论文标题:利用生防木霉基因提高水稻抗病性研究
Enhance Disease Resistance of Rice with Genes from Biocontrol Agent of Trichoderma
论文作者 刘梅
论文导师 徐同；孙宗修,论文学位 博士,论文专业 植物病理学
论文单位 浙江大学,点击次数 87,论文页数 123页File Size7448k
2003-05-01论文网 http://www.lw23.com/lunwen_935828762/ 转基因水稻;植物表达载体;根癌农杆菌介导法;水稻纹枯病;稻瘟病;哈茨木霉（Trichoderma harzianum）;几丁质酶;葡聚糖酶
transgenic rice; plant expression vector; Agrobacteriwn-mediated transformation; rice sheath blight; rice blast; Trichoderma harzianum; chitinase; glucanase
水稻纹枯病(Rhizoctonia solani)和稻瘟病(Magnaporthe grisea)是2种严重危害我国水稻生产的真菌病害。由于纹枯病抗源缺乏使该病的抗病育种工作进展缓慢，而稻瘟菌小种的复杂多样，常使育成的抗病品种在短期内失去抗性。利用外源几丁质酶基因和葡聚糖酶基因提高水稻对这两种病害的抗性被认为是一种有效的途径。在本实验室的前期工作中，将来源于生防真菌哈茨木霉(Trichoderma harzianum P1)的内切几丁质酶基因基因组序列ThEn-42(ech42)导入水稻，该基因受CaMV35S启动子调控，在水稻中组成型表达，经初步鉴定转基因水稻T_0代表现提高了对纹枯病的抗性。 本研究对上述基因在水稻中的遗传稳定性进行了检测，并对转基因水稻后代进行抗纹枯病和稻瘟病筛选。为进一步提高水稻对纹枯病和稻瘟病的抗性，采用Actl启动子取代CaMV 35S启动子，并利用3种木霉胞壁降解酶基因(ech42、nag70和gluc78)转化水稻，以期通过在水稻中高水平表达几丁质酶和葡聚糖酶活性，获得高抗纹枯病和稻瘟病的转基因水稻株系，为水稻的抗病遗传育种提供基础材料。 主要结果如下： 1．对ThEn-42基因的转基因水稻后代进行PCR鉴定和潮霉素抗性鉴定，结果表明大约70％的T_1代转基因株系符合3∶1(阳性：阴性)的分离比，表明ThEn-42在这些株系中有单个插入位点；大约20％的T_1代株系分离比符合15∶1，表明这些株系含有2个整合位点。选择部分T_1代潮霉素抗性植株进行Southern杂交鉴定，结果证实外源基因已经稳定遗传给T_1代。对部分T_2代株系进行潮霉素抗性鉴定和PCR鉴定，结果表明ThEn-42已经稳定遗传给T_2代植株。 2．检测了T_1代转ThEn-42植株对水稻纹枯病(Rhizoctonia solani)和两个分别属于A群和B群的稻瘟病(Magnaporthe grisea)小种的抗性，30％株系表现了对这两种病害抗性水平不同程度的提高。有7个株系的抗性与对照相比有很显著的提高，其中Tp64表现对两个稻瘟病小种完全免疫。对Tp64的T_2代纯合植株进行稻瘟病抗性鉴定，转基因植株仍然表现对所检测的2个稻瘟 菌小种完全免疫，但是该株系对纹枯病的抗性无明显提高。这些结果表明内 切见丁质酶基因ThEn－42已经在受体植物中准确表达，并且在提高水稻抗病 性方面起了重要作用。但并未获得对二种病原都具有高度抗性的植株。3．为进一步提高水稻对纹枯病和稻瘟病的抗性，利用来源于哈茨木霉 （Trichoderma h心ianum）PI菌株的3个胞壁降解酶基因ech42、n昭70与glue78 构建了包括不同基因及其所有组合的7个植物表达载体，每个基因受独立的 ACt］启动子调控。构建的7个载体不仅包含所有组合的3个外源基因，而且 具有双元载体本身携带的HTP基因与Gus基因，从而为研究不同T－DNA长 度、不同基因组合与不同基因排列方向对植物遗传转化效率以及外源基因在 转基因植株中表达的影响提供了一套比较完整的材料。4．利用本实验室的农杆菌高效转化体系，将3种胞壁降解酶基因ech42、nng70 与glue78所有组合的7个载体分别转入粳稻品种石狩白毛（OryZasativaLssp． 冲。n加。cv·Ism（ad－shk。ge）中，共获得再生植株180o 余株。对部分再生植 株进行了 PCR检测和 Southern杂交检测，证明 96％的植株携带有外源基因 中的至少一个基因，80％以上的植株整合有完整的外源基因片断，而且多数 基因在受体植株中有单个整合位点。对不同载体的分化率和绿苗率进行分析， 发现随着T－DNA区长度的增大，分化率和绿苗率都有降低的趋势。携带 glCC 78基因的载体导致分化率、绿苗率以及分化速度的显著降低。5．转基因再生植株移栽到土壤中以后，不同载体的转化植株成活率和正常植株 比例明显不同。转化单个glCC 78基因的植株成活率和正常植株比例最低，在 所有携带glCC78基因的载体的转化植株中都有部分植株出现不同程度的矮化 现象，这种植株的叶片在未接种任何病原的情况下出现褐色斑点，不能抽穗、 结实，所检测的矮化植株中都具有较高水平的p上，3一葡聚糖酶活性，而株高 基本正常的植株 p刁，3一葡聚糖酶活性较低，可见glCC78严重影响了水稻的生 长。转化ech42基因的水稻出现生长延缓现象，但是并不影响株高和结实。 转化载体上其它外源基因的存在会削弱gi讹 78或ech42对植株生长造成的影 响，可见同一T－DNA区某一个基因的表达会受其它基因的影响。6．对上述转化植株T。代生长正常植株进行纹枯病抗性鉴定，除转化单个葡聚糖 酶基因植株因为正常植株比例太少不做分析外，其余6种转化植株中都存在 工互互 5 部分植株的抗病性有明显提高，同时也存在部分植株的抗性与对照无差异。 对转化植株2种几丁质酶活性测定和抗病性检测的结果显示，ech42基因的高 水平表达导致植株对纹枯病抗性的显著提高，而nog70与抗病的关系不明显。 在所
Rice sheath blight (Rhizoctonia solani) and rice blast (Magnaporthe grisea) are two serious rice diseases in China. Rice cultivars with broad spectrum, high level and durable resistance to these two pathogens are not easy to obtained by traditional breeding methods due to the deficiency of disease-resistant germplasm to R solani and the complex genetic diversity of M grisea. Using chitinase and glucanase genes to alter rice resistance to fungal pathogens could be an effective approach to solve this problem. In the previous work of our laboratory, an endochitinase gene, ThEn-42(ech42), from a biocontrol agent, Trichoderma harzianum strain P1, was introduced into rice genome. The transgenic rice TO plants showed enhanced resistance to R. solani. The