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LI Yan, SHU Jinping, HUA Keda, ZHANG Yabo, YING Yue, ZHANG Wei. Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra[J]. Journal of Zhejiang A&F University, 2024, 41(4): 724-734. doi: 10.11833/j.issn.2095-0756.20240138
Citation: LI Yan, SHU Jinping, HUA Keda, ZHANG Yabo, YING Yue, ZHANG Wei. Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra[J]. Journal of Zhejiang A&F University, 2024, 41(4): 724-734. doi: 10.11833/j.issn.2095-0756.20240138
shu

Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra

doi: 10.11833/j.issn.2095-0756.20240138
  • 1.

    Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

  • 2.

    Hangzhou Fuyang District Agriculture and Rural Bureau, Hangzhou 311400, Zhejiang, China

  • Received Date: 2024-01-23
  • Accepted Date: 2024-04-22
  • Rev Recd Date: 2024-04-15
    • Available Online: 2024-06-04
  • Publish Date: 2024-07-12
  •   Objective  This study, with a comprehensive sequencing and analysis of the mitochondrial genome of Garella ruficirra as well as an investigation into the phylogenetic position of Noctuidae through mitochondrial genome sequencing, is aimed to offer a valuable reference for delving into the phylogenetic relationships and evolutionary processes within the Noctuidae as well as exploring the phylogenetic relationship and evolutionary process of Noctuidae.   Method  First, the Illumina sequencing technique was employed to sequence the complete mitochondrial genome of G. ruficirra, allowing for the analysis of its overall characteristics and base composition. Subsequently, a phylogenetic tree encompassing mitochondrial genomes from 5 genera of Noctuidae, comprising 12 species, was constructed using both maximum likelihood and Bayesian methods so as to facilitate the examination of the phylogenetic relationships within the Noctuidae.   Result  The circular mitochondrial genome of G. ruficirra spanned 15 294 base pairs and encompasses 13 protein synthesis-related genes, 22 transfer RNA genes, and two ribosomal RNA genes and its gene arrangement conformed to the standard configuration observed in Lepidoptera mitogenomes, featuring a region rich in adenine and thymine, an A+T content of 80.53% and a significant AT bias. The gene order of trnM-trnI-trnQ in G. ruficirra aligned with other Noctuidae species and the majority of Lepidoptera with ATN sequences serving as initiation codons for all 13 protein-coding genes. While trnS1 lacked a DHU arm, the remaining tRNA genes exhibited a typical cloverleaf-shaped structure. According to the study of mitochondrial genome, Garella was most closely related to Nycteola and least closely related to Pseudoips among the 5 genera.   Conclusion  Gene rearrangement was evident in the mitochondrial genome of G. ruficirra. Phylogenetic analyses, indicating that the clustering of G. ruficirra and G. musculana samples was within a single clade. This study has furnished valuable insights into the evolutionary patterns of Garella and offered a foundational framework for investigating the phylogeny and evolution of Noctuidae within the Lepidoptera order. [Ch, 4 fig. 4 tab. 47 ref.]
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Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra

doi: 10.11833/j.issn.2095-0756.20240138
  • 1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China
  • 2. Hangzhou Fuyang District Agriculture and Rural Bureau, Hangzhou 311400, Zhejiang, China
  • Received Date: 2024-01-23
  • Accepted Date: 2024-04-22
  • Rev Recd Date: 2024-04-15
  • Available Online: 2024-06-04
  • Publish Date: 2024-07-12

