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æŸžèš•çº¿ç²’ä½“åŸºå› ç»„ç ”ç©¶ 刘彦群 / åšå£« 1. 沈阳农业大å¦ç”Ÿç‰©ç§‘å¦æŠ€æœ¯å¦é™¢ 2. 西å—大å¦èš•æ¡‘å¦å†œä¸šéƒ¨é‡ç‚¹å¼€æ”¾å®žéªŒå®¤. The mitochondrial genome of the Chinese oak silkworm, Antheraea pernyi Dr. Yanqun Liu 1.College of Bioscience and Biotechnology, Shenyang Agricultural University
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柞蚕线粒体基因组研究 刘彦群/博士 1.沈阳农业大学生物科学技术学院 2.西南大学蚕桑学农业部重点开放实验室 The mitochondrial genome of the Chinese oak silkworm, Antheraea pernyi Dr. Yanqun Liu 1.College of Bioscience and Biotechnology, Shenyang Agricultural University 2.The Key Sericultural laboratory of Agricultural Ministry, Southwest University
柞蚕线粒体基因组研究The mitogenome of the Chinese oak silkworm, A. pernyi 汇报内容： 1. 柞蚕线粒体基因组的特征 2. 放养柞蚕与野生柞蚕的线粒体基因组比较 CONTENTS: 1. The mitochondrial genome of Antheraea pernyi 2. Comparative mitogenomics between domestic and wild A. pernyi
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 线粒体（图1）是细胞的动力工厂。Mt DNA是第2遗传系统，呈母性遗传。 动物mtDNA是双链环状DNA分子，一般含有37个基因（13个蛋白编码基因、22个tRNA基因、2个rRNA基因）。 线粒体基因和基因组已经广泛应用于分子进化研究，包括系统发育和种群结构研究。 图1 线粒体模式图 Fig.1 Module map of the mitochondria Animal mtDNA is a circular DNA molecule presenting a conserved set of 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and two ribosomal RNA (rRNA) genes. The control region is also known as the A+T-rich region in insects. The mito genes and genomes have been widely used as an informative molecular marker for diverse evolutionary studies of animals, including phylogenetics and population genetics.
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 鳞翅目是昆虫中仅次于鞘翅目的第2大目。最近，人们对鳞翅目昆虫线粒体基因组的序列测定投入了极大的兴趣。 目前，公共数据库中可以利用的鳞翅目昆虫线粒体基因组全序列有11个，其中有5个来自于蚕丝昆虫包括蚕蛾科的家蚕、中国野桑蚕、日本野桑蚕和大蚕蛾科的合目大蚕蛾、柞蚕。 测定新的蚕类昆虫线粒体基因组可以为我们理解蚕类昆虫线粒体基因组的多样性和进化提供更深入的帮助。 There has been a recent interest in mitogenome sequencing within Lepidoptera, which represents the second only to Coleoptera biodiversity in insects. To date, eleven complete mitogenomes have been currently available in the GenBank for lepidopterans, of which 5 complete mitogenomes are from silkworms (3 Bombycidae and 2 Saturniidae). Newly added silkworm mitogenomes can provide further insight into our understanding of insect mitogenome diversity and evolution.
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 柞蚕线粒体基因组（图2）长 15566 bp，其基因构成和排列与已测的鳞翅目昆虫的线粒体基因组一致。 The complete mitogenome of A. pernyi (Fig.2) is 15,566 bp, similar to other sequenced lepidopteran mitogenomes, and presents the typical gene content, gene order and orientation observed in metazoan mitogenomes. 图2 柞蚕(AY242996)的mt DNA基因组图谱 Fig.2 Map of the mitogenome of A. pernyi
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 柞蚕线粒体基因组主链AT的含量是80.16%，碱基T的含量高于碱基A （AT skewness为0.021）。 除了COI基因之外，其他蛋白编码基因均以最常见的ATN密码子作为起始子。我们推测COI基因以TTAG作为起始子，但还需要进一步在mRNA转录水平进行研究。 The genome composition of the major strand of the A. pernyi mitogenome is heavily biased toward As and Ts, which accounts for a total of 80.16%, and with a slightly negative AT skewness (0.021), showing the occurrence of more Ts than As. All PCGs are initiated by ATN codons, except for COI, which is proposed by the TTAG sequence as observed in other lepidopterans. More studies for mRNA transcripts are necessary to clarify the position of COI initiation.
