【作者】 谷守芹；

【导师】 韩建民； 董金皋；

【作者基本信息】 河北农业大学 ， 植物学， 2004， 硕士

【摘要】 以含有抗大斑病不同主效抗病基因的玉米自交系(B37、B37Ht1、B37Ht2)为材料，利用DDRT-PCR技术对接种玉米大斑病菌(0号小种和1号小种)后玉米叶片早期表达基因进行分析，以明确亲和性互作与非亲和性互作中差异表达基因的数目，并对可能与抗病性有关的差异表达片段进行回收、克隆和测序，为建立玉米抗大斑病表达序列标签并最终克隆这些基因，研究其功能奠定基础。 研究所获主要结果如下： 1、建立了适合玉米叶片mRNA差异显示的技术体系 (1)UNIQ-10柱式Trizol总RNA抽提试剂盒提取玉米叶片总RNA，反转录合成的cDNA在300bp-2000bp，适合于DDRT-PCR扩增。 (2)两步扩增法的PAGE检测结果优于一步扩增法，带型清晰，条带数量也较多，每对引物可获得扩增条带40-60条，大小为150-1000bp。 2、用12个随机引物、3个锚定引物组成的36个引物组合对接种(0号、1号小种)后的玉米叶片进行DDRT-PCR扩增，经PAGE电泳和银染检测，共获得差异显示片段148条，对其进行回收，二次扩增后，经1.2％琼脂糖电泳检测，发现138条能再次产生扩增条带，差异片段回收率达93.2％。 3、经反式Northern杂交检测，共获得阳性片段63条，阳性率为42.6％。亲和性互作中特异表达的基因片段为24条(其中可能与B37对0号小种感病有关的片段17条，可能与B37Ht1对1号小种感病性有关的片段7条)；而非亲和性互作中，特异表达的基因片段为21条(其中可能与B37Ht1、B37Ht2对0号小种抗病性有关的片段为16条，可能与B37Ht2对1号小种抗病性有关的片段5条)。 4、对特异于B37Ht2的5条基因片段克隆、测序、同源性分析表明，它们与某些植物、细菌、动物中氧化还原酶、合成酶、激素受体蛋白、转录调节因子、膜转运蛋白等有一定的同源性。对于这些片段的功能还需进一步获得基因全长和通过功能互补实验才能确定。更多还原

【Abstract】 Expressed genes in corns with major different resistant genes, B37,B37Htl and E37Ht2, had been separated and partially cloned after inoculated with race 0 and race 1 of Setosphaeria turcica by DDRT-PCR. The experimental results will help to elucidate the number of genes differentially expressed in compatible or incompatible interactions, and lay a solid foundation for establishing expressed sequence tags (ESTs) and study their function in the future by means of recovering, cloning and sequencing the differentially expressed genes related to corn disease-resistance.The main results in the study were as follow:1. An optimum protocol for corn leaf mRNA differential display was established.(1) The cDNA reverse-transcripted from total RNA isolated by UNIQ-10 column Trizol isolation kit ranged from 300bp to 1000bp. which was suitable for DDRT-PCR.(2) Compared with the one-step amplification method, the two-step amplification method was much better for DDRT-PCR, which can acquire clear amplification bands with the number of 40-60 and the length of 150-1000bp.2. Three-anchor primers and 12 random primers were used to DDRT-PCR amplification in corn cDNA, which was obtained from corn near isogenic lines induced by S. turcica. 148 differential expressed gene fragments were obtained by polyacrylamide gel electrophoresis (PAGE) and silver stain detection. 138 fragments out of the 148 differential bands were successfully recovered from PAGE gels and were effectively amplified again, and the recovery percent was up to 93.2%.3. Through reverse Northern blotting, 63 positive fragments were obtained with the positive percentage of 42.6%. Among them, 17 fragments from B37 were probably related to corn susceptibility to race 0, and 7 fragments from B37Htl related to corn susceptibility to race 1. 5 fragments specific to the resistance of B37Ht2 to race 1 and 16 fragments from both B37Htl and B37Ht2 probably related to corn resistance to race 0 were also found.4. Five fragments specific to the resistance of B37Ht2 to race 1 were cloned,regulation factors, and membrane transport protein to some extent. To make sure the definite function of the fragments, the full-length genes should be obtained and their functional complement experiments should be performed in the next step.更多还原