genetic stability analysis of this gene and further screening of resistant lines in transgenic offspring were carried out in this research. In order to acquire higher resistance to the pathogens, a stronger promoter, Actl, was used to replace the CaMV 35S promoter and three cell wall degrading enzyme genes(ech42, nag70 and glue78) in the different combination were transformed into rice.The results obtained are as following:1. T1 transgenic rice plants with ThEn-42 were detected with PCR and analyzed with hygromycin B resistance, and the results were the same. About 70 percent of the TI lines showed the segregation ratio of 3:1 (positive : negative), indicating that these lines had single integration locus. About 20 percent of the T1 lines displayed the segregation ratio of 15:1, indicating that these lines had two integration loci. Several lines showing resistance to hygromycin B were also analyzed by Southern blotting. The results further confirmed that ThEn-42 could be stably inherited toT1 generation. The PCR analysis and hygromycin B resistant analysis of some T2 plants also demonstrated that ThEn-42 gene had stably inherited to T2 generation.2. Comparing with the controls, most of the transgenic rice offspring lines showed enhanced resistance to .R. solani K(?)hn and M. grisea in greenhouse or nursery at different level. The selected Hne-Tp64 was even completely resistant to M. grisea but didn"t show enhanced resistance to R. solani. The results revealed that the endochitinase gene ThEn-42 could be exactly expressed in transgenic rice and play an important role in increasing the disease resistance.3. In order to attain rice with a higher resistance against the fungal pathogens, three different cell wall degrading enzyme(CWDE) genes, ech42, nag70 and gluc78, from T. harzianum P1 were placed respectively downstream of Act1 promoter. Based on this work, 7 different plant expression vectors were constructed by cloning each gene alone or either two genes in combination or three genes together into binary vector pCAMBIA1305.2. The constructed vectors also carried HPT gene and Gus gene which was already placed in two sides of the multiple cloning sites of binary vector pCAMBIA1305.2, that provided a suit of systematic materials for further study on the influence of T-DNA size, gene combination and gene direction on transformation frequency and gene expression.4. Seven combinations of CWDE genes were transformed into Oryza saliva L ssp. Japonica cv. Ishikari-shiroge respectively mediated by Agrobacterium tumefaciens. More than 1,800 independent regenerated plantlets of all seven populations were obtained. PCR and Southern blot analysis of part of transgenic plants revealed that about 96 percent of them integrated with at least one of three exogenous genes, more than 80 percent had intact exogenous fragment, and most of the transgenic plants had one insertion site. Differentiation and regeneration frequencies in transformation process appeared a decreasing tendency along with the increase of T-DNA size of the different vectors. The differentiation and regeneration frequencies of vectors carrying gluc78 gene were significantly low, indicating that gluc78 gene had negative effects on calli growth.5. Survival of tr
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