覃屏生等
摘要:采用PCR擴(kuò)增方法測(cè)定了鱷蜥
關(guān)鍵詞: 鱷蜥(Shinisaurus crocodilurus Ahl.);線粒體基因組;控制區(qū)
中圖分類號(hào):Q953.3 文獻(xiàn)標(biāo)識(shí)碼:A 文章編號(hào):0439-8114(2014)04-0925-04
Mitochondrial Genomes of Shinisaurus crocodilurus and Its Structure
QIN Ping-sheng,TAO Chun-rong,SHE Ying,HOU Li-xia,YANG Xiao-wen,WU Zheng-jun,QIN Xin-min
(College of Life Science, Guangxi Normal University/Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guilin 541004,Guangxi, China)
Abstract:The complete nucleotide sequence of the mitochondrial genome of Shinisaurus crocodilurus was determined by PCR. The complete mtDNA from S. crocodilurus was 16 585 bp in length and contained 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region(D-loop). The 22 tRNA genes can be fold in the typical cloverleaf structure except tRNA-Ser(AGY)gene. The average nucleotide composition was 31.99% A, 26.48% T, 27.96% C and 13.57% G. Among 13 protein-coding genes of the S. crocodilurus mitochondrial genome, the common start codon (ATG) could be assigned as the start codon for all protein-coding genes except for COI which started with ATC. Most of the protein-coding genes including ND1,ND2,COI,ATP6,ATP8,ND4L,ND4,ND6,Cytb had TAA, AGA or AGG as termination codon,while COⅡ, COⅢ, ND3 and ND5 had incomplete termination codons TA/T.
Key words: Shinisaurus crocodilurus Ahl.; mitochondrial genome; control region
鱷蜥(Shinisaurus crocodilurus Ahl.)隸屬有鱗目鱷蜥科(Shiniasuridae)鱷蜥屬(Shinisaurus),為獨(dú)科獨(dú)屬獨(dú)種動(dòng)物,分布于中國廣東、廣西以及越南北部[1]。鱷蜥為第四紀(jì)冰川末期殘留下來的古老爬行類動(dòng)物,有“活化石之稱”,其分類地位非常特殊,因此其在爬行綱動(dòng)物的起源、演化和分類等方面的研究上,有著重要的學(xué)術(shù)價(jià)值。近年來,由于人為捕捉,棲息環(huán)境被污染、破壞,鱷蜥種群數(shù)量急劇下降,目前在我國已不足1 000只,已處于極度瀕危的狀況,被國家列為Ⅰ級(jí)保護(hù)動(dòng)物,并被列入瀕危物種國際貿(mào)易公約(Convention on International Trade in Endangered Species)附錄Ⅱ。
目前,關(guān)于鱷蜥的研究主要集中在物種資源調(diào)查、生態(tài)、行為、生理和繁殖等方面[1-6]。動(dòng)物線粒體DNA是研究物種系統(tǒng)發(fā)育、種群遺傳變異和分化、起源與進(jìn)化的有效遺傳標(biāo)記。試驗(yàn)測(cè)定了鱷蜥的線粒體基因組全序列,以期為鱷蜥的遺傳結(jié)構(gòu)、起源進(jìn)化以及種質(zhì)資源保護(hù)等相關(guān)研究提供分子信息。
1 材料與方法
1.1 材料
鱷蜥死亡個(gè)體尾部肌肉。
1.2 基因組DNA的提取
基因組DNA的提取參照汪永慶等[7]的方法。
1.3 引物的設(shè)計(jì)
參考爬行動(dòng)物通用引物擴(kuò)增12S rRNA、16S rRNA、ND2、ND3、COI、COIII、ND5、ND6和Cytb基因的部分片段,根據(jù)上述片段的測(cè)序結(jié)果,利用Primer 5.0軟件設(shè)計(jì)引物,用以擴(kuò)增短片段之間的長(zhǎng)片段序列,所用引物見表1。
1.4 PCR擴(kuò)增測(cè)序
2.2 蛋白質(zhì)編碼基因
3 小結(jié)
目前,國內(nèi)外有關(guān)鱷蜥的研究主要集中在生態(tài)、行為、生理、和人工飼養(yǎng)等方面。本研究分析了鱷蜥線粒體基因組全序列,包括了13個(gè)蛋白質(zhì)編碼基因、22個(gè)tRNA基因、2個(gè)rRNA基因和1個(gè)控制區(qū)(D-loop)。結(jié)果表明,鱷蜥線粒體基因組中基因的組成、順序、編碼鏈的選擇、tRNA的結(jié)構(gòu)都與爬行動(dòng)物相同或相近。結(jié)果可為鱷蜥的分類、進(jìn)化和系統(tǒng)發(fā)育等方面提供分子依據(jù)。
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[4] 于 海,黃乘明,武正軍,等.鱷蜥生活習(xí)性的觀察[J].四川動(dòng)物,2006,25(2):364-366.
