四倍体野生种花生Ty1-copia类逆转座子逆转录酶基因的克隆与分析
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摘要
克隆与分析四倍体野生种花生的Ty1-copia类逆转座子逆转录酶基因序列,可为研究其转录活性、功能及调控提供序列基础。使用根据保守区设计的简并引物对,利用PCR技术对四倍体野生种花生(Arachis monticola)的基因组DNA进行扩增,经回收、克隆和测序,对目的序列进行生物信息学分析。结果显示,目的条带大小约260 bp,克隆获得了32条逆转录酶序列,序列长度范围为257～269 bp,A+T所占比例范围为54.47%～68.77%,A+T与G+C比例为1.20～2.20。核苷酸序列间相似性范围为46.4%～98.9%,存在较高异质性,表现为缺失突变与点突变;氨基酸序列间相似性范围为6.3%～100%,有12条序列发生了终止密码子突变,呈现高度异质性。绝大部分序列的保守基序一致,但各序列间的保守基序也存在一定差异,呈现一定程度的异质性。32条序列系统聚类为4个家族,家族Ⅰ和家族Ⅲ分别占总序列数的31.25%和37.50%。对四倍体野生种花生与其它物种植物同一类型逆转录酶的氨基酸序列构建系统发育进化树,结果显示所有序列被分为6类,其中,Ⅰ类和Ⅱ类分别包含15条和9条四倍体野生种花生逆转录酶序列,表明四倍体野生种花生逆转录酶序列具有比较高的保守性;同时四倍体野生种花生逆转录酶序列与葡萄、拟南芥、马铃薯、野茶树、辣椒、甜菜、烟草、番茄、绿豆以及欧洲云杉等具有较近的亲缘关系,表明它们之间可能存在横向传递。本研究获得的逆转录酶序列为基于LTR逆转座子的花生属分子标记开发和应用奠定了基础。
The reverse transcriptase sequences of Ty1-copia retrotransposons from tetraploid wild peanut species were cloned and analyzed, which could provide sequence basis for studying its transcriptional activity, function and regulation. The genomic DNA of Arachis monticola was amplified by PCR using a pair of degenerate primers designed according to the conservative region of reverse transcriptases of Ty1-copia retrotransposons. Then the target band was recovered, cloned and sequenced and the obtained sequences were analyzed through the bioinformatics method. The results showed that the size of the target band was approximately 260 bp and thirty-two reverse transcriptase sequences were obtained. The length of sequences ranged from 257 bp to 269 bp. The proportion of A+T ranged from 54.47% to 68.77%. The ratio of A+T to G+C was 1.20～2.20. The similarity between nucleotide sequences ranged from 46.4% to 98.9%. These indicated that the nucleotide sequences of reverse transcriptases existed higher heterogeneity in the form of deletion mutation and point mutation. The similarity between amino acid sequences ranged from 6.3% to 100% and twelve sequences had termination codon mutations, which indicated that the amino acid sequences of reverse transcriptase existed high heterogeneity. The conservative motifs of most sequences were identical, but also there were some differences between them, showing a certain degree of heterogeneity. The thirty-two reverse transcriptase sequences were clustered into four families. FamilyⅠand Family Ⅲ accounted for 31.25% and 37.50% of the total sequences, respectively. Phylogenetic tree was constructed according to the amino acid sequences of the same type of reverse transcriptases in tetraploid wild peanut species and some other plant species. The results showed that all sequences were classified into six categories. Among them, ClassⅠand ClassⅡ contained fifteen and nine reverse transcriptase sequences from tetraploid wild peanut species, respectively, which showed that the reverse transcriptase sequences of tetraploid wild peanut species were highly conservative. Meanwhile, the reverse transcriptase sequences of tetraploid wild peanut species had close relationship with grape, Arabidopsis, potato, Camellia sinensis, pepper, sugarbeet, tobacco, tomato, mung bean and Picea abies. It indicated that horizontal transmission might occur between these plant species. The reverse transcriptase sequences obtained in this study laid a foundation for the development and application of molecular markers in Arachis based on LTR retrotransposons.
