【作者】 卢燎勋；

【导师】 林拥军；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 纹枯病是水稻三大病害之一,对水稻生产造成非常大的损失。由于在现有的水稻种质资源中没有发现对纹枯病完全免疫的抗源材料,也没有克隆到抗纹枯病的主效QTL,而且水稻转基因抗纹枯病缺乏很好的新的基因资源,所以水稻抗纹枯病遗传育种进展缓慢,一直没有取得重大突破。本研究主要从以下两方面着手进行探索研究：(1)对水稻来源的对真菌病原具有潜在抗性的多聚半乳糖醛酸酶抑制蛋白基因(OsPGIP)家族进行生物信息学分析和功能验证；(2)将有效霉素A生物合成途径的8个必需基因转化水稻。希望一方面可以深入分析水稻内源抗病相关基因并从中挑选出对水稻转基因抗纹枯病育种有潜在应用价值的基因；另一方面打破固有的水稻转基因抗纹枯病育种思路,将农业生产上应用得十分成熟而且有效的有效霉素A生物合成途径转入水稻,以期得到更好的抗性效果。本研究获得的主要结果如下：1.通过生物信息学分析,在水稻中新找到2个OsPGIP基因,它们都具有典型的LRR结构域和膜定位信号肽以及保守的半胱氨酸残基。2.对不同物种中PGIP基因家族做进化树分析,发现所有的禾本科植物中的PGIP基因亲缘关系比较近,单子叶和双子叶植物中的PGIP基因进化关系比较远,表明PGIP基因在进化中具有较高的保守性。3.对水稻籼稻品种明恢63(Minghui63,MH63)中OsPGIP基因家族成员(除了OsPGIP3)在不同组织和器官的表达谱进行了分析,结果表明这6个基因的表达模式具有多样化的特性,这可能与这些基因的功能分化有密切联系。4.为了验证实验室芯片数据库的结果,我们对水稻籼稻品种MH63三叶一心期幼苗中OsPGIP基因家族在不同激素处理下的表达谱进行了分析,结果表明这7个基因都对GA3,KT和NAA处理有响应。进一步对水稻粳稻品种中花11(Zhonghua11,ZH11)三叶一心期幼苗中OsPGIP基因家族在更多激素处理下的表达谱进行了分析,结果表明在粳稻中,这7个基因对ABA,BR,GA3,IAA, JA,KT和SA处理的响应情况具有多样性。5.对水稻粳稻品种ZH11孕穗期植株中OSPGIP基因家族在纹枯病菌接种处理后的响应情况进行了分析,结果表明除了OsPGIP6的表达量在接种后下降了,其它6个基因的表达均不同程度地被纹枯病菌接种所诱导上调。6.对水稻中OsPGIP基因家族成员启动子区潜在的顺式元件进行了分析,结果表明大多数的基因启动子区都存在与激素响应或者是病原诱导相关的顺式原件,这也进一步验证了前期的实验结果。7.对OsPGIP1,2,3,4四个基因分析了其亚细胞定位。经过基因枪转化洋葱表皮细胞和PEG介导转化水稻原生质体实验,初步确定了这4个基因均定位于细胞膜上。8.挑选OsPGIP1,2,3,4这4个基因分别构建超表达载体,利用农杆菌介导的水稻遗传转化,在水稻品种ZH11中进行超表达,通过对转基因植株进行southren blot和northern blot检测,证实目标基因已经整合到水稻基因组上,而且多数家系目的基因表达量与对照相比有了显著升高。将单拷贝而且表达量高的转基因家系挑选出来进行后续研究。9.转基因植株经过2年大田接种纹枯病菌实验,结果表明OsPGIP1和OsPGIP4的部分转基因阳性株系与转基因阴性和野生型对照以及感病对照相比对水稻纹枯病菌的抗性都有显著提高,预示着这2个基因在抗纹枯病转基因育种中有潜在的应用价值。10.将有效霉素A生物合成途径的8个必需基因每一个都分别接上一个能在植物中表达的启动子,然后两两构建入一个表达载体(共构建4个载体),最后通过农杆菌介导的水稻遗传转化,将其转入水稻ZH11,得到了转基因植株。通过对转基因植株进行southern blot和northern blot检测,证实目标基因已经整合到水稻基因组上,而且多数家系目的基因表达量非常高。将单拷贝而且表达量高的转基因家系挑选出来作为杂交聚合的亲本。11.经过杂交聚合,得到了8基因聚合的植株,通过real-time PCR检测,显示在部分8基因聚合的植株中目标基因均有比较高的表达量,但是经过初步检测,在8基因聚合的阳性植株中没有检测到最终产物有效霉素A的存在。更多还原

【Abstract】 Sheath blight was one of the three major diseases of rice. It caused great losses in rice production every year. The breeding progress for sheath blight resistance was very slow and so far no break-through had been made. The major reasons could be:(1) no rice varieties had been found to be completely immune to Rhizoctonia solani;(2) no major resistance QTLs had been identified to sheath blight;(3) lack of new resistance genes for transgenic rice breeding to sheath blight. In this study, we focused on the following two aspects:(1) bioinformatic analysis and functional characterization of the rice-derived polygalacturonase-inhibiting protein (OsPGIP) gene family that could be resistant to rice sheath blight;(2) transformation of8val genes that were essential for the biosynthetic pathway of validamycin A into rice. On one hand, we expected that we could identify important endogenous defense-responsive genes, which had potential applications in transgenic breeding against rice sheath blight. On the other hand, we aimed to try a new approach for rice sheath blight resistance transgenic breeding. The main results obtained in this study were as follows:1.2OsPGIP genes were newly identified in rice by using bioinformatic analysis, which all have the typical LRR domain, signal peptides directed to the membrane and conserved cysteine residues.2. An unrooted phylogenetic tree was generated by using the alignments of the PGIP amino acid sequences from different species. The results showed that all the PGIP from the Gramineae among monocot plants were in the same group, while the PGIP from monocot and dicot had a far relation. All these results might provide an evidence for the genetic evolution of PGIP from different species were conserved.3. The expression profiles of OsPGIP gene family (OsPGIP’3not included) in different tissues and