【作者】 吴艳；

【导师】 何光存；

【作者基本信息】 武汉大学 ， 生物学、遗传学， 2017， 博士

【摘要】 水稻是最主要的粮食作物之一,属于双子叶植物。褐飞虱(Nilaparvatalugens Stal)是一种水稻专食性昆虫,通过口针鞘吸食韧皮部汁液,来获取营养。褐飞虱的取食能直接或间接地危害水稻的生长,严重的甚至导致死亡。对于褐飞虱的防治,最经济环保的方法是选育褐飞虱抗性水稻品种。从1969年第一个褐飞虱抗性基因BPH1被发掘开始,到现在已有三十多个抗性基因被鉴定出来,这些基因主要来自于栽培稻(如Mudgo,ASD7,Rathu Heenati)和野生稻(如Oryza officinalis,Oryza minuta,Oryza latifolia)。本文研究的对象为BPH15。BPH15来源于药用野生稻Oryza officinalis,位于4号染色体的短臂,抗虫鉴定表明其对褐飞虱具有很强的抗性。类受体激酶LecRK广泛存在于高等植物中,它的结构可以分为三部分,胞外lectin结构域,跨膜结构域以及胞内激酶结构域。有文献报道它参与了植物的发育调节,激素信号途径响应以及对生物胁迫和非生物胁迫的应答等过程。本文通过反向遗传学对LecRK的功能进行了研究,初步探索了它的抗性机理,得到的实验结果如下:1.通过亚细胞定位实验证实,LecRK是一个膜定位蛋白。通过对虫取食的感性水稻kasalath进行定量PCR实验发现,LecRK能够受到褐飞虱取食的诱导表达。同时PTI标志基因也会对褐飞虱的取食产生应答。接着我们构建了 LecRK超量表达和抑制表达的载体并别转入感性水稻品种Kasalath,获得超量和抑制表达的纯合T2代植株后对其进行抗虫和抗病实验。通过宿主选择性实验显示,褐飞虱更倾向于选择LecRK抑制表达植株,其次是野生型植株,最后是LLecRK过量表达植株,说明了 LecRK转基因植株对褐飞虱具有趋避性。在虫增重和蜜露量实验中,LecRK抑制表达植株上的褐飞虱虫增重和蜜露量最多,相较于野生型植株达到了显著性水平。而LecRK过量表达植株上的虫增重量和蜜露量则和野生型植株相比则无显著性差异。说明了Lec K抑制表达植株对褐飞虱的抗性最弱,LecRK过量表达植株最强,即LecRK正调控了水稻对褐飞虱的抗性反应。褐飞虱死亡率实验结果显示,无论是LecRK过量表达植株还是抑制表达植株上的褐飞虱死亡率和野生型植株相比都没有出现显著性差异,这表明LecRK基因并不会对褐飞虱产生致死性。利用剪叶法观察LecRK转基因植株对白叶枯病菌的抗性,结果显示LecRK抑制表达植株相较于野生型植株感病性增强,而LecRK过量表达植株相较于野生型植株表现出对白叶枯病菌抗病性增强。以上实验结果说明,LecRK同时参与抗虫和抗病反应。我们推测LecRK可能激活了水稻的PTI反应,虽然介导的抗性没有ETI那么强烈,但是可能是一个广谱抗性,参与了抗虫和抗病反应。2.表达LecRK的水稻原生质在受到褐飞虱和几丁质粉末处理后,LecRK基因的表达量相对于未处理组都有所上升。同时,处理组的PTI标志基因的表达量高于未处理组。这个结果说明了无论是褐飞虱处理还是几丁质处理都能够诱发PTI标志基因上调表达,而表达LecRK的水稻原生质在受到褐飞虱和几丁质处理后,PTI标志基因的表达量上调的更多。LecRK抑制表达植株和野生型植株制备的原生质体,在受到几丁质处理后,都能显示出氧爆发,而前者的氧爆发强度低于后者,说明在LecRK的表达量受到抑制时,由几丁质诱发的氧爆发也受到了抑制。3.利用酵母双杂交系统,我们使用LecRK的胞外结构域筛选褐飞虱文库,期望得到可能的褐飞虱体内的PAMP分子。经过两轮筛选,我们获得了 79个候选的互作蛋白,从中我们选取了 6个候选蛋白进行了后续验证和研究。我们通过酵母双杂交实验以及CoIP实验,证实了这6个候选蛋白和LecRK的胞外结构域以及全长都能够互作。在水稻原生质体中共表达这些候选蛋白,并没有影响LecRK的蛋白表达量,说明LecRK的表达不受候选蛋白的调控。利用phos-tag实验方法,发现这些候选蛋白也不会影响LecRK的磷酸化状态。4.在RH材料中发现的BPH3基因与本实验室在B5材料中发现的三个LecRK序列相同。为了探索B5中这三个LecRK与本文所研究的LecRK的关系,我们从B5的cDNA中扩增得到这三个LecRK序列并进行互作验证,发现这三个蛋白本身是能够相互作用的,而且三个蛋白之间两两都相互作用。也就是说LecRK1,LecRK2和LecRK3这三个蛋白自己可以形成二聚体,而且两两之间还可以形成多聚体。我们通过CoIP实验发现这四个LecRK可以直接和几丁质相互作用结合,所以我们推测这四个LecRK可能单独或者共同行使了 PRR分子的功能,可以识别几丁质,激发下游信号途径。5.MicroRNA是一种广泛存在于动物和植物体内的小分子RNA,虽然它的数目不多但是却有着很重要的调控作用。通过高通量测序的方法,我们从microRNA的角度分析了BPH1 的抗虫机理。找出了一些可能参与褐飞虱抗性应答的microRNA,它们可能参与了激素的调节,抗性反应以及信号转导。同时也分析了一些在抗性材料中可能起着关键抗性调控作用的microRNA,并对它的靶基因进行了预测和鉴定。更多还原