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 除tRNASer(AGN)之外，其余的tRNA基因均可形成典型的线粒体tRNA的三叶草结构，而tRNASer(AGN)基因DHU环不能形成稳定的茎环结构（图3）。 All tRNAs have a clover-leaf structure typical of mitochondrial tRNA, except for tRNASer(AGN), the DHU arm of which could not form a stable stem-loop structure (Fig.3). 图3 推测的tRNASer(AGN)基因结构 Fig. 3 Inferred structure of tRNASer(AGN)
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 非编码区总计202 bp，分布在18个区域，长度在1~56 bp之间。 这些非编码区的序列与分布在昆虫线粒体基因组中并不是保守的，表明它们并没有显著的功能意义。 18 non-coding regions ranging from 1 to 56 bp (=202 bp) were identified in the A. pernyi mitogenome. The location and sequence of the non-coding regions are not conserved in other insect sequences, which would imply that these regions have no functional significance.
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 从AT富集区（图4）鉴定出了~38 bp的重复单元。该重复单元目前仅在柞蚕属中发现。可能用于种群遗传分析以及柞蚕属系统发育研究。 The repeat region in Saturniidae and Bombycidae may have independent origins after these families diverged. 图4 AT富集区的结构示意图Fig. 4 Map of the A+T-rich region In the A+T-rich region (Fig.4), a repeat region composed of six 38 bp tandem repeat units was identified. The repeat unit is unique only to Antheraea genus. The repeat region could be potentially be useful in analyzing the genetic diversity of populations as well as phylogenetic studies of genus Antheraea. (Aunkumar et al..2006.Molecular Phylogenetics and Evolution)
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 重复单元（图5）在A. pernyi和A. proylei中均是重复6次；而在A. roylei和新发现的云南野柞蚕中是5次重复。Aunkumar et al.（2006）将A. roylei鉴定为6次重复。 图5 重复区的对齐结果 Fig. 5 Alignmentof the repeat region We identified the repeat region composed of 38 bp tandemly repeated six times in the A+T-rich region of A. pernyi and A. proylei, whereas five times in A. roylei and a new subspecies of A. pernyi (A. pernyi wild; see below) (Fig.5). In the paper by Aunkumar et al.(2006) the repeat region in A. roylei was identified to be tandemly repeated six times.
1. 柞蚕线粒体基因组的特征 The mitochondrial genome of Antheraea pernyi 柞蚕线粒体基因组的全序列可以作为一个参考序列，在野蚕进化和种群遗传研究方面具有重要意义，尤其为开展其它野蚕线粒体基因组研究奠定了基础。 The complete mitogenome of A. pernyi can be used as a resource on the evolutionary history and population genetics of wild silkmoths to help generate and support additional mitogenome research in the Antheraea species.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 2001年发现于云南曲靖，形态学上与饲养柞蚕一致，称之为“云南野柞蚕”(A. pernyi wild) 。 DNA条形编码技术对其分类学地位的探讨表明，可以考虑成为柞蚕种的一个亚种——野柞蚕亚种 （朱绪伟和刘彦群等，2008）。 The wild Chinese oak silkworm was collected from Yunnan Province in China. It was identified as the wild type of A. pernyi according to the high similarity with the domestic populations by morphology. Analysis of the DNA Barcodingindicated that A. pernyi wild collected in Yunnan could be considered as a new subspecies of A. pernyi.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 野生柞蚕: 青黄蚕体色 ; 1化和2化的混合群体；个别个体可以保持长达2年的滞育状态 ；飞翔能力强，交配能力差。 放养柞蚕: 青黄蚕、黄蚕、白蚕、蓝蚕四种体色，黄蚕品种分布于南方，青黄蚕品种分布于北方；化性基本稳定；飞翔能力弱，交配能力强。 Wild population：a yellow-cyan skin color for larvae; both univoltine and bioltine in nature; some individuals keeping in pupal diapause through two years Domestic counterparts: four kinds of skin color including yellow-cyan, yellow, white, and blue; either univoltine or bivoltine; no individuals delaying in the diapause break.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 放养柞蚕种群和野生柞蚕种群均起源于一个共同的祖先（古柞蚕），并在不同的选择压力下生存。 因此，调查同一种内的放养类型和野生类型的线粒体全基因组的碱基变异和进化模式就显得非常有意义。 