水稻抗褐飞虱基因Bph14的分离和功能分析-生物学、遗传学

中文摘要褐飞虱（Nilaparvata lugens Stl.，BPH ）是水稻种植地区为害水稻最严重的害虫之一。褐飞虱为害水稻程度较轻时，导致水稻植株矮小、生长活力降低、分蘖减少和产量下降；而褐飞虱为害水稻严重时，将导致植株整体死亡。由于目前种植的水稻大部分都是感褐飞虱品种，农民只能使用农药来防治褐飞虱。但是农药的使用增加了农业生产的成本，并造成环境的污染。因此，最经济的、对环境最友好的防治褐飞虱的方法是种植抗褐飞虱的水稻品种。迄今为止，科学家已经在栽培稻和野生稻中定位了 21 个抗褐飞虱基因，但并没有一个抗褐飞虱基因被克隆出来，并且水稻抗褐飞虱的分子机制也不清楚。在早期研究中，我们在水稻品种 B5 中定位了两个抗褐飞虱基因 Bph14 和Bph15。其中，Bph14 基因位于水稻第三染色体长臂，Bph15 基因位于水稻第四染色体短臂。为了克隆 Bph14 基因，我们从 B5 与明恢 63 的重组自交系中选择了抗性品种 RI35，它只含有抗褐飞虱基因 Bph14。我们用 RI35 和感褐飞虱水稻品种台中本地 1 号（TN1）杂交，得到的 F1 代再自交，从而构建了 F2 作图群体。我们随机选择了 100 株 F2 植株进行抗虫鉴定，结果显示抗性和感性植株的分离比为 3：1（72:28； 2=0.48），这表明在 RI35 中单个的 Bph14 基因提供了水稻对褐飞虱的抗性。为了定位 Bph14 基因，我们用侧翼分子标记 RM514 和 SM4 筛选了 3700份 F2 植株，得到了 49 株重组单株。我们分析了这些重组单株的基因型和表型，将 Bph14 基因定位于分子标记 RM570 和 G1318 之间 120kb 的区段。为了进一步精细定位 Bph14 基因，我们从 F2 植株中选取 Bph14 区域为杂合，而其他区域来源于 TN1 的单株。这些单株自交后再选择，一直到 F5 代群体。我们用 5000份的 F5 植株来构建高密度的定位图谱，并最终将 Bph14 定位在标记 SM1 和G1318 之间 34kb 的区段。该区段测序后，我们发现有两个预测的编码抗性蛋白的基因，分别命名为 Ra 和 Rb。为了最终确定哪个基因是 Bph14 基因，我们将预测的 Ra 和 Rb 基因以及携带自身启动子的转化载体，分别转入感褐飞虱水稻品种 Kasalath。然后，我们对转基因的 T2 代做了抗虫鉴定。鉴定结果表明：所有表达 Ra 基因的转基因 T2 代植株均为对褐飞虱的抗性，而 Rb 的转基因 T2 代植株仍是感虫的。另外，我们利用 RNAi 技术抑制了抗虫品种 RI35 的 Ra 基因的表达，对 RNAi 转基因 T2 代植株的抗虫鉴定结果表明 Ra 基因被抑制的植株II丧失了对褐飞虱的抗性。因此，我们认为是 Ra 提供了水稻对褐飞虱的抗性，它就是 Bph14 基因。Bph14 基因编码一个 1,323 氨基酸的蛋白质，其具有一个螺旋 -螺旋（Coiled-coil，CC） ，核结合位点（Nucleotide-binding，NB ）和亮氨酸富集重复（Leucine-rich，LRR ）的基序。进化树分析表明，Bph14 蛋白与水稻中的其他抗病蛋白的关系较近，而与其他植物已知的抗病蛋白亲缘关系较远。我们分析了在 21 个水稻品种的 Bph14 等位基因的表达。这些品种中， Bph14 的等位基因均有转录，且表达量在大部分品种中都没有差异。但是，通过序列比较，我们发现 Bph14 蛋白的 CC 和 NB 区的功能基序在这些水稻品种中是保守的，不同水稻品种之间没有差异，而 LRR 区则存在 54 个独有的氨基酸以及两个氨基酸的缺失。Bph14 基因在水稻根、叶鞘和叶片中组成型表达，且表达量随褐飞虱取食时间而增强。为了检测 Bph14 基因的表达部位，我们构建了 Bph14 基因的启动子驱动 GUS 基因的转化载体，并转入水稻 Kasalath。我发现 Bph14 基因主要在根、叶鞘和叶片的导管和筛管周围的薄壁细胞表达。我们将 Bph14 基因与绿色荧光蛋白融合，并在洋葱表皮细胞瞬时表达。我们发现 Bph14 蛋白分布在细胞质中。为了研究 Bph14 转基因植株的抗性机制，我们观察了在抗性的 Bph14 转基因植株以及感性的野生型植株中，褐飞虱的不同表现。这些研究包括褐飞虱对宿主的选择，褐飞虱的取食行为、蜜露量、群体生长率、存活率以及产卵力。在宿主选择性实验中，我们观察到分布在转基因和野生型植株上的褐飞虱没有显著性差异。褐飞虱在不同的抗、感植株上的产卵数目也没有太大差异。我们用电子刺探仪记录了褐飞虱在水稻上的取食行为。数据结果表面，当褐飞虱取食 Bph14 转基因以及野生型植株时，褐飞虱第一次穿刺到达韧皮部的时间，以及第一次在韧皮部的消化时间并没有差异。而在取食 Bph14 转基因植株时，褐飞虱的非刺探时间以及穿刺时间有明显增加，并且在韧皮部的消化时间显著减少。与此相同的是，褐飞虱取食 Bph14 转基因植株后，分泌的蜜露也明显降低。同时，在 Bph14 转基因植株中褐飞虱的群体生长率仅为在感性品种的 1/5，并且褐飞虱在 Bph14 转基因植株上的存活率也明显比在野生型植株上低。这些结果表明 Bph14 基因介导的抗性是抗生性，能降低褐飞虱的取食，生长和存活率。III我们观察到，褐飞虱取食 Bph14 转基因植株后，Bph14 转基因植株的筛板以及维管组织的细胞壁都有大量胼胝质沉积的现象。我们调查了胼胝质相关基因的表达模式，结果发现：在褐飞虱取食后的野生型和 Bph14 转基因植株中，三种胼胝质合成酶基因（GSL1、GSL5 和 GSL10）的表达量都有上调。然而，编码胼胝质水解酶（-1, 3-葡聚糖酶）的基因（GNS5 和 GNS9）仅在 Bph14 转基因植株的表达量下调。因此，我们认为褐飞虱取食后，主要是 Bph14 转基因植株中胼胝质水解酶基因表达的下调导致了转基因植株中胼胝质的沉积和筛管的堵塞。另外，我们发现褐飞虱取食转基因植株后，转基因植株的 Bowman-Birk 胰岛素抑制剂基因的表达量上调更明显，而褐飞虱体内的胰岛素基因则受到更多的抑制。这些结果表明，Bph14 转基因植株中胼胝质的沉积以及胰岛素抑制剂产物阻止了褐飞虱的持续取食和韧皮部汁液的消化。植物防御昆虫取食的信号途径包括激活水杨酸和茉莉酸/乙烯信号分子。我们检测了褐飞虱取食后，水杨酸和茉莉酸/乙烯信号途径基因在 Bph14 转基因植株和野生型植株的表达量。在转基因植株中，茉莉酸/ 乙烯信号途径基因（LOX 、AOS2 和 EIN2）的表达比野生型明显降低，而水杨酸信号途径基因（EDS1 ，PAD4，ICS1， PAL 和 NPR1）的表达比野生型有明显上升。这些结果表明褐飞虱取食后，Bph14 基因可能激活了水杨酸依赖的抗性途径。 PR 基因被认为在植物中能有效抑制病原菌生长并阻止其扩散，使植物产生系统获得性抗性。在 Bph14 转基因植株中，PR 基因（PR1b ，PR4，PR5 和 PR10）的表达量明显比野生型低。因此，褐飞虱取食后，Bph14 激活的是一个不依赖于 PR 基因的抗性途径。Bph14 基因是第一个成功克隆的水稻抗褐飞虱基因。它的研究为水稻抗褐飞虱的分子机制奠定了基础。在 Bph14 基因介导的水稻对褐飞虱的抗性机制中，早期宿主/昆虫识别，胼胝质沉积，胰岛素抑制剂产生以及植物激活水杨酸防御信号途径的机制与植物的抗病机制具有很大的相似性。