The reverse transcriptase sequences of Ty1-copia retrotransposons from tetraploid wild peanut species were cloned and analyzed, which could provide sequence basis for studying its transcriptional activity, function and regulation. The genomic DNA of Arachis monticola was amplified by PCR using a pair of degenerate primers designed according to the conservative region of reverse transcriptases of Ty1-copia retrotransposons. Then the target band was recovered, cloned and sequenced and the obtained sequences were analyzed through the bioinformatics method. The results showed that the size of the target band was approximately 260 bp and thirty-two reverse transcriptase sequences were obtained. The length of sequences ranged from 257 bp to 269 bp. The proportion of A+T ranged from 54.47% to 68.77%. The ratio of A+T to G+C was 1.20～2.20. The similarity between nucleotide sequences ranged from 46.4% to 98.9%. These indicated that the nucleotide sequences of reverse transcriptases existed higher heterogeneity in the form of deletion mutation and point mutation. The similarity between amino acid sequences ranged from 6.3% to 100% and twelve sequences had termination codon mutations, which indicated that the amino acid sequences of reverse transcriptase existed high heterogeneity. The conservative motifs of most sequences were identical, but also there were some differences between them, showing a certain degree of heterogeneity. The thirty-two reverse transcriptase sequences were clustered into four families. FamilyⅠand Family Ⅲ accounted for 31.25% and 37.50% of the total sequences, respectively. Phylogenetic tree was constructed according to the amino acid sequences of the same type of reverse transcriptases in tetraploid wild peanut species and some other plant species. The results showed that all sequences were classified into six categories. Among them, ClassⅠand ClassⅡ contained fifteen and nine reverse transcriptase sequences from tetraploid wild peanut species, respectively, which showed that the reverse transcriptase sequences of tetraploid wild peanut species were highly conservative. Meanwhile, the reverse transcriptase sequences of tetraploid wild peanut species had close relationship with grape, Arabidopsis, potato, Camellia sinensis, pepper, sugarbeet, tobacco, tomato, mung bean and Picea abies. It indicated that horizontal transmission might occur between these plant species. The reverse transcriptase sequences obtained in this study laid a foundation for the development and application of molecular markers in Arachis based on LTR retrotransposons.
引文
[1] Kumar A,Pearce S R,Mclean K,et al.The Tyl-copia group of retrotransposons in plants:genomic organisation,evolution and use as molecular markers[J].Genetica,1997,100(1/2/3):205-217.
[2] Kumar A,Bennetzen J L.Plant retrotransposons:annual review of genetics[J].1999,33:479-532.
[3] Feschotte C,Jiang N,Wessler S R.Plant transposable elements:where genetics meets genomics[J].Nature Reviews Genetics,2002,3(5):329-341.
[4] Bonchev G,Parisod C.Transposable elements and microevolutionary changes in natural populations[J].Molecular Ecology Resources,2013,13(5):765-775.
[5] Voytas D F,Cummings M P,Konieczny A,et al.Copia-like retrotransposons are ubiquitous among plants[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(15):7124-7128.
[6] Kumekawa N,Ohtsubo E,Ohtsubo H.Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom[J].Genes & Genetic Systems,1999,74(6):299-307.
[7] Kalendar R,Grob T,Regina M,et al.IRAP and REMAP:two new retrotransposon-based DNA fingerprinting techniques[J].Theoretical and Applied Genetics,1999,98(5):704-711.
[8] Waugh R,Mclean K,Flavell A J,et al.Genetic distribution of bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP) [J].Molecular and General Genetics,1997,253(6):687-694.
[9] Patel M,Jung S,Moore K,et al.High-oleate peanut mutants result from a MITE insertion into the FAD2 gene[J].Theoretical and Applied Genetics,2004,108(8):1492-1502.
[10] Gowda M V C,Bhat R S,Motagi B N,et al.Association of high-frequency origin of late leaf spot resistance mutants with AhMITE1 transposition in peanut[J].Plant Breeding,2010,129(5):567-569.
