马尾松毛虫线粒体全基因组的测定与分析

doi:10.11707/j.1001-7488.20191217

摘要/Abstract

摘要：

目的: 测定和分析马尾松毛虫线粒体基因组的特征，从线粒体基因组水平探究蛾类昆虫高级阶元的系统发育关系。方法: 采用HiSeq X Ten测序仪测定马尾松毛虫线粒体的全基因组序列，参考鳞翅目昆虫已知线粒体基因组的全序列对其各基因进行定位和注释。采用tRNA Scan-SE 2.0软件预测tRNA基因的二级结构。基于线粒体全基因组的核苷酸序列构建鳞翅目蛾类13个科50种昆虫的系统发育关系。结果: 马尾松毛虫线粒体基因组全长15 417 bp，A+T含量为79.6%，包括13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和一段长度为320 bp的A+T富含区，无基因重排。13个蛋白质编码基因中，氨基酸使用较频繁的依次包括Phe、Leu、Ile、Tyr和Asn（Count>290），而Ala和Arg的使用相对较少（Count < 50）；在其密码子使用中，除COI以CGA为起始密码子外，其余基因的起始密码子均为昆虫典型的ATN（ATA、ATT、ATG）；终止密码子除COI、COII和ND4基因为T外，其他基因的均为TAA。在所测得22个tRNA基因中，tRNA^Ser（AGN）缺少DHU臂，其余tRNA均为典型的三叶草结构。A+T富含区存在1段由ATAGAA引导保守长度为14 bp的多聚T结构和2段长度为13 bp的重复单元以及1个（AT）₇结构。马尾松毛虫与其他松毛虫属的线粒体基因组全序列及13个蛋白质编码基因的核苷酸序列进行比对分析，结果表明，马尾松毛虫与文山松毛虫和油松毛虫三者间具有较高的同源性。系统发育树的结果显示：马尾松毛虫与油松毛虫亲缘关系最近，思茅松毛虫和赤松毛虫亲缘关系最近。结论: 马尾松毛虫线粒体基因结构稳定，基于线粒体基因组构建的鳞翅目蛾类昆虫的系统发育关系与传统的形态分类学结果基本一致。

关键词: 鳞翅目, 枯叶蛾科, 马尾松毛虫, 线粒体基因组, 系统发育

Abstract:

Objectve: In this study, we sequenced the mitochondrial genome of Dendrolimus punctatus, and analyzed the mitochondrial genome characteristics and the higher-level phylogenetic relationships in moths of Lepidoptera. Method: The complete genome sequence of the mitochondrial genome of D. punctatus was determined by Hiseq X Ten sequencer. The genes were located and annotated based on the known complete mitochondrial genomes of Lepidopteran species. The tRNA Scan-SE 2.0 was used to predict the secondary structure of the tRNA genes online. Based on the whole nucleotide sequence of mitochondrial genome, the phylogenetic relationships of 50 insect species from 11 families of Lepidoptera were reconstructed. Result: The results showed that the complete mitochondrial genome of D. punctatus was a circular molecule of 15 417 bp, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and one control region without gene rearrangement. Among the 13 protein-coding genes, amino acids of Phe, Leu, Ile, Tyr, and Asn (Count>290) were used more frequently, while Ala and Arg were uesd less frequently (Count < 50). In the use of its codon, except CGA as the starting codon of COI gene, the starting codons of the other genes were all typical ATN (ATA, ATT, ATG). The stop codon of COI、COII and ND4 was T, while that of the other genes was TAA. All tRNAs showed the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNA^Ser(AGN) formed a simple loop. The (A+T)-rich region contained some conserved structure including the motif ATAGAA, 14 bp ploy (T) stretch and two repeating units with a length of 13 bp and one (AT)₇ structure. The complete mitochondrial genome sequence and the nucleotide sequence of 13 protein-coding genes of D. punctatus were compared with other species of Dendrolimus genus, and the results showed that there was high homology between D.punctatus, D.wenshanensis, and D.tabulaeformis. Phylogenetic tree results show that D. punctatus is closely related with D. tabulaeformis, and D. spectabilis is closely related with D. kikuchii. Conclusion: The mitochondrial genes structure of D. punctatus are stable, the phylogenetic relationship of some species from Lepidoptera based on mitochondrial genome is consistent with the traditional morphological classification.

Key words: Lepidoptera, Lasiocampidae, Dendrolimus punctatus, mitochondrial genome, phylogeny

中图分类号:

S718.7

杜会聪,王瑶,方加兴,张珍荫,张苏芳,刘福,张真,孔祥波. 马尾松毛虫线粒体全基因组的测定与分析[J]. 林业科学, 2019, 55(12): 162-172.

Huicong Du,Yao Wang,Jiaxing Fang,Zhenyin Zhang,Sufang Zhang,Fu Liu,Zhen Zhang,Xiangbo Kong. Sequencing and Analysis of the Complete Mitochondrial Genome of Dendrolimus punctatus (Lepidoptera: Lasiocampidae)[J]. Scientia Silvae Sinicae, 2019, 55(12): 162-172.