The domestic and wild populations are believed to have originated from a common ancestor (ancient Chinese oak silkworm) in the past and undergone different selection pressure. Therefore, it is very interesting to ascertain the nucleotide variation and evolutionary patterns of complete mitogenomes between domestic and its wild type within a single species.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 野生柞蚕的线粒体基因组长 15 537 bp，比放养柞蚕小29 bp（15 566 bp）。 基因构成和排列与放养柞蚕一致。 在核苷酸和氨基酸序列上的同源性分别高达98%和99%，说明二者的分化时间时比较短的。 The wild A. pernyi mitogenome was determined to be 15537 bp in length, thus 29 bp smaller than that of domestic A. pernyi（ 15566 bp ）. Its organization and composition are identical to that of domestic. The mitochondrial nucleotides and peptides comparatively show 98% and 99% identity, respectively, indicating that the time divergence after domestication is relatively short.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 野生柞蚕的AT富集区长516 bp，比放养柞蚕小36 bp（552 bp），并且二者的核苷酸序列相似性高达97%。 最大的差别在于，野生柞蚕的AT富集区的重复区（图5）只包含5个重复单元（184 bp），而放养柞蚕则包含6个（221 bp） 。 我们还测定了11个柞蚕品种的AT富集区，结果确认均包含6个重复单元。 The wild A. pernyi A+T rich region is 516 bp, thus 36 bp shorter than that of domestic. The nucleotide sequence similarity reach 97% between them. The wild A. pernyi A+T rich region contains a repeat region composed of five tandem repeat units (Fig.5), but six tandem repeat units in domestic. We also sequenced the A+T region from the eleven cultivars, confirming the presence of six repeat elements.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 13个PCGs的Ka/Ks比值非常低（0.0640），这表明PCGs承受着纯选择的压力，并且各个PCGs承受的纯选择压力也不同。 图6 13个PCGs的Ka/Ks比值 Fig. 6 The Ka/Ks ratio of 13 PCGs The low Ka/Ks ratio of 13 PCGs (0.0640) indicates that the PCGs are under purifying selection, and these PCGs may reflect different purifying selection pressure.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 在线粒体基因组上放养与野生柞蚕表现出较低的序列变异（1.70%）和较高的转换突变（ts:tv = 4.13），但家蚕与野桑蚕之间则表现出较高的序列变异（3.11%）和较高的颠换突变（ts:tv = 1.42）。 相对过量的转换突变也发生在Drosophila melanogaster亚族的成员（Ballard, 2000 ）和玉米螟（Coates et al., 2005 ）。 Comparative analysis revealed a relatively lower level of sequence divergence (1.70%) and an excess transition mutation (ts:tv = 4.13) between the two mitogenomes, whereas a relatively higher level of sequence divergence (3.11%) and an relatively higher transversion mutation (ts:tv = 1.42) between B. mori and Chinese B. mandrinia. Relatively excess transition mutation was also reported between Drosophila melanogaster subgroup members (Ballard, 2000), and between Ostrinia nubilalis and O. furnicalis (Coates et al., 2005).
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 统计分析表明，野生柞蚕与放养柞蚕线粒体基因组的序列变异显著小于家蚕与野桑蚕之间的（1.70% vs 3.11%; χ2 = 70.79, d.f. = 1, P < 0.001）,而转换突变比率则显著高于家蚕与野桑蚕之间的（4.13 vs 1.42；χ2 = 27.67, d.f. = 1, P < 0.001）。 因此，柞蚕与桑蚕的线粒体基因组进化模式是明显不同的。 The sequence divergence between wild and domestic A. pernyi is significantly smaller compared to the data observed between B. mori and Chinese B. mandarina(1.70% vs 3.11%; χ2 = 70.79, d.f. = 1, P < 0.001), however, the transition mutation ratio is significantly higher (4.13 vs 1.42；χ2 = 27.67, d.f. = 1, P < 0.001). The evolutionary pattern between wild and domestic A. pernyi mitogenomes is distinct from that between the truly domesticated Bombyx mori and its probable ancestor Chinese B. mandarina.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 在线粒体基因组水平上柞蚕与桑蚕具有明显不同的进化模式，这说明它们承受着不同的进化压力。 我们推测，柞蚕线粒体基因组的过量转换突变可能主要是由于环境选择压力所造成的，而桑蚕线粒体基因组的相对过量颠换突变则可能主要是由于人工选择压力所造成的。 The distinct nucleotide substitution pattern between Antheraea and Bombyx showed they are attributed to different evolutionary forces. We suggested that an excess transition mutations between the two Antheraea mitogenomes are mainly subjected to the environment selection pressure, whereas a relatively excess transversion mutation in mitogenomes from Bombyx is mainly subjected to the artificial selection pressure.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 图7 放养与野生柞蚕线粒体基因的核苷酸序列替换 Fig. 7 Nucleotide differences betweendomestic and wild A. pernyi mitochondrialgenes
