【作者】 吴杨；

【导师】 潘大仁；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 本研究以43份未经线虫病常规鉴定的甘蔗质资源为供试材料，首先提取供试甘蔗基因组DNA，然后根据番茄抗根结线虫病基因和甜菜抗胞囊线虫病基因的保守序列区域，分别设计了两条上游引物、两条下游引物，对引物组合后，进行了PCR特异扩增，从中分别筛选出一对特异引物，并优化了PCR扩增反应体系中的各个影响因素。用抗根结线虫基因设计的引物进行扩增最终获得了一条大约460bp大小的特异片段；用抗胞囊线虫基因设计的引物进行扩增最终获得了一条大约690bp大小的特异片段，并进一步做了PCR-Southern杂交，以确认这两条特异片段的同源性程度和真实性，本实验所得结果如下： 1．建立了适合于甘蔗PCR特异扩增的反应体系：反应总体积为20μl，含10mmol／L Tris-HCl（pH8.3），50mmol／L KCl，2.0mmol／L MgCl₂，100μmol／L dNTP，0.2μmol／L引物，40ng模板DNA，1.0U Taq酶；反应程序为：94℃，5min，1 cycle；94℃，40s，60℃，50s，72℃，90s，35 cycles；72℃延伸10min。 2．对43份未经线虫病常规鉴定的甘蔗种质资源根据不同的引物进行PCR检测。其中，用抗根结线虫病基因设计的引物进行扩增，结果有37份甘蔗出现特异条带，6份未出现任何条带；用抗胞囊线虫病基因设计的引物进行扩增，结果有39份甘蔗出现特异条带，4份未出现任何条带。 3．对两种PCR特异产物进行了PCR-Southern杂交，结果显示它们有杂交信号，说明这两个片段分别与两个抗线虫病基因有较高的同源性，为克隆甘蔗抗线虫病基因奠定了基础。更多还原

【Abstract】 The nematode resistance of forty-three sugarcane cultivars (without conventional identification of nematode resistance) was tested in the experiment. Firstly, the sugarcane genomic DNA was extracted. Secondly, according to the sequence of root-knot nematode resistant gene in tomato and the sequence of nematode resistant gene in sugar beet, two forward primers and two reverse primers were produced separately by synthetic, 4 pairs of primers that were in .each kind were composed, and a pair of primers in each was selected among them by PCR. Thirdly, conditions for PCR in sugarcane were optimized, the results showed that a special fragment of about 460bp and a special fragment of about 690bp were appeared, and PCR-Southern blotting about them was made further in order to affirm the homogeneous sequences of nematode resistant gene. Finally , 43 sugarcane cultivars were primarily detected by PCR technique. The results were summarized as following :1. An optimal reaction system for PCR in sugarcane was established : in a 20ul reaction solution, the optimal composition included 10 mmol/L Tris-HCl(pH8.3), 50 mmol/L KC1,1.5 mmol/L MgCl2,100umol/L dNTP ,0.2umol/L primers ,40ng of template DNA and 1.0 U Tag DNA Polymerase.The reaction program was devised in to 3 processes: The first one is denaturation at 94℃ for 5 min. The second one is 35 cycles of denaturation at 94 ℃ for 40s, annealing at 60℃ for 50s and extension at 72 ℃ for 90 s. The last one is extension at 72℃ for 10 min.2. Among 43 sugarcane cultivars, 37 cultivars appeared a special fragment of about 630bp, and 6 cultivars had nothing by using root-knot nematode resistant primers. 39 cultivars appeared a special fragment of about 630bp, and 4 cultivars had nothing by using cytocyst nematode resistant primers.3. The two kinds of special PCR products were hybridized by PCR-Southern blotting. The blotting signals appeared, which proved that there was a great homogeneous sequence between 460bp special fragment and root-knot nematode resistant gene in tomato and there was a great homogeneous sequence between 690bp special fragment and nematode resistant gene in sweet potato. They were the basis for cloning nematode resistant gene in sugarcane. 4.The results will be tested by field identification in the further.更多还原