[5] 王振興,武正軍,于 海,等.廣東羅坑自然保護(hù)區(qū)鱷蜥的體溫調(diào)節(jié)及靜止代謝率的熱依賴性[J].動(dòng)物學(xué)報(bào),2008,54(6):964-971.
[6] 王振興,武正軍,蔡鳳金,等. 鱷蜥人工飼養(yǎng)技術(shù)[J].廣東林業(yè)科技,2010,26(5):51-55.
[7] 汪永慶,王新國,徐來詳,等.一種動(dòng)物基因組DNA提取方法的改進(jìn)[J].動(dòng)物學(xué)雜志,2001,36(1):27-29.
[8] SCHATTNER P, BROOKS A N, LOWE T M. The tRNA scan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs[J]. Nucleic Acids Research,2005,33(s2):686-689.
[9] TAMURA K, DUDLEY J, NEI M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution,2007,24(8):1596-1599.
[10] QIN X M, QIAN F, ZENG D L, et al. Complete mitochondrial genome of the red-spotted tokay gecko(Gekko gecko, Reptilia: Gekkonidae): Comparison of red-and black-spotted tokay geckos[J]. Mitochondrial DNA,2011,22(5/6):176-177.
[11] JANKE A, ARNASON U. The complete mitochondrial genome of Alligator mississippiensis and the separation between recent Archosauria (Birds and Crocodiles)[J]. Mol Biol Evol, 1997, 14(2):1266-1272.
[3] 寧加佳,黃乘明,于 海,等.廣東羅坑自然保護(hù)區(qū)鱷蜥夏季生境特征[J].動(dòng)物學(xué)研究,2006,27(4):419-426.
[4] 于 海,黃乘明,武正軍,等.鱷蜥生活習(xí)性的觀察[J].四川動(dòng)物,2006,25(2):364-366.
[5] 王振興,武正軍,于 海,等.廣東羅坑自然保護(hù)區(qū)鱷蜥的體溫調(diào)節(jié)及靜止代謝率的熱依賴性[J].動(dòng)物學(xué)報(bào),2008,54(6):964-971.
[6] 王振興,武正軍,蔡鳳金,等. 鱷蜥人工飼養(yǎng)技術(shù)[J].廣東林業(yè)科技,2010,26(5):51-55.
[7] 汪永慶,王新國,徐來詳,等.一種動(dòng)物基因組DNA提取方法的改進(jìn)[J].動(dòng)物學(xué)雜志,2001,36(1):27-29.
[8] SCHATTNER P, BROOKS A N, LOWE T M. The tRNA scan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs[J]. Nucleic Acids Research,2005,33(s2):686-689.
[9] TAMURA K, DUDLEY J, NEI M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution,2007,24(8):1596-1599.
[10] QIN X M, QIAN F, ZENG D L, et al. Complete mitochondrial genome of the red-spotted tokay gecko(Gekko gecko, Reptilia: Gekkonidae): Comparison of red-and black-spotted tokay geckos[J]. Mitochondrial DNA,2011,22(5/6):176-177.
[11] JANKE A, ARNASON U. The complete mitochondrial genome of Alligator mississippiensis and the separation between recent Archosauria (Birds and Crocodiles)[J]. Mol Biol Evol, 1997, 14(2):1266-1272.