导　　师： 何光存

学科专业： 071007

授予学位： 博士

作　　者： ;

机构地区： 武汉大学

摘　　要： 水稻(oryza sativa l.)是世界上最重要的粮食作物,褐飞虱(nilaparvata lugens stal.)是水稻的主要害虫之一.b5是药用野生稻(o.officinalis wall ex watt)与栽培品种珍汕97b杂交得到的转育材料,带有qbp1和qbp2两个抗褐飞虱基因,对褐飞虱表现为高抗.该文选用b5为材料,对水稻抗虫反应中基因表达的调控进行了研究,主要结果如下:1.构建了褐飞虱取食后b5的正、反向抑制差减杂交(suppression substrative hybridization;ssh)文库.2.筛选得到136个可能的抗虫相关的表达序列标签(expressed sequencetags;est).3.构建了一个褐飞虱诱导的b5 cdna文库.以经褐飞虱取食的三叶期b5为材料,构建了褐飞虱诱导的全长b5 cdna文库,该文库含有1.1×106个克隆,平均插入片断长度为1.5kb,符合筛选低丰度cdna的要求.4.分离了三个受褐飞虱诱导的特异表达基因的全长cdna.5.原位杂交分析.选取了3个经northern杂交证实受褐飞虱诱导特异表达的基因进行mrna原位杂交实验,结果表明,基因os-nc1在未受褐飞虱取食的水稻根、叶鞘、叶中没有杂交信号.6.对基因os-nc1的相关特性分析.基因os-nc1在northern杂交中表现为受褐飞虱强烈诱导,因此,对此基因的相关特性进行了具体分析.该研究所揭示的大量信息为今后进一步研究抗刺吸式昆虫反应机理和反应途径提供了许多有价值的线索.水稻抗褐飞虱相关基因的分离,为认识水稻抗褐飞虱反应的复杂机理奠定了重要的基础;对一些基因相关特性的分析也使我们对植物与刺吸式昆虫之间复杂的关系有了初步的了解;另一方面差异表达基因中紧邻抗褐飞虱主效基因的est的获得,也为该室的抗褐飞虱基因的克隆提供了新的分子标记. Brown planthopper /(Nilaparvata lugens Stal., abbrievated as BPH/) is one of the most serious pests of rice /(Oryza sativa L./), which is a typical specialist herbivore of rice that sucks assimilates from the phloem of plants and causes severe lost of rice grain yield. In genomic study, rice is a model organism, because of its small genome size, availability of high-density genetic and physical map, and high degrees of synteny with other cereal plants. Rice and brown planthopper has become an ideal model system for studying interaction between plants and sucking insects. Scientists all over the world have tried to study the mechanism and the biochemical and physiological basis of plant defense to BPH, which include the feeding action and physiology of BPH; The influence of different resistant rice cultivars to the development of BPH; Impacts of BPH feeding upon rice plant, and so on. Understanding the mechanism of rice-BPH interaction will help to develop insect-resistant rice variety and novel environment-nonpolluting insecticides. However, all in all, limited knowledge is shared about the mechanisms of plant-insect interactions and the molecular and physiological changes in plant after the sucking insect infestation in rice. B5 is an introgression line derived from a cross between an accession of O. officianlis collected in China and cultivated, carrying two main effect BPH resistance genes Qbp1 and Qbp2, and showing high resistance to BPH. In this study, B5 was used as objective materials, and the extremely sensitive plants from recombinant inbred lines as materials to eliminate the background. By combining the technique of SSH and cDNA array, we systemically analysed the gene expression of B5 responding to BPH. The results are as follows: 1. Construction of the forward- and reverse- suppression substrative hybridization /(SSH/) library. The forward SSH was performed using poly /(A/)+ RNA from B5 plants infested with BPH as 'tester' and those mixed from the BPH-infested extremely susceptible bulk and the uninfected B5 as 'driver'; whereas, the reverse SSH was conducted by using the mixed poly /(A/)+RNA as 'tester' and the BPH-infested B5 as 'driver'. The forward and reverse SSH cDNA libraries contain 3,900 and 1,800 independent clones, respectively. 2. One hundred and thirty-six possible resistant BPH genes were screened. 5,700 rice cDNA clones from the SSH cDNA libraries were arrayed on Hybond-N+ membranes in two identical copies by Q-Pix /(Genetix, UK/). RNA samples from 'Tester' and 'Driver' were used to synthesize cDNA as probes for screening the SSH libraries, respectively. Clones showing difference two-fold or even more /(=2/) stronger in signal intensity between two kinds of probes were considered as BPH-regulated genes. A total of 120 clones showed increased expression and 34 presented decreased expression after BPH infestation. After sequencing, 136 are unique genes and the other 18 are duplicated. Sequence analysis revealed that 66 of the 136 sequences had various degrees of homology with known genes in databases, and could be classified into several functional groups according to the predicted functions. 61 are homologous to the unknown protein or cDNA clones from rice or other plants, and 9 were novel ESTs. The results indicated that the responses between rice to BPH and the other biotic and abiotic stresses are crosstalk; the important genes involved in the JA signal pathway weren't induced by BPH. In addition, many of these genes presented first time in the interactions of plant and insect, which suggested that some specific responses to BPH infestation existed in rice plant. 3. Construction of BPH-induced cDNA library of rice. Three-leaf stage B5 plants fed by BPH were used as original materials to construct cDNA library. The library contains 1.1 X 106recombinants, and the average length of inserts is about 1.5 kb. The titer of the amplified cDNA library is 1.1 X 1011pfu//ml. This library is suitable for screening low abundance genes. 4. The cDNA library was screened with EST BPHiw001, BPHiw042

关 键 词： 褐飞虱 水稻 抑制差减杂交 抗褐飞虱相关基因 点阵技术

分 类 号： [S511.034 Q943.2]

领　　域： [农业科学] [生物学]