Abstract:   Objective  This study, with a comprehensive sequencing and analysis of the mitochondrial genome of Garella ruficirra as well as an investigation into the phylogenetic position of Noctuidae through mitochondrial genome sequencing, is aimed to offer a valuable reference for delving into the phylogenetic relationships and evolutionary processes within the Noctuidae as well as exploring the phylogenetic relationship and evolutionary process of Noctuidae.   Method  First, the Illumina sequencing technique was employed to sequence the complete mitochondrial genome of G. ruficirra, allowing for the analysis of its overall characteristics and base composition. Subsequently, a phylogenetic tree encompassing mitochondrial genomes from 5 genera of Noctuidae, comprising 12 species, was constructed using both maximum likelihood and Bayesian methods so as to facilitate the examination of the phylogenetic relationships within the Noctuidae.   Result  The circular mitochondrial genome of G. ruficirra spanned 15 294 base pairs and encompasses 13 protein synthesis-related genes, 22 transfer RNA genes, and two ribosomal RNA genes and its gene arrangement conformed to the standard configuration observed in Lepidoptera mitogenomes, featuring a region rich in adenine and thymine, an A+T content of 80.53% and a significant AT bias. The gene order of trnM-trnI-trnQ in G. ruficirra aligned with other Noctuidae species and the majority of Lepidoptera with ATN sequences serving as initiation codons for all 13 protein-coding genes. While trnS1 lacked a DHU arm, the remaining tRNA genes exhibited a typical cloverleaf-shaped structure. According to the study of mitochondrial genome, Garella was most closely related to Nycteola and least closely related to Pseudoips among the 5 genera.   Conclusion  Gene rearrangement was evident in the mitochondrial genome of G. ruficirra. Phylogenetic analyses, indicating that the clustering of G. ruficirra and G. musculana samples was within a single clade. This study has furnished valuable insights into the evolutionary patterns of Garella and offered a foundational framework for investigating the phylogeny and evolution of Noctuidae within the Lepidoptera order. [Ch, 4 fig. 4 tab. 47 ref.]

LI Yan, SHU Jinping, HUA Keda, ZHANG Yabo, YING Yue, ZHANG Wei. Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra[J]. Journal of Zhejiang A&F University, 2024, 41(4): 724-734. doi: 10.11833/j.issn.2095-0756.20240138
Citation: LI Yan, SHU Jinping, HUA Keda, ZHANG Yabo, YING Yue, ZHANG Wei. Sequencing and analysis of the complete mitochondrial genome of Garella ruficirra[J]. Journal of Zhejiang A&F University, 2024, 41(4): 724-734. doi: 10.11833/j.issn.2095-0756.20240138
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  • 线粒体广泛存在于动物和植物的每个个体之中，作为真核细胞中一种半自主的细胞器，能够满足生命过程如生长发育、新陈代谢及移动所需要的能源需求，对诸如细胞功能调节、老龄衰退或死亡等现象发挥着重要的作用[1−2]。相较于其他类型的基因而言，线粒体结构简单且遵循母系遗传，同时演化速度更快，变异频率也更高[3]。昆虫线粒体基因组是一串长约14~19 bp的闭合双链环状DNA分子，共有37个基因，由13个蛋白质编码基因(PCGs)、22个转运RNA(tRNAs)以及2个核糖体RNA(rRNAs)构建而成[4]。通过线粒体基因组研究昆虫的进化规律和系统发育关系已经成为成熟的手段[5]。

    线粒体基因组技术主要应用于昆虫起源、分子进化、系统发育等研究领域，涉及到包括鞘翅目Coleoptera、半翅目Hemiptera、膜翅目Hymenoptera等在内的昆虫[6−10]。鳞翅目Lepidoptera作为昆虫纲中第二大目，害虫种类繁多，因此在昆虫研究中具有重要的代表性[11]。暗影饰皮夜蛾Garella ruficirra隶属于鳞翅目夜蛾科Noctuidae丽夜蛾亚科Chloephorinae[12]，在河北、河南、山东、江苏、浙江和江西等省均有不同程度的危害发生[13]。暗影饰皮夜蛾是薄壳山核桃Carya illinoinensis、板栗Castanea mollissima等经济林的重要害虫[12, 14]，薄壳山核桃害虫种类繁多[15]，近年来暗影饰皮夜蛾在薄壳山核桃林内新发生且呈现逐年上升的趋势，现有的研究多数集中在发生情况的简报和防治建议方面[16−17]。对其遗传特征、与同类昆虫之间的亲缘关系研究还未见报道。因此，对暗影饰皮夜蛾进行全面的线粒体全基因组分析，除了能丰富对夜蛾科尤其是丽夜蛾亚科昆虫线粒体基因组的了解外，还能为暗影饰皮夜蛾的分子系谱、群落遗传特性以及分子生态环境等研究提供数据支持。