Bph14 的克隆能推动水稻抗虫基因的研究，为培育抗虫水稻打下基础。Bph14 的克隆有效控制褐飞虱，并减少了农药的使用，降低了生产成本和环境的污染。关键词：褐飞虱，Bph14，图位克隆，CC-NB-LRR 蛋白，抗生性，信号途径IVIsolation and functional analysis of a brown planthopper resistance gene Bph14 in riceDoctoral Candidate: Du BoSupervisor: Prof. He Guangcun(College of Life Sciences, Wuhan University, Wuhan 430072)AbstractThe brown planthopper (Nilaparvata lugens Stl, BPH) is one of the most destructive pests in crop production worldwide. Light planthopper infestation reduces plant height, growth vigor and the number of productive tillers, while heavy infestation cause complete drying of the crop. As the popular rice varieties are susceptible to planthoppers, farmers depend solely on chemical pesticides to control this insect, which is expensive in terms of labor, cost and environment. The most economical and environment-friendly strategy to control this insect is to grow genetically resistant rice varieties. To date, 21 BPH-resistance genes have been identified from cultivated and wild rice species. However, none of these genes has been previously cloned and the molecular mechanisms of rice resistance to the BPH have not yet been elucidated.Previously, we mapped two major BPH-resistance genes in rice B5, Bph14 on the long arm of chromosome 3 and Bph15 on the shorter arm of chromosome 4. To identify the Bph14 gene, we developed an F2 mapping population derived from a cross between RI35, a recombinant inbred line containing the Bph14 locus from B5, and Taichuang Native 1 (TN1), a BPH-susceptible indica variety.The resistant and susceptible plants segregated in a 3:1 ratio (72:28; 2=0.48) in the F2 population, indicating that a single Bph14 gene conferred the BPH-resistance in RI35.To identify the Bph14 gene, we used the flanking markers RM514 and SM4 to screen 3,700 F2 plants and obtained 49 recombinants. We analyzed the genotype and biotype of these recombinants and located the Bph14 gene in a 120-kb region between the markers RM570and G1318. For further fine mapping, we selected the F2 plants in which the region around Bph14 was heterozygous and other regions were derived from TN1. These plants were self-pollinated and selected until the F5 generation. The F5 population consisting of 5,000 plants was used for the high-resolution mapping and Bph14 was defined on a 34-kb region between SM1 and G1318. Two predicted genes encoding putative resistance proteins, designated Ra and Rb respectively, were Videntified after sequencing this region. To determine which gene was Bph14, the predicted Ra and Rb genes with their native promoters, respectively, were transferred into the BPH-susceptible indica variety Kasalath. Then we examined the T2 families for BPH-resistance using the bulked seeding test and found that all of the T2 transgenic lines expressing Ra showed high resistance to the BPH and the transgenic plants carrying Rb were also susceptible. In addition, we used RNA interference (RNAi) to suppress the expression of Ra in the RI35 rice plants and the RNAi-transgenic lines were susceptible. Thus, we concluded that Ra confers the resistance phenotype and is the Bph14 gene.The Bph14 gene encodes