三种蝗虫线粒体基因组测序与直翅目比较线粒体基因组学分析

【作者】 刘念；

【导师】 黄原；

【作者基本信息】 陕西师范大学 ， 动物学， 2011， 博士

【摘要】 采用L-PCR结合二次嵌套PCR技术对蝗总科的3种蝗虫中华蚱蜢(Acrida cinerea)、永登短鼻蝗(Filchnerella yongdengensis)和东亚飞蝗(Locusta migratoria manilensis)的线粒体基因组全序列进行测定、拼接和注释,比较分析了这3种蝗虫以及NCBI中已收录的、本研究小组其他成员所测定的共计58种直翅目昆虫线粒体基因组全序列,构建了这些线粒体基因组22种tRNA和2种rRNA的通用二级结构模型。获得的主要结果如下：(1)所测得的3种蝗虫的线粒体基因组长度分别为东亚飞蝗15895bp、中华蚱蜢15599bp和永登短鼻蝗15674bp。它们均具有典型的昆虫线粒体基因组基因组成：13种蛋白质编码基因、22种tRNA编码基因以及2种rRNA编码基因。3种蝗虫与已报道蝗亚目昆虫拥有相同的基因次序,相较于已发表的螽亚目昆虫的线粒体基因组,其trnK和trnD发生了转位,这一线粒体基因组基因排序特征有可能是蝗亚目昆虫特有特征。(2)中华蚱蜢和永登短鼻蝗线粒体基因组的cox1都是非常规的ATN起始密码子。而3种蝗虫线粒体基因组均有不完全的终止密码子T或TA,特别是在nad5基因中。同义密码子的使用与密码子第三位点的碱基组成紧密相关,而与基因组所编码的tRNA反密码子匹配情况关系不大,通常,密码子第三位点为A/T的密码子使用频次高于G/C的密码子。(3)3种蝗虫线粒体基因组的tRNA二级结构均存在一定数量的碱基错配,且均以G-U弱配对为主。tRNA基因的保守程度与与其相关的密码子使用频率无关,但是与其所在的编码链有关,J-链编码的tRNA要比N-链编码的tRNA保守。通过借鉴其他发表的rRNA二级结构,并与其他直翅目昆虫rRNA一级序列进行比较分析得到这3种蝗虫12S rRNA和16S rRNA二级结构。(4)直翅目昆虫线粒体基因组长度从疑钩额螽的14971bp到Physemacris variolosa的17004bp之间变化,平均值是15684±265bp。蛋白质编码基因的平均长度为11154±37bp,密码子数平均值为3718+12；22个tRNA基因总平均长度1447±7bp；2个rRNA基因总平均长度为2098±14bp,A+T丰富区的平均长度为835±278bp。(5)直翅目昆虫的线粒体基因组A+T%含量平均值为73.42%+2.45。螽亚目昆虫的A+T%要比蝗亚目的低。直翅目昆虫中A+T碱基含量最高的区域是A+T丰富区(平均值为80.9%)以及PCG的第三位点(84.8%),蛋白质编码基因第二位点最低(平均值为65.59%)。(6)从构建的58种直翅目昆虫的22种tRNA的通用二级结构模型上看出,除了trnS (agn),所有tRNAs序列都能够形成典型的三叶草结构,由J-链编码的tRNA通常更保守,具有更多的保守位点。而A、T碱基含量以及密码子使用频率对于tRNA序列的保守性似乎没有太大的影响。(7)从构建的58种直翅目昆虫的12S rRNA的通用二级结构模型上看出,蝗虫的12S rRNA由四个结构域(即结构域Ⅰ、Ⅱ、Ⅲ和Ⅳ)的28个螺旋构成。(8)从构建的58种直翅目昆虫的16S rRNA的通用二级结构模型上看出,16S rRNA由5个结构域(分别标记为Ⅰ、Ⅱ、Ⅳ、Ⅴ和Ⅵ),缺结构域Ⅲ。蝗虫的结构域Ⅰ简化了很多,只剩下5个螺旋,并且变异很大。(9)从58种直翅目昆虫的A+T丰富区的比较中可以看出,在不同昆虫中可能存在两种不同的序列结构参与线粒体基因组的复制起始调控。在所有蝗亚目、蟋蟀科和蝼蛄科昆虫A+T丰富区的中间偏’rnI基因的区域中都能形成一个约16bp左右的茎环结构,除了基部的三个碱基对外发生补偿性突变外,其他的螺旋对都非常保守,在茎环结构的5’端相邻区域有’TATA’基序,3’端有‘G(A)nT’基序。在螽亚目的其他昆虫中,在H-链靠近rrnS处超过17bp的T-簇,在T-簇的两端分别有一个嘌呤。更多还原