    本研究通过对暗影饰皮夜蛾线粒体的碱基组成、起始密码子、终止密码子、二级结构等进行研究，将其与已经公布的鳞翅目昆虫的线粒体基因组相比较，通过最大似然法和贝叶斯法联合构建系统发育树，以期分析暗影饰皮夜蛾线粒体基因组特征与系统发育关系，为暗影饰皮夜蛾的演变历程和系统发展提供分子证据。

    • 1.   材料与方法

      1.1.   供试昆虫及DNA提取方法

    • 2022年10月，在江苏省东台市(32°47′42″N，120°31′08″E)薄壳山核桃林中采集暗影饰皮夜蛾幼虫危害的薄壳山核桃果实，带回实验室。解剖出来的幼虫放置在−80 ℃超低温冰箱保存，用于提取DNA。选择龄期一致的幼虫个体作为研究对象，利用DNA提取试剂盒(德国Qiagen公司)提取暗影饰皮夜蛾的总DNA，使用质量浓度为1%的琼脂糖凝胶检测该幼虫DNA的纯度和浓度。

      • 1.2.   线粒体基因测序

    • 检测总DNA质量后，采用全基因组鸟枪法(WGS)，使用Illumina Miseq平台进行双端测序(PE)构建文库，在得到原始序列后，通过FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc)平台对原始序列进行质量剪切和过滤：包括去除读长(reads)中的接头(adapter)序列，剪切去除5′端包含有非腺嘌呤(A)、胸腺嘧啶(T)、胞嘧啶(C)、鸟嘌呤(G)的碱基；修剪低质量的reads末端；去除无法确定的碱基信息(N)含量大于5的reads；去掉adapter以及经过质量修剪之后长度不足25 bp的小片段，最终得到高质量的干净数据(clean data)[18−19]。

      • 1.3.   线粒体基因组拼接及注释

    • 采用A5-miseq v20150522以及SPAdes v 3.10.0软件对获得的高质量clean data进行从头组装，从而得到重叠群(contig)和支架(scaffold)序列[20−22]。使用Mummer v 3.1软件进一步剔除线粒体基因组序列中模糊核苷酸和质量平均值低于G30(质量值大于等于30的碱基所占比例)的reads，进行数据重组，得到contig和scaffold。将完整的线性contigs上传到MITOS网页服务器(http://mitos2.bioinf.uni-leipzig.de/index.py)进行功能注释[22−23]。选择“Genetic Code”的可选设置为05-inverterbrate，其余设置按照默认参数进行选择。使用Organella Genome Draw网络服务器工具(http://ogdraw.mpimp-golm.mpg.de/)对样本的环状线粒体基因组进行可视化处理[22, 24]。按照拼接序列的测序深度，使用美国国家生物技术信息中心(NCBI)中的nt文库进行高测序深度的BLAST分析，并将其与拼接后的序列进行比较，获得预测基因的注释信息[22]。将序列上传至NCBI数据库中，获得登录号为ON125428。

      • 1.4.   线粒体基因组的比较分析

    • 使用https://www.bioinformatics.org/cgview/gallery网址进行暗影饰皮夜蛾线粒体基因组圈图的绘制，接着使用MEGA 6软件计算G. ruficirra包括AT含量、AT偏斜以及GC偏斜等在内的核苷酸组成情况，同时对蛋白质编码基因的氨基酸使用情况和相对同义密码子使用度(RSCU)进行分析[22]。应用tRNAscan-SE Search Server v1.21[25]预测暗影饰皮夜蛾tRNA基因的二级结构模型，分析暗影饰皮夜蛾的碱基组成差异。结合区域、螺旋、螺旋跨膜区域和其他不规则区域，对ATP8蛋白的二级结构进行预测，同时对ATP8蛋白的氨基酸组成和编码序列组成进行分析。

