【作者】 孙钦霞；

【导师】 张雅林；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2004， 硕士

【摘要】 运用线粒体DNA中编码rRNA蛋白质的基因16S rRNA作为分子标记，选取半翅目Hemiptera蝽科Pentatominae蝽亚科Pentatominae7族18属22个代表种，即：斑须蝽Dolycoris baccarum (Linnaeus)，尖角普蝽Priassus spiniger Haglund，斑莽蝽Placosternum urus St?l，麻皮蝽Erthesina fullo (Thunberg)，赤条蝽Graphosoma rubrolineata (Westwood)，二星蝽Stollia guttiger (Thunberg)，玉蝽Hoplistodera fergussoni Distant，角肩真蝽Pentatoma angulata Hsiao et Cheng，褐真蝽Pentatoma armandi Fallou，中纹真蝽Pentatoma distincta Hsiao et Cheng，斑真蝽Pentatoma mosaicus Hsiao et Cheng，全蝽Homalogonia obtuse (Walker)，小斑岱蝽Dalpada nodifera Walker，宽碧蝽Palomena viridissim (Poda)，菜蝽Eurydema dominulus (Scopoli)，凹肩辉蝽Carbula sinica Hsiao et Cheng，辉蝽Carbula. obtusangula Reuter，珀蝽Plautia fimbriata (Fabricius)，稻绿蝽Nezara viridula Linnaeus，宽胫格蝽Cappaea tibialis Hsiao et Cheng，卵圆蝽Hippota dorsalis (St?l) 和茶翅蝽Halyomorpha Picus (Fabricius) 共27个个体对其亲缘关系进行初步研究。通过特异引物扩增和基因测序的研究方法获得它们的线粒体DNA（mtDNA）的16S rRNA基因片段，并分析了其遗传多态性。在扩增的长为400bp的基因片段中，去掉两端引物，通过排序，有350个碱基可用于这27个个体的比较。通过测定分析该基因片段的序列发现，27个个体具有27种基因型，不同种群存在丰富的DNA序列多态性，同一种的不同个体差异较小。DNA序列应用ClustalX1.8、DnaSP、Bioedit 5.09、MEGA软件进行排序、比较和分析，并计算核苷酸组成、多态性位点以及遗传距离，用邻位相连法构建Neighbor-joining（NJ）系统进化树，从而确定各类群之间的系统关系。通过分析得出以下结论：1．在国内首次使用特异引物扩增蝽亚科昆虫的mtDNA-16S rRNA基因片段，通过测序获得了蝽亚科7族18属22种27个个体的基因序列片段。研究发现16S rRNA基因片段适合于族、属之间亲缘关系的研究。2．无水乙醇浸制标本可应用于总DNA的提取、16S rRNA基因片段的特异引物扩增、提纯和测序。3．本实验中，标本DNA的轻微降解不影响实验结果。4．在扩增的16S rRNA基因片段中，共检测到核苷酸变异位点191个，占总序列的54.57％，除去具缺失或插入的位点后，序列变异为42.57％。A、T、C、G的平均含量为33.84％、43.24％、6.78％、16.14％，A+T的含量为77.08％，G+C的含量为22.92％，这一结果与昆虫中A+T的含量高于G+C的含量这一结论是一致的。5．以“Bootstrap Test of Phylogeny”1000次重复作出各个分枝的置信度。形成的Neighbor-Joining (NJ)、Minimum Evolution (ME)和Maximum Parsimony (MP)分子系统树表明，NJ树的置信度最高。6．以蝽亚科昆虫27个个体的16S rRNA基因序列构建的分子系统树显示：麻皮蝽<WP=6>属与菜蝽属的遗传关系较近，二者位于进化树的底部，说明这两个种比其它类群起源较早；绿蝽属和碧蝽属的亲缘关系比较近，二者又与玉蝽属互为姐妹群；条蝽属与斑须蝽属互为姐妹群；二星蝽属与辉蝽属的亲缘关系较近，之后二者又与卵圆蝽属互为姐妹群；蝽族的真蝽属与岱蝽属的关系比全蝽属的亲缘关系近，全蝽属与格蝽属互为姐妹群。这与传统分类中属的关系有较大差异。更多还原

【Abstract】 Relationship of 18 genera of some tribes of Pentatominae was primarily studied by using 16S rRNA gene in mitochondrial DNA as molecular marks. With the PCR and DNA sequencing, 27 16S rRNA gene sequences were obtained from 22 species of 18 genera of 7 tribes. The examined species were as follows: Dolycoris baccarum (Linnaeus), Priassus spiniger Haglund, Placosternum urus St?l, Erthesina fullo (Thunberg), Graphosoma rubrolineata (Westwood), Stollia guttiger (Thunberg), Hoplistodera fergussoni Distant, Pentatoma angulata Hsiao et Cheng, Pentatoma armandi Fallou, Pentatoma distincta Hsiao et Cheng, Pentatoma mosaicus Hsiao et Cheng, Homalogonia obtuse (Walker), Dalpada nodifera Walker, Palomena viridissim (Poda), Eurydema dominulus (Scopoli), Carbula sinica Hsiao et Cheng, Carbula. obtusangula Reuter, Plautia fimbriata (Fabricius), Nezara viridula Linnaeus, Cappaea tibialis Hsiao et Cheng , Hippota dorsalis (St?l), Halyomorpha Picus (Fabricius).The genetic polymorphism in mitochondrial DNA of 27 samples of the 22 species was analyzed and 400bp was amplified with the PCR (polymerase chain reaction) and sequenced. From the consensus sequence of the two strands from each specimen, an alignment of 350 nucleotides coulde be used for comparison of the 27 specimens. The genetic polymorphism among populations and 27 haplotypes had been found by analysing genetic sequences. DNA sequences were aligned, compared and analysed by ClustalX1.8, DnaSP, Bioedit5.09 and MEGA, to calculate residue composition, polymorphic sites and genetic distance, to create Neighbor-joining (NJ) phylogenetic tree and determine phylogeny of the taxa involved in the research. The results were as follows:1. The mtDNA-16S rRNA gene sequences of Pentatominae were amplified by specific primer for the first time in China.Gene sequences of 27 pentatomine specimens from 22 species of 18 genera of 7 tribes are obtained by DNA sequencing. The gene is good for revealing the relationship of tribes or genera. 2. The spemcimens deposited in alcohol can be used to extract DNA, amplify 16S rRNA gene sequences by specific primer.3. The slight degradation of DNA specimen does not affect the results of experiment.4. In the gene , 191 nucleotide polymorphic positions（54.57%of the whole sequence） have been checked. The sequence variation was 42.75% except the sites of deletion or insertion.The average contents of A,T,C,G are 33.84％,43.24％,6.77％,16.14％ respectively, and A+T 77.08％ is higher than G+C 22.92％ Which is consistent with other insects.5. The Bootstrap confidence values of each branch were obtained by 1000 repeats. Molecular phylogenetic tree was constructed by Neighbor-Joining (NJ), Minimum Evolution (ME) and Maximum Parsimony (MP), and show that the confidence values of NJ is the highest. <WP=8>6. Molecular phylogenetic tree was constructed by using 16S rRNA gene sequences of 27 specimens. It showed that Erthesina and Eurydema are closer, and they appeared earlier at the root of the tree than the other genera. The relationship between Nezara and Palomena, Graphosoma and Dolycoris, Stollia and Carbula are closer respectively. Pentatoma and Dalpada are closer than to Homalogonia, which is different from the genera phylogeny at generic level of traditional taxonomy.更多还原