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 柞蚕线粒体基因组中13个PCGs基因（除ND4L外）之间的核苷酸序列替换比率（图7）并没有显著的差异（χ2 = 10.13, d.f. = 11, P < 0.5） 。 桑蚕线粒体基因组中有五个基因（ND1、COI、COII、ATP6、ND2）平均替换比率显著小于另外四个基因（COIII、ND4、ND5、cytB）；果蝇线粒体基因组中则有四个隔开的区域表现不同的碱基替换模式。 说明柞蚕线粒体基因组所承受的进化压力与桑蚕和果蝇是不同的。 In Antheraea mitogenome, there is no significant differences in nucleotide substitution rate (Fig.7) for the 13 PCGs except for ND4L gene (χ2 = 10.13, d.f. = 11, P < 0.5). However, in Bombyx mitogenomes the averge substitution rate of the five genes (ND1,COI,COII,ATP6,ND2) is significantly smaller than that of the four genes(COIII,ND4,ND5,CytB), whereas in Drosophia mitogenomes four disjunct regions have a significantly different nucleotide substitution process. The Antheraea mitogenome is not under the common evolutionary process to Drosophia and Bombyx.
2. 放养柞蚕与野生柞蚕的线粒体基因组比较 Comparative mitogenomics between domestic and wild A. pernyi 根据线粒体基因COI和COII的序列，估计的野生柞蚕与放养柞蚕线粒体的分歧时间是在0.74~0.97百万年前。 这个时间远大于历史记载的柞蚕放养时间（3000年）。我们推测是驯化后人工选择对线粒体基因组所造成的影响（加速了线粒体基因组的进化）。 The divergence time between the two mitochondria was estimated to be about 0.74 ± 0.13 ~ 0.97 ± 0.17 MYA, based on the genes COI+COII sequences. This time course is much larger than that (3000 years) in historic records. The excess divergence time is supposed to be attributed to the artificial selection which might act on the mitogenome during the domestication (Pan et al.,2008). We used the substitution rate of 7.8–10.2×10–9 per site per year from the mitochondrial genes COI+COII of the swallowtail butterflies of the genus Papilio (Lepidoptera: Papilionidae) as a molecular clock.
附 1：蚕类昆虫线粒体基因组研究 Sequencing of the mitogenome of the important silk-producing insects 正在进行线粒体基因组全序列测定的蚕类昆虫包括大乌桕蚕、樗蚕、栗蚕、柳蚕、樟蚕、波洛丽蚕等。 该工作是与合肥工业大学魏兆军博士（教授）合作进行的。 蚕类昆虫资源丰富，希望各位同仁合作。 The gene content and order for the available lepidopteran mitogenomes are highly conserved, allowing us to conclude that the mitogenomes from other silk-producing insects have an identical gene content and order as observed in A. pernyi. Thus, more complete mitogenome sequences from Antheraea species must be determined in order to reconstruct their phylogenetic relationships. We are sequencing the Attacus atlas, Philosamia cyntia, Dictyopoea japonica, Actias selene, Eriogyna pyretoum, Antheraea proylei, et al.
附 2：柞蚕功能基因组研究 The functional genomics of Chinese oak silkworm, A. pernyi 家蚕基因组框架图的完成和功能基因组研究的实施，为柞蚕相关研究跨越到基因组水平和开展与家蚕的比较基因组研究奠定了基础。 与辽宁省蚕业科学研究所、西南大学合作构建了柞蚕蛹的全长cDNA文库。 已经完成了3*96个ESTs测定，正在进行100个柞蚕功能基因全长cDNA的序列测定工作，下一步将进行这些基因的基因组结构和表达模式研究。 Functional genomics has particular promise in silkworm biology for identifying genes involved in a variety of biological functions. We have constructed a full-length cDNA library from pupae of Chinese oak silkworm, A. pernyi. We will obtain about 2000 ESTs from this library. About 300 EST sequences have been generated from Chinese oak silkworm, of which about 100 ESTs have been chosen to provide the full-length cDNA for the functional studies.