      • 1.5.   系统发育分析

    • 基于夜蛾科13个种昆虫线粒体全基因组的核苷酸序列，选择天蛾科Sphingidae 2个种作为外群，使用http://www.phylo.org网址联合建立最大似然法(ML)和贝叶斯法(BI)系统发育树。

      以NCBI下载的12种夜蛾科昆虫的序列作为参考，与3种天蛾科昆虫的序列联合进行系统发育分析。通过贝叶斯框架和BEAST v 1.6.1软件包对克隆进化的分化时间进行估算，同时选用J model test软件得到最佳替代模型GTR+I+G (表1)。

      类群总科属物种
      内群夜蛾科NoctuidaeGarella暗影饰皮夜蛾G. ruficirra
      G. musculana
      G. rotundipennis
      G. nilotica
      G. curiosa
      皮夜蛾属NycteolaN. indica
      亚皮夜蛾N. asiatica
      饰纹夜蛾属Antoculeora饰银纹夜蛾A. ornatissima
      Ctenoplusia白条夜蛾C. albostriata
      C. ogovana
      银纹夜蛾C. agnata
      饰夜蛾属PseudoipsP. prasinana
      外群天蛾科Sphingidae蛀野螟属Conogethes桃蛀螟C. punctiferalis
      秆野螟属Ostrinia玉米螟O. nubilalis

      Table 1.  Information of the insect species for phylogenetic analysis in mitochondrial genome

    2.   结果与分析

      2.1.   线粒体基因组的序列与结构

    • 暗影饰皮夜蛾线粒体基因组全长为15 294 bp，呈闭合环状的双链结构，整个线粒体基因组编码了37个基因，其中包括13个蛋白质编码基因(PCGs)、22个tRNA基因、2个rRNA基因(rrnL和rrnS)以及1个A+T富含区域。其中，9个蛋白质编码基因以及14个tRNA基因编码在N链上，其余14个基因编码在J链上。其线粒体基因组在25个不同的位置存在长度约1~66 bp的基因间间隔区，最长间隔序列位于trnE和trnF基因之间，其中有6对基因为重叠状态，重叠长度在1~7 bp，5对基因相邻，包括trnV-rrnS (图1和表2)。

      Figure 1.  Structure of the mitochondrial genome of G. ruficirra

      基因编码链位置长度/
      bp      		起始密
      码子      		终止密
      码子      		反密
      码子      		基因间隔
      核苷酸      		基因编码链位置长度/
      bp      		起始密
      码子      		终止密
      码子      		反密
      码子      		基因间隔
      核苷酸
      trnMN1~6868CATtrnNN6 062~6 12867GTT3
      trnIN69~13365GAT−3trnS1N6 132~6 19766GCT
      trnQJ131~19969TTG57trnEN6 198~6 26366TTC65
      nad2N257~1 2701 014ATTTAA−2trnFJ6 329~6 39264GAA2
      trnWN1 269~1 33668TCA−8nad5J6 395~8 1371 743ATTTAA
      trnCJ1 329~1 39163GCA2trnHJ8 138~8 20366GTG−1
      trnYJ1 394~1 45966GTA2nad4J8 203~9 5431 341ATGTAA−1
      cox1N1 462~3 0001 539ATGTAA−5nad4lJ9 543~9 836294ATGTAA2
      trnL2N2 996~3 06267TAAtrnTN9 839~9 90466TGT
      cox2N3 063~3 747685ATGT(AA)−3trnPJ9 905~9 96965TGG6
      trnKN3 745~3 81571CTTnad6N9 976~10 509534ATTTAA6
      trnDN3 816~3 88267GTCcobN10 516~11 6671152ATGTAA13
      atp8N3 883~4 044162ATTTAA−7trnS2N11 681~11 74666TGA19
      atp6N4 038~4 715678ATGTAA−1nad1J11 766~12 704939ATGTAA1
      cox3N4 715~5 503789ATGTAA2trnL1J12 706~12 77368TAG36
      trnGN5 506~5 57065TCCrrnLJ12 810~14 099129027
      nad3N5 571~5 924354ATCTAA6trnVJ14 127~14 19165TAG−1
      trnAN5 931~5 99868TGC−1rrnSJ14 191~14 972782−13
      trnRN5 998~6 06164TCG