a putative 1,323 amino acid protein containing a coiled-coil, nucleotide-binding and leucine-rich repeat (CC-NB-LRR) motif. Phylogenetic analysis revealed that Bph14 is closely related to other rice disease resistance proteins and is divergent from the majority of known plant disease resistance proteins in other species. We analyzed the expression of the Bph14 alleles for 21 rice varieties. The results showed that the transcripts were present in all of the rice varieties and the transcript levels were not significantly different in most of the varieties. Through comparison of the coding sequences between Bph14 and its alleles, we found that the central motifs of the CC and NB domain are well conserved among these rice varieties, but in the LRR domain 54 residues and two deletions of Bph14 were unique.The Bph14 gene was expressed constitutively in leaf sheaths, leaf blades and roots and was enhanced by BPH feeding. Then we examined Bph14 activity using transgenic plants carrying the fusion constructs of the Bph14 promoter region and the GUS reporter gene and we found that Bph14 was expressed mainly in the parenchyma cells bordering xylem vessels and sieve tubes of various organs, including leaf sheaths and leaf blades. By translation a Bph14 and green fluorescent protein (GFP) fusion construct in onion epidermal cells, we found that Bph14 protein is localized in the cytoplasm.To explore the resistance mechanism of the Bph14-transgenic plants, we investigated the responses of the BPH insects feeding on the resistant Bph14-transgenic and susceptible wild-type plants in terms of host choice, feeding activities, honeydew excretion, population growth rate, nymph survival and fecundity. In host choice tests, there was no significant difference in numbers of BPH nymphs that settled on the plants between the transgenics and wild-type. Neither was it different in number of eggs found on the plants. We also recorded in detail the feeding behavior VIof BPH on rice plants in real time employing the electronic penetration graph (EPG) analysis. The EPG data showed that there were no significant differences between the transgenic plants and the wild-type in the time from the beginning of plant penetration to the first phloem ingestion and the duration of the first phloem ingestion. However, the duration of non-probing and penetration was significantly longer and the duration of phloem ingestion was clearly shorter on the transgenic plants, compared with the wild-type plants. Honeydew excretion was found to be lower on the transgenic plants compared with that on the wild-type plants. The population growth rate of the insects on the transgenic plants was only one-fifth of that on the wild-type plants. There was a pronounced decrease in the survival rate of the insects on the transgenic plants. Taken together, these results demonstrate that Bph14 confers an antibiotic resistance that reduces the feeding, growth rate and longevity of the BPH insects.We observed that callose was deposited abundantly on sieve plates and the cell walls of vascular tissue in the Bph14-transgenic plants after BPH infestation. Then we