【作者】 陈德西；

【导师】 李仕贵；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 Ⅰ抗稻瘟病基因pi-d2导入水稻提高稻瘟病抗性的研究水稻稻瘟病是水稻生产中的最严重病害之一，俗称水稻“癌症”。控制稻瘟病害的方法主要是使用杀菌剂和利用抗病品种。植物基因工程的兴起为病害的控制提供了更广泛的选择余地。利用该技术可向现有栽培品种中导入外源抗病基因而不受种属限制，拓展了可利用基因的来源，这无疑为作物抗病育种工作开辟了一条崭新而有效的途径和发展空间。在水稻中已克隆了六个抗病R基因Pi-b、Pi-ta、Pi-9、Pi-2、Piz-t和Pi-d2。前5个基因蛋白属NBS-LRR类蛋白，而Pi-d2具有一个独特的B-lectin而作为新的一类抗病蛋白。本研究首次利用农杆菌介导法将Pi-d2基因转入水稻感病品种丽江新团黑谷、日本晴和抗病品种中花9号等，进行分子生物学检测、GUS活性检测和抗病性鉴定，得到了对稻瘟病抗性提高的水稻材料，为水稻以及其它禾本科植物抗病分子育种奠定基础，并为有性杂交育种提供了基因资源。主要研究结果如下：1以不同水稻品种的幼胚和成熟胚作为外植体，通过农杆菌介导法将目的基因三种不同的表达载体（含有长为3.2kb自身启动子驱动的Pi-d2基因组序列的pCB6.3k、含有长为2.2kb自身启动子驱动的Pi-d2基因组序列的pCB5.3k和35S驱动的Pi-d2基因全长cDNA序列的表达载体pZH01-2.72kb以及阴性对照载体pZH01-tp309）导入到水稻中，获得一批转Pi-d2基因的潮霉素抗性植株。2在农杆菌介导过程中首次将PSK添加在共培养基中提高了转化效率。PSK浓度为10nmol/L时，抗性愈伤出愈率比对照提高最高达11％；当PSK为200noml／L时，几乎完全抑制抗性愈伤的生长。PSK还与筛选培养基中的2，4-D浓度有关，2，4-D为2mg／L比1mg／L更有利抗性愈伤生长。3 PCR、RT-PCR、Southern blot杂交等分子检测结果证明外源基因以1-4个拷贝随机插入水稻基因组中，并在转基因植株中稳定遗传。对转基因植株后代的潮霉素抗性分析表明转基因的整合情况大多为单位点、单拷贝。潮霉素的抗性已稳定传至T₂代，获得转基因纯合株系。4对35个独立转基因株系的T₁代植株接种结果表明转基因植株对稻瘟病生理小种ZB15的抗性提高，病斑大小和数量减少，抗性呈孟德尔和非孟德尔分离。目的基因三种表达载体在受体中所表现的对ZB15抗性无明显差异。T₂代潮霉素抗性植株接种结果表明转基因株系仍然对稻瘟病ZB15具有抗性，3个株系内存在抗感分离，且T₁代抗性较好的株系在T₂代对稻瘟病仍具有较好的抗性，T₁和T₂代植株抗稻瘟病性结果较为一致。5连续三年的田间试验表明转基因植株抗穗颈瘟的能力大部分增强；抗叶瘟的转基因株系不一定抗颈瘟。通过田间试验，筛选到高抗稻瘟病株系。6用39个稻瘟病菌株测定转Pi-d2基因9个高代材料的抗谱表明不同转基因株系的抗病频率不同，目的基因在感病植株中的表达抗病频率提高十分明显，最高提高达91.7％。而在抗性品种中花9号中抗病频率基本未提高。用中国农科院收集的58个菌株对四个转基因早代纯合株系的抗谱测定表明转基因品系对81.48％以上菌株表现抗性，具有广谱抗性特点。7通过对目的基因不同三个表达载体在转基因植株中的表达分析，表明Pi-d2基因长为2.2kb或3.2kb自身启动子在转基因植株中具有启动子功能。具有Pi-d2基因基因组序列的转基因植株与具有cDNA序列转基因植株对稻瘟病的抗性都有提高。8对转基因农艺性状考查结果表明除少部分农艺性状发生变化外，大部分转基因植株农艺性状没发生显著变化。对随机抽取的一份转基因进行的稻米营养成分测定，结果表明转基因稻米中的营养成分未发生明显改变。Ⅱ一份条斑和颖花异常水稻突变体研究水稻是单子叶植物的模式植物。对水稻的发育过程进行研究具有重要的理论意义和应用价值。本实验在水稻遗传转化过程中发现一个不含外源基因的条斑和颖花异常的突变体，并对突变体进行了形态和组织学鉴定及遗传分析，为突变基因的克隆打下基础。其研究结果如下：1突变体表型特征：该突变体的茎、叶、穗出现条斑。在分蘖盛期，一些叶片始分岔或卷曲；花器官数目增多，表现为多内外稃，叶片状浆片，或浆片增大，雌蕊增多，颖花开裂。极端表型为同一颖花中长出几朵小花或小花枝梗伸长为花序。2生理特征：叶绿素总含量和净光合速率明显低于野生型。在分蘖中期、开花前期和开花后期三个时期，SOD、POD、CAT与性状表达呈一定的关系。3细胞学特性：透射电镜对叶片白色组织细胞超微结构观察，发现细胞壁内陷，质体结构异常，不能发育出正常叶绿体所具有的片层和类囊体。过渡区的紧靠绿色部分的细胞较大，叶绿体发育相对正常，而靠近白色区的细胞很小，叶绿体的基粒排列紊乱。突变体绿色组织部分中的细胞生长正常。利用扫描电镜对花器官形态发生过程进行观察，内外稃原基正常形成，其后突变体表现出与野生型颖花原基发育明显不同的特征。突变体开始形成额外的内／外稃原基。颖花原基出现不均等分裂，一般为二裂原基，较大部分分化较早，甚至有些颖花原基出现三个不等分裂。在雄蕊原基和内外稃之间，有膨大的浆片原基出现，后发育成类稃状浆片。雄蕊原基发育不同步，原基大小也不一样；花分生组织的较野生型大，并且一些具有永久的活性。4组织学特性：石蜡切片观察生长扭曲的叶片发现有维管束鞘细胞的过度生长，或一些泡状细胞增大。5遗传学特性：通过突变体与4个叶色正常的材料杂交和回交遗传分析，突变性状呈细胞质遗传方式。提取水稻叶绿体基因组DNA，并用180个RAPD引物PCR扩增，共扩增出1100个左右条带，其中两个有片段在突变体和野生型中存在差异。更多还原

【Abstract】 Ⅰ. Enhanced resistance to blast fungus Magnaporthe grisea by expressionof a resistance gene Pi-d2 in transgenic riceRice blast, which is caused by the fungal pathogen Magnaporthe grisea, is one of the mostdevastating diseases of rice, so it is called cancer of rice. The measures of control to the fungi diseaseare mainly to use the bactericide and the resistant varieties. Practice proved that the most economicefficient and essential way to fight against blast is breeding and use of resistant varieties. The rise ofplant genetic engineering offers the wide selection to control the fungi disease. The technologyintroduces the foreign resistant genes into cultivar and develops the gene source, which provides a newway to crop resistant breeding.In rice, six R genes including Pi-b、Pi-ta、Pi-9、Pi-2、Piz-t and Pi-d2 had been cloned. The former5 resistant gene proteins belong to the NBS-LRR type proteins, while the Pi-d2 protein with a specialstructure B-lectin is regarded a new type protein. In the research, we firstly introduce the Pi-d2 geneinto susceptible varieties LTH and Nipponbare and resistant variety Zhonghua 9 withagrobacterium-mediated