      Table 2.  Organization of the mitochondrial genome of G. ruficirra

      • 2.2.   线粒体基因组核苷酸组成

    • 暗影饰皮夜蛾线粒体基因组A、T、C、G含量分别为39.02%、41.51%、11.55%和7.92%，线粒体基因组的A+T含量为80.53%，表现为A、T碱基偏向性(表3)，蛋白质编码基因的A+T含量在71.42% (cox1)~90.74% (atp8)范围内[22]。

      基因序列碳基含量/% AT偏斜GC偏斜
      ACGTA+TG+C
      全基因组39.0211.557.9241.5180.5319.47−0.0309−0.1867
      蛋白质编码基因33.389.9511.1545.5278.9021.10−0.15380.0566
      密码子第1位36.139.4916.7837.6073.7326.27−0.01990.2777
      密码子第2位22.0816.1713.4248.3370.4129.59−0.3728−0.0930
      密码子第3位41.944.203.2350.6392.577.43−0.0938−0.1295
      tRNA基因42.337.5311.2338.9081.2318.770.04220.1971
      rRNA基因44.164.9710.1440.7384.8915.110.04040.3419
      控制区47.143.701.0148.1595.294.71−0.0106−0.5714

      Table 3.  Nucleotide composition of the complete mitochondrial genome

      整个核苷酸链显示轻微的AT偏斜和中度的GC偏斜(表3和图2)。在鳞翅目其他昆虫的线粒体基因组中同样存在AT偏斜和GC偏斜的情况[25−28]。暗影饰皮夜蛾的蛋白质编码基因和密码子第1位的AT偏斜为负，GC偏斜为正，密码子第2位和第3位产生的AT偏斜与GC偏斜均为负，tRNA和rRNA产生的AT偏斜和GC偏斜全部为正，蛋白质编码基因中T和G的占比高于A和C。

      Figure 2.  Percentage of each amino acid of proteins coded

      • 2.3.   蛋白质编码基因、密码子使用度和蛋白质符合率

    • 在暗影饰皮夜蛾线粒体基因组编码的13个蛋白质基因中，有9个基因编码在N链上，另外4个基因编码在J链上(图2)，蛋白质编码基因总长为11 224 bp，占全基因组的73.39% (表2)。

      暗影饰皮夜蛾13个蛋白质编码基因都是以ATN作为起始密码子，多数都是以ATG作为起始密码子，nad3以ATC作为起始密码子，而nad2、atp8、nad5、nad6以ATT作为起始密码子(表2)。大多数蛋白质编码基因都是以TAA作为终止密码子，只有cox2以不完整的T作为终止密码子，这与多数昆虫线粒体基因组以TAA或TAG作为终止密码子的情况相同[29]。

      对暗影饰皮夜蛾蛋白质编码基因的氨基酸使用情况和相对同义密码子使用度(RSCU)的分析结果(表4)表明：除终止密码子外，暗影饰皮夜蛾线粒体基因组共有3 745个密码子，与多数昆虫线粒体编码出来的蛋白质密码子在3 585~3 746的范围一致。线粒体基因组中使用频率最高的4个密码子分别是UUU (苯丙氨酸Phe)、UUA (亮氨酸Leu)、UAU (酪氨酸Tyr)、AUA (异亮氨酸Ile)，这4个密码子在线粒体基因组中的使用次数分别为488、474、315、304次，因此，蛋白质编码基因中编码最频繁的氨基酸依次为Leu (17.0%)、Ile (16.3%)、Phe (11.0%)、Tyr (8.0%)。使用最少的氨基酸是色氨酸Trp (0.52%)，以C和G为结尾的密码子出现频率较低，以U和A为结尾的密码子通常RSCU大于1，说明U和A为基因组密码子中的偏爱碱基，C和G为基因组密码子中的非偏爱碱基。

