功能研究主要集中在拟南芥、水稻和杨树上,其它植物上的相关研究很少。烟草是重要经济作物,也是植物与病原菌互作研究的经典植物材料。本研究分析了普通烟(Nicotiana tabacum)和本氏烟(N.benthamiana)DCL的抗病调控功能,获得以下主要研究结果：(1)获得了4个烟草DCL基因的RNAi转基因烟草植株。本研究构建了适用于烟草转化的、分别包含4个烟草DCL基因片段发夹结构的RNAi载体。利用组织培养和转基因技术,获得了各NtDCL基因RNAi(RNAi-NtDCL)转基因普通烟植株：RNAi-NtDCL1植株67株,RNAi-NtDCL2植株33株,RNAi-NtDCL3植株27株,RNAi-NtDCL4植株31株。对插入的T-DNA上NPTⅡ基因片段和发夹结构内含子pBS-IN片段的PCR检测分析得出,4类NtDCL基因RANi结构的转基因阳性率约为55%。通过实时荧光定量PCR检测各转基因株系中DCL基因沉默效果,发现NtDCL(1-4)表达量分别受到不同程度的抑制,沉默效果较好的株系中,其相应DCL表达量只有对照的1/10左右。对RNAi-NtDCL植株的表型分析结果显示,NtDCL1基因的RNAi影响植株的生长发育,但NtDCL2、 MDCL3、NtDCL4的RNAi对烟草生长发育的影响不大。NtDCL1基因沉默后,抑制植物的生长,导致植株矮小,叶片畸形(新叶增生),老叶细长、破损,花叶和根系不发达等。(2)通过实时荧光定量PCR分析了DCL基因在烟草不同组织中的表达情况。结果表明：在烟草中4个不同DCL基因表达具有组织特异性。同一DCL基因在同种植物不同组织中表达不同：在花器中表达水平较高,而在营养器官如茎、叶肉、叶脉中表达水平较低。不同DCL基因在同一植物组织中表达量不同：本氏烟茎、叶肉、叶脉中DCL基因的表达量高低次序依次为：DCL2>DCL1>DCL4>DCL3,且DCL2表达量显著高于其它DCL基因。同一基因在不同植物相同组织中,其表达量也存在一定差异。在茎和叶脉等维管束组织中,普通烟DCL(1-4)基因表达量均高于本氏烟,在叶肉和花器中,这一规律并不明显。(3)采用瞬时RNAi技术研究了DCL基因对植物过敏反应(Hypersensitive response,HR)和抗病性的调控功能。与(?)RNAi处理部位相比,DCL(1-3)的RNAi处理部位中,表达Cf-4/Avr4的农杆菌浸润组织绿色更深；接种烟草野火病菌(Pseudomonas syringae pv. tabaci, Pst)后发病更快、病害症状更严重；但接种菌核病菌(Sclerotiniasclerotiorum, Ss)后病害症状减轻。但DCL4作用并不明显。研究结果表明,DCL(1-3)基因在烟草对Cf-4/Avr4介导的HR和Ss抗病性中起负调控作用,而对Pst抗病性中起正调控作用,因而是植物抗病反应的重要调控因子。更多还原

【Abstract】 DCL(Dicer-like) proteins are the members of the conservative dsRNA-specific RNase III family. They are key components in small RNA biogenesis and RNA interference-related processes, and play a pivot role in anti-pathogens defense. These information is mainly from Arabidopsis and rice. In tabacoo, however, DCLs remain uncharacterized. Tobacco is an economically important crop, as well as a model plant species to study plant-pathogen interactions. In this study, we performed functional and expression analyses of the tobacco DCLs, and obtained results mainly as follows:(1) Transgenic tobacco plants carrying RNAi constructs for the four tobacco DCL genes were generated. To this end, RNAi constructs harboring a harpin of DCL gene fragments and suiting for tobacco transformation were made. Employing tissue culture procedure, transgenic tobacco plants carrying RNAi constructs for NtDCL genes (RNAi-NtDCL) were generated,67,33,27and31plants for RNAi-NtDCL1through RNAi-NtDCL4in turn. Detection by PCR for a NPTII fragment in T-DNA and the intron sequence in the harpin of the RNAi constructs revealed that the positive rate of gene transformation is around55%. Analysis of quantative Real-time PCR (qRT-PCR) for DCL gene expression demonstrated that expression of NtDCLs was repressed to a various extent in minimum of only1/10in comparisom with the non-RNAi controls. Investigation of phenotype of the RNAi-NtDCL plants showed that RNAi of NtDCL1, but not other DCLs, affected plant growth and development, resulting in stunted plants, abnormal leaves (slender only-main-vein leaf, curly leaf etc.) and underdeveloped roots. However, silencing of NtDCL2, NtDCL3and NtDCL4in N. tabacum have no obvious developmental defects under normal growth conditions.(2) Expression of DCL genes in avariety of plant tissues was analyzed by qRT-PCR. DCLs are ubiquitously but not evenly expressed in different tissues. The same DCL gene was expressed differently in the different tissues, highly in flower tissues, while lowly in the vegetative organs such as stem, leaf mesophyll, and vein. DCL genes expressed to a different level in same tissues. In the vegetative organs of N. benthamiana, Expression level of DCL2was significantly higher than the other DCLs, followed by DCLI and DCL4, DCL3the lowest. In addition, expression of DCLs differed in the same tissues of the two Nicotiana species. In the stems, veins and other vascular tissues, expression of all the DCL genes was higher in N. tabaccum than in N. benthamiana, while it is not obvious in the leaves and flowers.(3) Functions of DCLs in regulation of plant hypersensitive response (HR) and disease resistance were analyzed employing transient RNAi assays. Compared with non-RNAi control leaves, in RNAi leaves, areas infiltrated with agrobacterium suspension expressing Cf-4/Avr4were deeper green, areas inoculated with Pseudomonas syringae pv. tabaci (Pst) developed wild-fire symptom more quickly and into more severe symptoms, however, areas inoculated with Sclerotinia sclerotiorum (Ss) displayed less severe symptoms. However, there is no obvious difference between the control and RNAi-DCL4leaves, These data revealed that DCL1to DCL3, but not DCL4probably, negatively regulate Cf-4/Avr4-dependent HR and resistance to Ss, while positively regulate resistance to Pst in tobacco, and thus act as important regulators of plant disease resistance.更多还原