【Abstract】 Rice is one of the most important food crops.In the main cultivation area of rice,brown planthopper(BPH)is the most destructive pests to rice.BPH(Nilaparvata lugens Stal)is a monophagous,phloem-sucking herbivore.It sucks the sap from the rice phloem using its stylet,causes direct and indirect damage to rice growth,seriously,even causes rice death.The most economical and environmentally friendly method is to breed resistant rice varieties to against BPH attacks.Since the first BPH resistance gene BPH1 was identified from 1969,more than 30 BPH resistance genes have been genetically defined,most of which are from cultivated rice(e.g.Mudgo,ASD7,Rathu Heenati)and wild rice(e.g.Oryza officinalis,Oryza minuta,Oryza latifolia).The objective of this study is the candidate gene of BPH15 resistance gene,named LecRK.BPH15 derives from wild rice(Oryza officinalis).It was mapped on the short arm of rice chromosome 4 and exhibits high resistance to BPH.Receptor-like kinase LecRK exists widely in higher plants,whose structure can be divided into three parts,extracellular lectin domain,transmembrane domain and intracellular kinase domain.It has been reported that LecRK was involved in the regulation of plant development,responding for hormonal signaling pathway and biotic stress and abiotic stress.In this study,we explored the function of LecRK by reverse genetics.The resistance mechanism was preliminarily explored and the experimental results are as follows:1.Subcellular localization experiments confirmed that LecRK is a membrane targeting protein.After the susceptible rice Kasalath was fed by BPH,the expression of LecRK could be induced,as well as the response of PTI marker genes.Then,LecRK overexpression and inhibited expression vectors were constructed and transformed into the BPH-susceptible rice variety Kasalath.The overexpression and inhibited expression homozygous T2 transgenic lines were selected for BPH-resistance and disease-resistance evaluation.Host choice tests revealed that BPH was more likely to choose LecRK inhibited expression plants,followed by wild type plants,and finally LecRK overexpression of plants,suggesting that LecRK do act via antixenosis.BPH growth weight and honeydew excretion tests revealed that BPH insects feeding on LecRK inhibited expression plants produced significantly higher BPH growth weight and honeydew excretion than those on WT plants,Conversely,BPH growth weight and honeydew excretion produced by BPH Insects feeding on LecRK overexpression plants showed no significant differences compared with those on WT plants.These suggested that the resistance of LecRK inhibited expression plants to BPH was the weakest,while the resistance of of LecRK overexpression plants was the strongest,which indicated that LecRK played a positive regulated role for the resistance of rice to BPH.Experimental results of BPH mortality rate showed that there was no significant difference in the mortality rate of the BPH in LecRK overexpression plants or in the inhibited expression plants when compaired with that in WT plants,which indicated that LecRK gene expression was not lethal to BPH.A leaf cutting inoculation method was used in LecRK transgenic plants to examine the resistance to bacterial blight.The LecRK inhibited expression plants show increased susceptibility to bacterial blight than WT plants,while The LecRK overexpression plants show increased resistance to the disease than WT plants.Those results above indicated that LecRK was involved in both insect resistance and disease resistance.We hypothesized that LecRK should activate the PTI response in rice,although the resistance is not so strong as that of ETI,it may be a broad-spectrum resistance,which is involved in insect resistance and disease resistance.2.After the rice protoplasts expressed LecRK is treated with BPH and chitin powder,the expression level of LecRK was higher than that of untreated group.Additionally,the expression of PTI marker gene in treatment group was higher than that in untreated group.These results showed that both BPH treatment and chitin treatment could induce the expression of PTI marker genes,and the expression of PTI marker gene was up-regulated more in the rice protoplasts expressed LecRK.After chitin treatment,the protoplasts prepared from LecRK inhibited expression plants and WT plants both showed ROS burst,and the ROS level of the former is lower than tha of the latter.The results indicated that the ROS induced by chitin was inhibited along with the inhibitation of LecRK expression3.By yeast two hybrid system,we use extracellular domain of LecRK to screen the BPH library in order to identify some putative PAMP molecules in BPH.After two rounds of screening,we obtained 79 candidate interacting proteins,from which we selected 6 candidate proteins for further validation and investigation.By one-to-one verification in yeast cells and CoIP confirmation in rice protoplasts,we found these 6 candidate proteins could interact with both LecRK extracellular domain and full length.The protein expression of LecRK was not affected by the co-expression of these candidate proteins with LecRK.It is suggested that the expression of LecRK was not regulated by the candidate protein.Besides,we found these candidate proteins also had no effect on the phosphorylation status of LecRK by Phos-tag experimental method.4.The BPH3 gene found in RH material is the same as the three LecRK sequences found in our laboratory in B5 rice variety.In order to explore the relationship between the three LecRKs in B5 and the LecRK in this paper,the three LecRK sequences were amplified from B5 cDNA,and their coding proteins were verified to interact with themselves and each others.We found these four LecRK could associate with chitins,so we speculated that these four LecRK may exercise the function of the PRR molecule,which can identify chitin and stimulate the downstream signaling pathway.5.MicroRNA is a kind of small molecule RNA which widely existing in animals and plants.Although their amounts are not much,but they play a very important rolein regulation.From the perspective of microRNA,we analyzed the mechanism of insect resistance of BPH15.MicroRNA,which may be involved in the resistance response to BPH,was found to be involved in the regulation of hormones,resistance and signal transduction.Additionally,some microRNAs which act as the key regulatory roles in resistant materials were analyzed.Also,the target genes of these microRNAs were predicted and identified更多还原