      氨基酸密码子使用次数/次RSCU氨基酸密码子使用次数/次RSCU
      丙氨酸(Ala) GCU
      GCC
      GCA
      GCG      		 48
      5
      24
      12      		 2.16
      0.22
      1.08
      0.54      		 脯氨酸(Pro) CCG
      CCA
      CCU
      CCC      		 9
      24
      38
      16      		 0.41
      1.10
      1.75
      0.74
      半胱氨酸(Cys) UGU
      UGC      		 39
      13      		 1.50
      0.50      		 谷氨酰胺(Gln) CAG
      CAA      		 24
      70      		 0.51
      1.49
      天冬氨酸(Asp) GAU
      GAC      		 96
      15      		 1.73
      0.27      		 精氨酸(Arg) CGU
      CGC
      CGA
      CGG      		 10
      0
      12
      5      		 0.47
      0.00
      0.56
      0.23
      谷氨酸(Glu) GAG
      GAA      		 38
      76      		 0.67
      1.33
      苯丙氨酸(Phe) UUU
      UUC      		 488
      97      		 1.67
      0.33      		 酪氨酸(Tyr) UAU
      UAC      		 315
      38      		 1.78
      0.22
      甘氨酸(Gly) GGG
      GGA
      GGU
      GGC      		 36
      56
      48
      2      		 1.01
      1.58
      1.35
      0.06      		 丝氨酸(Ser) UCU
      UCC
      UCA
      UCG      		 77
      27
      66
      17      		 1.91
      0.67
      1.64
      0.42
      甲硫氨酸(Met) AUG 68 1.00 色氨酸(Trp) UGG 23 1.00
      天冬氨酸(Asn) AAU
      AAC      		 295
      33      		 1.80
      0.20      		 苏氨酸(Thr) ACG
      ACA
      ACU
      ACC      		 7
      30
      56
      25      		 0.24
      1.02
      1.90
      0.85
      赖氨酸(Lys) AAG
      AAA      		 61
      250      		 0.39
      1.61
      亮氨酸(Leu) UUG
      UUA
      CUG
      CUA
      CUU
      CUC      		 77
      474
      28
      53
      97
      22      		 0.62
      3.79
      0.22
      0.42
      0.77
      0.18      		 缬氨酸(Val) GUG
      GUA
      GUU
      GUC      		 18
      70
      75
      12      		 0.41
      1.60
      1.71
      0.27
      组氨酸(His) CAU
      CAC      		 72
      11      		 1.73
      0.27
      异亮氨酸(Ile) AUA
      AUU
      AUC      		 304
      373
      42      		 1.27
      1.56
      0.18      		 终止(End) UGA
      UAG
      UAA      		 77
      82
      285      		 0.52
      0.55
      1.93

      Table 4.  Usage of amino acids and the relative synonymous codon usage

      • 2.4.   tRNA和rRNA基因

    • 暗影饰皮夜蛾线粒体基因组的22个tRNA基因总长度为1 458 bp (表2)，在暗影饰皮夜蛾线粒体全基因组中占比为9.53%，在N链上编码14个tRNA基因，J链上编码8个tRNA基因。暗影饰皮夜蛾线粒体基因组的22个tRNA基因个体长度分布在63 bp (trnC)~71 bp (trnK)范围。rrnL分布在trnL和trnV之间、J链上的rrnS则分布在trnV和线粒体控制区(CR)之间。rrnL长度为1 290 bp，rrnS长度为782 bp，rrnL的A+T含量为84.81%，rrnS的A+T含量为85.04%。从表3可以看出：AT偏斜和GC偏斜都表现为正数，说明暗影饰皮夜蛾在A和G碱基具有偏向性。