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基因 Gene	方向 Direction	起始位置 Start position	终止位置 Stop position	长度 Length/bp	反密码子 Anticodon	起始密码子 Start codon	终止密码子 Stop codon	基因间隔 IGS/bp
trnM	F	1	67	67	CAT
trnI	F	75	135	61	GAT			7
trnQ	R	133	201	69	TTG			-3
ND2	F	260	1 267	1 008		ATT	TAA	58
trnW	F	1 266	1 334	69	TCA			-2
trnC	R	1 327	1 392	66	GCA			-8
trnY	R	1 393	1 460	68	GTA			0
COI	F	1 495	3 025	1 531		CGA	T	34
trnL2	F	3 026	3 092	67	TAA			0
COII	F	3 093	3 774	682		ATA	T	0
trnK	F	3 775	3 845	71	CTT			0
trnD	F	3 849	3 916	68	GTC			3
ATP8	F	3 917	4 078	162		ATT	TAA	0
ATP6	F	4 072	4 749	678		ATG	TAA	-7
COIII	F	4 764	5 552	789		ATG	TAA	14
trnG	F	5 555	5 620	66	TCC			2
ND3	F	5 621	5 974	354		ATG	TAA	0
trnA	F	5 974	6 040	67	TGC			-1
trnR	F	6 056	6 119	64	TCG			15
trnN	F	6 124	6 189	66	GTT			4
trnS1	F	6 201	6 268	68	GCT			11
trnE	F	6 268	6 332	65	TTC			-1
trnF	R	6 337	6 402	66	GAA			4
ND5	R	6 405	8 147	1 743		ATT	TAA	2
trnH	R	8 148	8 215	68	GTG			0
ND4	R	8 216	9 554	1 339		ATG	T	0
ND4L	R	9 579	9 872	294		ATG	TAA	24
trnT	F	9 880	9 943	64	TGT			7
trnP	R	9 944	10 008	65	TGG			0
ND6	F	10 017	10 547	531		ATA	TAA	8
CytB	F	10 552	11 700	1 149		ATG	TAA	4
trnS2	F	11 704	11 770	67	TGA			3
ND1	R	11 770	12 723	954		ATG	TAA	-1
trnL1	R	12 725	12 792	68	TAG			1
rrnL	R	12 793	14 253	1 461				0
trnV	R	14 254	14 318	65	TAC			0
rrnS	R	14 319	15 097	779				0
D-loop	F	15 098	15 417	320				0

基因序列 Gene sequences	T (%)	C (%)	A (%)	G (%)	A+T (%)	C+G (%)	AT偏斜 AT-skew	GC偏斜 GC-skew
全基因组Whole genome	38.6	12.5	41.0	7.9	79.6	20.4	0.030	-0.223
蛋白质编码基因Protein-coding genes	44.1	10.8	33.7	11.4	77.8	22.2	-0.134	-0.027
密码子第1位1^st codon site	45.7	9.1	35.6	9.6	81.3	18.7	-0.124	-0.024
密码子第2位2^nd codon site	41.9	11.2	34.0	12.9	75.9	24.1	-0.105	-0.070
密码子第3位3^rd codon site	44.8	12.1	31.4	11.7	76.2	23.8	-0.176	-0.028
tRNA基因tRNA genes	39.1	8.5	41.8	10.6	80.9	19.1	0.033	0.110
rRNA基因rRNA genes	43.7	4.3	41.2	10.8	84.9	15.1	-0.029	0.430
控制区Control region	49.4	5.0	42.8	2.8	92.2	7.8	-0.072	-0.282

物种Species	全基因组 Whole genome		PCG		tRNA		A+T-rich region		登录号 GenBank Accession No.
物种Species	大小 Size/bp	A+T (%)	大小 Size/bp	A+T (%)	大小 Size/bp	A+T (%)	大小 Size/bp	A+T (%)	登录号 GenBank Accession No.
马尾松毛虫D. punctatus	15 417	79.6	11 214	77.8	1 465	80.9	320	92.2	MK886799
文山松毛虫D. punctatus wenshanensis	15 418	79.4	11 214	77.6	1 468	81.0	320	92.5	KJ_913812
油松毛虫D. tabulaeformis	15 411	79.5	11 211	77.7	1 468	81.0	320	92.8	KJ_913817
思茅松毛虫D. kikuchii	15 370	78.8	11 211	77.0	1 473	80.7	319	92.5	KY_000412
落叶松毛虫D. superans	15 417	80.1	11 217	78.5	1 475	81.2	316	90.2	KY_000413
云南松毛虫D. houi	15 381	80.0	11 219	78.3	1 477	81.2	319	92.2	KJ_913817
赤松毛虫D. spectabilis	15 410	79.4	11 211	77.5	1 468	81.2	320	93.4	KJ_913816

物种 Species	马尾松毛虫 D. punctatus	文山松毛虫 D. punctatus wenshanensis	油松毛虫 D. tabulaeformis	思茅松毛虫 D. kikuchii	落叶松毛虫 D. superans	云南松毛虫 D. houi	赤松毛虫 D. spectabilis
马尾松毛虫D. punctatus	100.00	98.84	98.89	90.85	91.31	93.45	95.12
文山松毛虫D. punctatus wenshanensis	98.99	100.00	99.46	90.78	91.14	93.29	95.00
油松毛虫D. tabulaeformis	98.99	99.55	100.00	90.78	91.23	93.34	95.09
思茅松毛虫D. kikuchii	91.49	91.48	91.49	100.00	91.28	91.11	90.78
落叶松毛虫D. superans	91.75	91.66	91.71	91.85	100.00	91.86	91.41
云南松毛虫D. houi	93.84	93.76	93.79	91.73	92.36	100.00	93.38
赤松毛虫D. spectabilis	95.64	95.59	95.64	91.53	91.87	93.83	100.00

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