organs throughout the entire life cycle of indica rice variety MH63were analyzed. The results showed that the expression patterns of these6genes were variable, which indicated that these6genes might play different roles in the development of rice.4. In order to confirm the results of the microarray database, we treated indica rice MH63with GA3, KT and NAA at trefoil stage. The expression profiles of these7genes were analyzed. The results showed that all7genes could respond to GA3, KT and NAA treatments. We also detected the responses of OsPGIP genes in japonica rice Zhonghua11to ABA, BR, GA3, IAA, JA, KT and SA treatments. The results showed that the responses of OsPGIP genes in japonica rice to different phytohormone treatments were complicated and diversified.5. In order to obtain the information about responses of the OsPGIP gene family in rice to Rhizoctonia solani, we inoculated rice japonica variety ZH11with Rhizoctonia solani at booting stage. The results showed that except of the decreased expression of OsPGIP6, all the other six genes were up-regulated after inoculation.6. To evaluate the different expression patterns and various responses to stresses of OsPGIP genes in japonica rice, putative cis-elements in the promoter regions were checked. The results showed that most OsPGIP promoters contained at least one cis-acting regulatory element associated with pathogen or phytohormone responses, which was in accordance with the different expression patterns of OsPGIP genes.7. We analyzed the subcellular localization of OsPGIP1,2,3and4by bombardment of the onion epidermal and transformation rice protoplasts by using PEG. The results showed that all four genes were localized on the cell membrane.8. We transformed OsPGIP1,2,3and4into rice and analyzed the transgenic plants by using southern blotting and northern blotting. The results showed that all four genes were integrated into the rice genome, and in some transgenic plants, the expression of each gene was significantly increased compared to the control plants. We selected highly-expressed transgenic plants with a single copy for follow-up studies.9. The resistance level of transgenic plants to Rhizoctonia solani was tested for two years in the field trial. Results showed that some of the OsPGIP1and4transgenic lines were more resistant to sheath blight compared to the negative transgenic control, the wild-type control and the susceptible variety control. This indicated that OsPGIPI and4might be useful for application in transgenic breeding against rice sheath blight.10. In addition, in order to build a novel validamycin A biosynthesis pathway in rice, eight necessary genes were transformed into the rice genome. Those eight genes, each driven by a rice derived promoter, were first constructed, and then two expression cassettes were put into a transformation vector, resulting four transformation vectors. All these vectors were transformed to rice with Agrobacterium-mediated rice transformation method individually to obtain transgenic plants. Transgenic plants were then analyzed by southern blotting and northern blotting. Results showed that all eight genes had been successfully integrated into the rice genome, and the expression levels of target genes in some transgenic lines were very high. We selected highly-expressed transgenic plants with a single copy as hybridization parents.11. We pyramided all eight genes by genetically crosses and tested the hybrids by real-time PCR. Results showed that all eight genes were abundantly expressed in some transgenic lines, however, we could not detect the existence of validamycin A using present available methods.更多还原