      通过http://mitos.bioinf.uni-leipzig.de/网址分析暗影饰皮夜蛾tRNA基因的二级结构模型，其中21个tRNA均具有典型的三叶草形态，只有trnS1的DHU臂被环结构取代，无法形成三叶草形态(图3)。暗影饰皮夜蛾的tRNA二级结构中，有22对碱基出现了错配现象，在普遍的U-G错配中，氨基酸接受臂上存在4对，TΨC臂上存在2对，反密码子臂存在4对，DHU臂上存在10对，其余2对错配分别为DHU臂上存在1对A-A错配，TΨC臂上存在1对A-A错配。

      Figure 3.  Comparison on the secondary structure of tRNA genes

      • 2.5.   系统发育关系

    • 系统发育树结果(图4)表明：5个夜蛾属Garella与皮夜蛾属Nycteola亲缘关系最近，饰纹夜蛾属Antoculeora与Ctenoplusia属亲缘关系最近，与Garella和Nycteola稍远，这4种夜蛾与饰夜蛾属Pseudoips亲缘关系最远。Garella属的5种夜蛾之间，暗影饰皮夜蛾与G. musculana亲缘关系最近，G. rotundipennis与G. nilotica亲缘关系最近，与暗影饰皮夜蛾与G. musculana稍远，这4种夜蛾与G. curiosa亲缘关系最远。

      Figure 4.  Phylogenetic tree of based on protein-coding genes sequences of Noctuidae species

    3.   讨论

    • 20世纪80年代暗影饰皮夜蛾在河南、山东等省已有发现，分布范围逐步扩大，在多个地区已成为板栗的主要害虫[30−32]，近年来在薄壳山核桃主栽区逐年持续暴发。本研究表明：暗影饰皮夜蛾线粒体基因组长度为15 294 bp，在鳞翅目昆虫线粒体基因组报道的长度范围内[33]。其线粒体基因组所有区域表现出AT偏向性，与网纹蟒Reticulated python、绒螨目Trombidiformes螨类、对虾科Penaeidae物种相同[34−36]。而trnM-trnI-trnQ的基因排列方式在鳞翅目多个物种中均有存在[37−40]，这种重排方式十分典型[41−43]。暗影饰皮夜蛾线粒体基因组所有蛋白质编码基因均以ATN为起始密码子，这与大多数鳞翅目昆虫一致[29]。暗影饰皮夜蛾cox1的起始密码子均为ATG，ATG是最常用的起始密码子，这与多数夜蛾科昆虫一致，与果蝇Drosophila melanogaster的起始密码子(ATT)不同[44−45]。除cox2以不完整的T为终止密码子外，其余13个蛋白质编码基因的终止密码子都是TAA，蛋白质编码基因中使用不完全终止密码子在无脊椎动物中十分常见[46−47]。其线粒体基因组有21个tRNA为典型的三叶草构造，只有tRNASer(AGN)缺少DHU臂，这在多种昆虫中均有出现，是夜蛾科的普遍特征[25−26]。

      夜蛾科是鳞翅目中最大的一个科，而目前对夜蛾科线粒体基因组的研究种类较少，研究内容不全面，尤其是Garella属，目前除暗影饰皮夜蛾以外，其他物种均未进行线粒体基因组测定，因此需要补充大量夜蛾科各属昆虫线粒体全基因组序列的信息，为进一步探讨夜蛾科各属之间的系统发育关系提供基础数据。

    4.   结论

    • 暗影饰皮夜蛾的线粒体全基因组的碱基含量为T＞A＞C＞G，表现为AT偏向性，基因组排列顺序为trnM-trnI-trnQ，存在基因重排现象。暗影饰皮夜蛾线粒体基因组的二级结构为典型的三叶草构造。系统发育分析表明：暗影饰皮夜蛾和G. musculana的亲缘关系最近。

Reference (47)

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