【作者】 梁定东；

【导师】 郑经武；

【作者基本信息】 浙江大学 ， 植物病理学， 2004， 硕士

【摘要】 本研究对不同的大豆胞囊线虫(Heterodera glycines)群体和中华异皮线虫(H.sinensis)、禾谷胞囊线虫(H.avenae)及寄生于烟草根部的胞囊线虫等种从形态学和分子生物学两方面进行了比较和分析。 通过制作永久玻片和临时玻片，对一些重要的胞囊线虫种及种下群体进行了详细的形态学观察，通过扫描电镜观察了二龄幼虫唇区结构、侧区侧线数等重要特征，同时对二龄幼虫的体长、口针长、尾长及透明尾长等主要鉴别特征进行了形态测量。结果表明：寄生于烟草根部的胞囊线虫无论从唇区结构、侧线数目、还是从各项测量数值上，与大豆胞囊线虫群体均无明显差别。中华异皮线虫与其他种相比差异较大。 对全部样品rDNA的ITS区进行了PCR扩增，并用AluⅠ，Bsh1236Ⅰ，BsurⅠ，CfoⅠ，DdeⅠ，HinfⅠ，MspⅠ及RsaⅠ等8种限制性内切酶对扩增产物进行了酶切分析及序列测定。PCR扩增结果表明：所有样品扩增片段长度均为1，030bp左右；RFLPs分析表明：经AluⅠ，CfoⅠ，RsaⅠ及MvaⅠ等酶切后，各大豆胞囊线虫群体均得到了相同的酶切图谱。经AvaⅠ酶切后，部分来自山西太谷的大豆胞囊线虫群体产生两类酶切图谱，显示出群体内存在一定的差异。另外，中华异皮线虫经除CfoⅠ酶以外的7种酶酶切后，得到的酶切图谱与其它种存在明显的差异。 通过对我国主要的胞囊线虫种群rDNA中ITS区的序列测定及同源性分析明确了不同种群的系统学关系。其中大豆胞囊线虫种内群体间rDNA中ITS区间差异极小；寄生于烟草的胞囊线虫与大豆胞囊线虫各群体差异不明显，相似度高达99％，结合形态学特征，初步将寄生于烟草的胞囊线虫鉴定为大豆胞囊线虫；中华异皮线虫同大豆胞囊线虫差异明显，相似度仅为77％，而与甘蔗胞囊线虫(H.sacchari)的同源性较高，相似度为86％，这也是国际上首次报道中华异皮线虫的分子信息。另外，根据所测定的序列比较而得出的大豆胞囊线虫种内群体及胞囊线虫种间亲缘关系与Subbotin et al.(2001)根据形态学差异所进行的分组是完全相符的。 本研究表明：利用rDNA ITS区对大豆胞囊线虫进行种间水平上的分子鉴定是稳定和可行的，但其在种内群体水平上无明显差异，鉴别意义较小。更多还原

【Abstract】 Morphological and molecular comparisons were made among different soybean cyst nematode populations (Heterodera glycines}, cereal cyst mematode (H. avenae), H. sinensis and a cyst forming nematode population parasitizing tobacco used in this study.Some important morphological characters, such as the shape of lip region and the number of lateral lines of J2 of intra- and inter-species of Heterodera were observed with both Light microscope and Scanning Electron Microscope (SEM). Some morphometrics of Ji, for example, the body length, stylet, tail and clear tail were measured, and it revealed that the cyst forming nematode population parasitized on the root of tobacco (Heterodera sp.) was identical to that of H. glycines, and distinct differences were detected among H. sinensis with the other cyst forming nematode species occurring in China.The ITS regions of rDNA of all the cyst forming nematodes, including H. glycines, H. avenae, H. sinensis and the cyst forming nematode populations parasitizing tobacco, were amplified and digiested with 8 different restriction enzymes, AM, Bsh1236I, BsurI, CfoI, DdeI, HinfI., MspI and RsaI. It indicated that all the inter- or intra-species of cyst forming nematodes yield identifical amplified ITS region products, with the sequencing length about l,030bp. PCR-RFLPs of the ITS region of 11 intra-populations of soybean cyst forming nematodes were made with five restriction enzymes. Four of which, Alu I, Cfo I , Rsa I and Mva I , produced the same digest pattern among all the 11 populations. Two digiest phenotypes were detected in the population from Taigu, Shanxi when digeseted with Ava I, which revealed the heterogeneity of the species in intra-population. Great differences were revealed between inter-species of H.sinensis and H.glycines after the digestion of 7 restriction enzymes, except for Cfol, and the cyst forming nematode population parasitizing on the tobacco produced the identical digest patterns with H. glycines when the 8 restraction enzymes were used.Sequence alignments of the ITS region of inter-population of soybean cystnematodes from different area of China showed little variation, and the cyst forming nematode population parasitizing tobacco, from Xuchang, Henan province also presents mini-difference with that of soybean cyst nematodes, and the sequence similarity reaches 99%. Comparing to both morphological and the rDNA characteristics, the cyst forming nematode population parasitizing tobacco was identified as soybean cyst nematode, H. glycines. It was found that there are clear sequence differences based on ITS region between H. sinensis and H. glycines, and the similarity is only 77%. This was the first report about the molecular data of H. sinensis, which show high similirity to that of H. sacchari. The phylogenetic relationship based on rDNA sequencing was identical to the grouping according to the morphological characters made by Subbotin et al. (2001).The present research indicates that it is stable and accessible for the identification of inter-species of Heterodera by the ITS region of rDNA, but it could not be used for the differentiation of the inter-population of the same species.更多还原