transformation. After the molecular test, GUS histochemical assay andresistance test, some plants with high resistance to rice blast have been obtained, which establish thefoundation of molecular breeding for rice and other gramineous plants and provide gene resources forhybrid breeding. The main results were as follows:1 Through agrobactetium-mediated transformation with the callus from immature embryos and matureembryos of different rice varieties, three difference expression vectors including pCB6.3kb whichharbored the 3.2kb-length native promoter and genome sequence of Pi-d2, pCB6.3kb that contained the2.2kb-length native promoter and genome sequence of Pi-d2, in pZH01-2.72kb full length of cDNAsequence of was driven by promoter 35S and the negative control vector pZH01-Tp309 were introducedinto rice. Some hygromycin resistant plants containing Pi-d2 were obtained.2 PSK-a, a biologically active peptide acting as a growth factor, was added into co-culture medium.The results showed that PSK-a indeed affected the recovery of resistant calli and the transformationfrequency of rice varieties. PSK-a with the concentration of 10 nmol/L could increase resistant calliand efficiency of transformation, with a 11% top increase, than the control. However, PSK-a with theconcentration of 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effectof PSK-a on agrobacterium-mediated transformation is related with the concentration of 2, 4-D inselection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSKplus 2 mg/L 2, 4-D than those treated with 1 mg/L 2, 4-D.3 PCR, RT-PCR, Southern blotting testing indicated that the foreign target gene randomly inserted into rice genome with 1-4 copy and stably inherited. The hygromycin resistance analysis of transgenic plantsprogeny showed that the hygromycin marker in many transformats was inserted into rice genomes withsingle loci and one copy, and already inherited to T₂ generation, and some pure hygromycin-resistancelines were obtained.4 The results of the inoculation with the rice blast race ZB15 in field and indoors showed that thetransgenic plants from T₁ enhanced the resistance to rice strain ZB15, the size and number of the lesionon leaf decreased, some populations showed the resistant and susceptible separation with Mendeliansegregation or non- Mendelian segregation. The resistant expression of foreign gene from threedifference expression vectors to ZB15 showed no significant difference. The transgenic lines from T₂progeny also showed susceptible-resistant segregation to ZB15. The results are corresponding withthat of T₁ progeny.5 Continuous field evaluation in three years showed that some transgenic plants enhanced the resistanceto neck blast, and obtained some high resistance lines to rice leaf blast and neck blast.6 The receptor cultivars and 9 transgenic stable lines derived from at less T₅ generation transgenic plantsharboring Pi-d2 gene were challenged in greenhouse by inoculating 39 isolates of Magnaporthe griseafrom Sichuan Province. The different transgenic rice lines had different resistant spectrum. The Pi-d2gene expressed in susceptible receptors evidently enhanced the disease-resistance frequency at above90%, while the disease-resistance frequency of transgenic plants from ZH9 almost didn’t increase. Fourtransgenic homozygous lines harboring full length cDNA sequence of Pi-d2 gene were inoculated with58 isolates selected by Agricultural Science Research Institute of China. The results showed thattransgenic lines resisted more than 81.48% isolates.7 Analysis of resistant