【Abstract】 The complete mitogenome of Acrida cinerea, Filchnerella yongdengensis and Locusta migratoria manilensis which belong to superfamily Acridoidea of Orthoptera were determined and compared with that of other 55 orthoperans which determined by our research group and recorded in NCBI. The universal secondary structure of the mitochondrial 22 tRNA and 2 rRNA were predicted based on these mitochondrial genomes. Conclusions were showed below:(1)The length of the complete mitochondrial of L. m. manilensis, A. cinerea and F. yongdengensis are 15,895bp,15,599bp and 15,674bp respectively. They all display a typical gene composition found in insect mitogenomes:13 PCGs,22 tRNA genes,2 rRNA genes and an A+T-rich region. The gene arrangement of these three grasshoppers’ mitogenomes is identical to those of other recorded sequences from Caelifera, which different from the ensiferans in the translocation from trnK-trnD to trnD-trnK. This arrangement of mitogenome may be the unique characteristic of caeliferans.(2)The typical initiation codon ATN not occurred in the mitogenomes of A. cinerea and F. yongdengensis. All the three mitogenomes have incomplete termination codon (T/TA) especially for nad5. The usage of synonymous codon markedly correlated with the nucleotide composition at third codon position, and not the anticodon of tRNA. Generally, the usage of codon that is A/T at the third codon position is higher than G/C.(3)The mismatches exist in secondary structure of the A.cinerea, F. yongdengensis and L. m. manilensis, mainly for wobble pair G-U. The tRNAs encoded by the J-strand generally contain more conservation sites than those encoded by the N-strand. The conservation of tRNA genes was not associated with the frequency of codon usage and A+T content. The model of secondary structure of 12S rRNA and 16S rRNA were predicted by comparative approach.(4)The average length of analyzed mtDNAs set is 15684±265bp, ranging from 14,971bp of R. dubia to 17,004bp of P. variolosa. The average length of PCGs is 11154±37bp, that the average codon usage number is 3718±12. The average length of 22 tRNAs gene,2 rRNAs and the A+T-rich region are 1447±7bp,2098±14bp and 835±278bp, respectively.(5)The average A+T% value for the analyzed mtDNAs set is 73.42%±2.45. In Orthoptera, the A+T contents of ensiferans are lower than those of caeliferan but have higher difference among the species, especially in the regions which have high A+Tcontent. Nevertheless, the second position of PCGs (average 65.59%) has the relative constant A+T concentration than other regions, whereas the A+T-rich (average 80.9%) and the third position of PCGs (84.8%) are highest.(6)A11 tRNAs from 58 orthopterans have the typical clover leaf structure except for trnS (agn) The tRNAs encoded by the H-strand generally contain more conservation sites than those encoded by the L-strand. The conservation of tRNA genes was not associated with the frequency of codon usage and A+T content.(7)The secondary structure and conserved consensus sequence of the 58 orthopterans rrnS is presented in this paper. It consists of 28 helices and subdivided into four principal domains (labeledⅠ,Ⅱ,Ⅲ, andⅣ), similar to the secondary structure of small ribosomal RNA subunits in prokaryotes.(8)The rrnL of orthopterans is divided into six domains (labeledⅠ,Ⅱ,Ⅲ,Ⅳ,ⅤandⅥ), each separated by a single stranded region. DomainⅢis absent in arthropods mitochondrion. Compared to the E.coli model, considerable degeneration in domainⅠof Orthoptera leads to only five remaining helices.(9)A stem-loop structure,16 bp or longer, also exists in the same position in all of the taxa from Caelifera, Gryllidae and Gryllotalpidae. Furthermore, at the basal three couplets was well established by CCSs and conserved in other pairs of the stem-loop structure. The flanking regions, including "TATA" on the 5’end and "G (A)nT" on the 3’end, are also conserved in Caelifera. In other ensiferans exist a common feature with a long T-stretch (>17 bp) next to rrnS on the H-strand, bounded by a purine at either end.更多还原