examined the expression patterns of callose-related genes. Three callose synthase-encoding genes, GSL1, GSL5 and GSL10, were clearly up-regulated in both the wild-type and transgenic rice plants after BPH infestation. However, the expression of the genes, GNS5 and GSN9, encoding callose-hydrolyzing enzyme -1, 3-glucanase, was slightly down-regulated in the transgenic plants, which prevented callose decomposing and kept the sieve tubes occluded. In addition, the expression of the Bowman-Birk trypsin inhibitor genes was enhanced in the transgenic plants, while the expression of the trypsin gene was suppressed to a greater degree in the BPH insects fed on the transgenic plants than on the wild-type plants. These results indicate that the callose deposition and trypsin inhibitor production prevent BPH insects from continuously ingesting and digesting phloem sap in the Bph14-transgenic plants.Plant defense responses to insects include the activation of pathways dependent on SA (salicylic acid) and JA (jasmonic acid) / ethylene signaling molecules. We examined transcript levels of defense-responsive genes involving in SA- and JA/ethylene-dependent pathways. The transcript levels of the genes, LOX, AOS2 and EIN2, involving in the JA/ethylene- dependent pathway were substantially lower in the transgenics. However, transcript levels of the genes, EDS1, PAD4, ICS1, PAL and NPR1, involving in the SA-dependent pathway were higher in the transgenic plants after BPH infestation. These results suggest that Bph14 may activate an SA-dependent resistance pathway after BPH feeding. PR genes are suggested to be effective in VIIinhibiting pathogen growth, multiplication and/or spread, and are responsible for the SAR (systemic acquire resistance) in plants. The transcript levels of PR genes, PR1b, PR4, PR5 and PR10, were substantially lower in the transgenics than in wild-type plants. These results demonstrate that Bph14 activates a PR-independent resistance pathway after BPH feeding.To our knowledge, this is the first report of a BPH-resistance gene, Bph14, in rice. Our results provide evidence of the molecular mechanism of rice resistance to the BPH. In the Bph14-mediated resistance of rice against the BPH, early host/insect recognition, callose deposition, trypsin inhibitor production and the activation of plant defenses through the SA signal transduction pathway are fundamentally similar to defense mechanisms against pathogens. The identification of the Bph14 gene should facilitate searches for and the study of more insect-resistance genes in rice, and presents enormous possibilities for breeding or engineering rice varieties. The identification of this gene will improve control of the BPH, while simultaneously reducing pesticide usage and decreasing economic and environmental costs.Key word: Brown planthopper, Bph14, Map-based cloning, CC-NB-LRR protein, Antibiosis, SA signaling pathway目 录中文摘要 .IABSTRACT.IV第一章 前 言 .11. 褐飞虱研究概况 .11.1 褐飞虱的形态特征及其生活史 .11.2 褐飞虱的生物型 .11.3 褐飞虱的取食行为及其对水稻的为害 .31.4 水稻抗褐飞虱资源的挖掘和抗褐飞虱基因的研究 .32. 植物对昆虫抗性研究进展 .62.1 植物对昆虫的直接防御 .72.1.1 植物中具有抗虫作用的抑制剂 .82.1.2 植物中具有抗虫作用的次生化合物 .92.2 植物对昆虫的间接防御作用 .