expression of transgenic plants haboring the intetest gene from three differentvectors showed that the native promoters about 2.2kb or 3.2kb of Pi-d2 had the fuction of promoter as35S. The resistance between transgenic plants with genome sequence of Pi-d2 and transgenic plantswith Pi-d2 full length cDNA had no difference.8 The results of investigation of the agronomic traits of transgenic plants showed that variation occurredin a few transformants and many plants didn’t obviously change. Nutrition test of a transgenic plantsselected randomly indicated that the nutrition in transgenic rice didn’t significantly change.Ⅱ. The study of a striped mutant with abnormal floral organs in riceRice is the model of monocotyledon. The study of rice development has important values in theoryand applications. In the study, rice double mutant without the alien gene derived from the transgenicprocess was found and researched in morphology, histological characters and genetic analysis, so foundation is constructed for the cloning of the mutant gene. The results were as follows:1 The phenotype characters of mutant: The mutant showed white stripe on stem, leaf and spikelet. Insome growing stage, the leaf started to produce fork or curliness. The floret number increased,showing the multi-lemma/palea, palea-like and lemma-like lodicules or enlarged lodicules,additional pistil and stamen and the spited floret. The extreme types showed that the a few newflowers or spikelets derived from floret.2 The physiological characters: The contents of chlorophyll and net photosynthesis rate in the mutantwere obviously lower than in the wild type. At the three growing stages including tilling stages,before heading and after heading stages, the activity of SOD, POD and CAT related the expressionof mutant characters of mutant.3 Cytological identity: With observation of cell ultra structure using electron microscope, the whitetissue showed concaved cell wall and abnormal plastid which can not develop normal lamellae andthylakoid. The transition tissues aside the green tissues had the bigger cells with almost normalchloroplasts, while the tissues aside the white tissues showed that the cells were small and thechloroplast grana had inordinate arrangement. The cells in green sectors grow normally. Themorphogenesis of floral organs was observed by using the scanning electron microscopy （SEM）.Results showed that palea and lemma normal developed, then the development programmes weredifferent between wild type and mutant. Additional palea and lemma begun to format and theflower primordium splited or formatted trifid primordium. The large part of primordium firstdeveloped. The lodicuale primordium appeared between stament primordium and palea/lemmaprimordium, and developed into palea/lemma-like lodicules. Stamen development was notsynchronal and the sizes of stamen primordium were different in mutant. The size of the floretmeristem was bigger than that of wild type, and some floret meristem had permanent activation.4 Histological characters: the mesophyll cells of white tissue were dyed light, and the contortedleaves had the excess growing vascular bundle cells or increscent bulliform cells.5 Genetic analysis: the results of the analysis with the progeny derived from the mutant crossing withnormal varieties showed that the mutant characters controlled by cytoplasts gene and inhered withmaternal inheritance way. Chloroplast genome DNA was extracted, and was amplified with 180RAPD primers. Two difference fragments between mutant and wild type were obtained.更多还原