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社会性昆虫抗菌肽基因的分子进化研究
Studies on Molecular Evolution of the Antimicrobial Peptide Genes of Social Insects
【作者】 徐鹏；
【导师】 陈学新；
【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2009， 博士
【摘要】 社会性昆虫在上亿年的进化过程中,形成了许多行之有效的分子进化机制,为我们研究分子进化提供了一个良好的模式系统。我们选取三种白蚁(黑翅土白蚁Odontotermes formosanus(Shiraki)、黄翅大白蚁Macrotermes barneyi Light和黑胸散白蚁Reticulitermes chinensis Snyder)和一种蜜蜂(中华蜜蜂Apis ceranaFabricius)为实验材料。根据已知的抗菌肽cDNA基因设计引物,通过反转录PCR的方法从构建的cDNA文库中扩增获得抗菌肽cDNA基因,并运用各种生物信息学软件进行遗传进化分析。本研究得到的结果和结论如下:(1)黑翅土白蚁Termicin家族基因共计含56个不同的cDNA基因,编码46个不同的抗菌肽;黄翅大白蚁Termicin家族基因共计含54个不同的cDNA基因,编码37个不同的抗菌肽;黑胸散白蚁Termicin家族基因共计含38个不同的cDNA基因,编码21个不同的抗菌肽。(2)黑翅土白蚁和黄翅大白蚁Termicin前体基因dN/dS值大于1,表明两种白蚁Termicin基因采取正选择的进化方式,两种白蚁受到的环境选择压力为正选择压力。黑胸散白蚁前体基因dN/dS值小于1,表明黑胸散白蚁Termicin基因采取负选择的进化方式,黑胸散白蚁受到的环境选择压力为负选择压力。黑翅土白蚁和黄翅大白蚁Termicin前体基因成熟肽区dN/dS值大于前体基因dN/dS的值,表明Termicin前体基因面临的环境选择压力主要体现在成熟肽区。(3)中华蜜蜂总计含87个不同的抗菌肽基因和26个不同的抗菌肽。其中Defensin家族基因含29个不同的cDNA基因,编码7个Defensin肽;Abaecin家族基因含11个不同的cDNA基因,编码2个Abaecin肽;Apidaecin家族基因含13个不同的cDNA基因,编码4个Apidaecin肽;Hymenoptaecin家族基因含34个不同的cDNA基因,编码13个Hymenoptaecin肽。(4)通过与意大利蜜蜂Apis mellifera L.的比较分析发现,中华蜜蜂比意大利蜜蜂含更丰富的抗菌肽基因(87∶16)和抗菌肽(26∶11),尤其是具有更丰富的Hymenoptaecin基因(34∶2)和更多数量的Hymenoptaecin肽(13∶1)。这可能是因为长期的家养驯化使意大利蜜蜂基因退化的缘故。(5)系统发育分析表明,几种社会性昆虫的抗菌肽基因均为同源基因。根据基因的“自私性”法则,我们预测上述社会性昆虫的抗菌肽基因家族可能存在一些在长期的进化过程将被淘汰的假基因或不必要基因。
【Abstract】 Social insects have beening exsisted on the earth for more than 100 million years, which provide an excellent model for us to study the molecular evolution.We chose four species of social insect as materials,including three termite.species {Odontotermes formosanus(Shiraki),Macrotermes barneyi Light and Reticulitermes chinensis Snyder) and one honeybee species(Apis cerana Fabricius).We designed primers according to the reported antimicrobial peptide cDNA genes,and amplified the cDNA genes from the constructed cDNA libraries using Reverse Transcript PCR (RT-PCR),and then analyzed these sequences data using bioinformatics softwares.The results and conclusions are summarized as follows:(1) Totally O.formosanus has 56 different Termicin cDNA genes encoding 46 different Termicin peptides,M.barneyi has 54 different Termicin cDNA genes encoding 37 different Termicin peptides,and R.chinensis has 38 different Termicin cDNA genes encoding 21 different Termicin peptides.(2) The values of dN/dS of the Termicin precursor genes are > 1 in O.formosanus and M.barneyi,indicating that the Termicin genes undergo positive selection in these two termte species,and the microenvironmental pressure of these two kinds of termites are positive microenvironmental pressure.The value of dN/dS in R.chinensis is < 1,indicating that the Termicin gene in R.chinensis is under negative selection, and the microenvironmental pressure of this kind of termite is negative microenvironmental pressure.The values of dN/dS of the Termicin mature region of O.formosanus and M.barneyi are bigger than those of the Termicin precursor genes, which suggest that the microenvironmental pressure are mainly on the mature region.(3) A.cerana has totally 87 different antimicrobial peptide cDNA genes encoding 26 different antimicrobial peptides,among which,29 different genes are Defensin genes encoding 7 different Defensin peptides,11 different cDNA genes are Abaecin genes encoding 2 different Abaecin peptides,13 different cDNA genes are Apidaecin genes encoding 4 Apidaecin peptides and 34 different cDNA genes are Hymenoptaecin genes encoding 13 different Hymenoptaecin peptides.(4) As compared with the Western honeybee Apis mellifera L.,the Asiatic honeybee has more variable antimicrobial peptide genes(87 versus 16) and antimicrobial peptides(26 versus 11),especially the Hymenoptaecin genes(34 versus 2) and the Hymenoptaecin peptides(13 versus 1).This suggests that,compared to the Western honeybee that has a longer history of domestication,selection on the Asiatic honeybee has favored the generation of more variable antimicrobial peptides as protection against pathogens.(5) The results of phylogenetic analysis show that all the genes in the same family are homogenetic genes.According to the rule of selfish-gene,we predicted that there may be pseudogenes or unnecessary genes in the antimicrobial peptide genes,and these genes would be eliminated in the long term of evolution.