[11] Gowda M V C,Bhat R S,Sujay V,et al.Characterization of AhMITE1 transposition and its association with the mutational and evolutionary origin of botanical types in peanut (Arachis spp.) [J].Plant Systematics and Evolution,2011,291(3/4):153-158.
[12] Shirasawa K,Hirakawa H,Tabata S,et al.Characterization of active miniature inverted-repeat transposable elements in the peanut genome[J].Theoretical and Applied Genetics,2012,124 (8):1429-1438.
[13] Shirasawa K,Koilkonda P,Aoki K,et al.In silico polymorphism analysis for the development of simple sequence repeat and transposon markers and construction of linkage map in cultivated peanut[J].BMC Plant Biology,2012,12(1):80-92.
[14] Shirasawa K,Bertioli D J,Varshney R K,et al.Integrated consensus map of cultivated peanut and wild relatives reveals structures of the A and B genomes of arachis and divergence of the legume genomes[J].DNA Research,2013,20(2):173-184.
[15] Mondal S,Hande P,Badigannavar A M.Identification of transposable element markers for a rust (Puccinia arachidis Speg.) resistance gene in cultivated peanut[J].Journal of Phytopathology,2014,162(7/8):548-552.
[16] Hake A A,Shirasawa K,Yadawad A,et al.Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.) [J].PLoS ONE,2017,12(10):e0186113.
[17] Gayathri M,Shirasawa K,Varshney R K,et al.Development of AhMITE1 markers through genome-wide analysis in peanut (Arachis hypogaea L.) [J].BMC Research Notes,2018,11(1):10-15.
[18] 王洁,李双铃,王辉,等.利用AhMITE1转座子分子标记鉴定花生F1代杂种[J].花生学报,2012,41(2):8-12.
[19] 王辉,李双铃,任艳,等.利用AhMITE转座子分子标记研究花生栽培种及高世代材料的亲缘关系[J].农业生物技术学报,2013,21(10):1176-1184.
[20] 尹亮,任艳,石延茂,等.利用AhMITE1转座子分子标记鉴定栽培花生杂交F1代种子真伪[J].山东农业科学,2015,47(12):1-5.
[21] 许梦琦,李双铃,任艳,等.花生作图亲本间分子标记的多态性分析[J].湖北农业科学,2015,54(11):2763-2766.
[22] 吴琪,曹广英,尹亮,等.利用AhMITE转座子分子标记鉴定花生杂交F1代真假杂种[J].花生学报,2017,46(3):48-53.
[23] 刘婷,王传堂,唐月异,等.利用近红外技术和转座子标记鉴定花生杂交F1真杂种[J].分子植物育种,2017,15(9):3592-3598.
[24] Nielen S,Campos-Fonseca F,Leal-Bertioli S,et al.FIDEL-a retrovirus-like retrotransposon and its distinct evolutionary histories in the A- and B-genome components of cultivated peanut[J].Chromosome Research,2010,18(2):227-246.
[25] Nielen S,Vidigal B S,Leal-Bertioli S C M,et al.Matita,a new retroelement from peanut:characterization and evolutionary context in the light of the Arachis A-B genome divergence[J].Molecular Genetics and Genomics,2012,287(1):21-38.
[26] 熊发前,刘俊仙,贺梁琼,等.花生LTR和MITE转座子及其分子标记开发利用研究进展[J].分子植物育种,2017,15(2):640-647.
[27] 熊发前,刘俊仙,刘菁,等.花生DNA的五种改良CTAB提取方法的比较分析及其应用[J].分子植物育种,2019,17(7):2207-2216.
[28] Steinhauer D A,Holland J J.Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA[J].Journal of Virology,1986,57(1):219-228.
[29] Gabriel A,Willems M,Mules E H,et al.Replication infidelity during a single cycle of Ty1 retrotransposition[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(15):7767-7771.
[30] Song Y,Ji D,Li S,et al.The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean[J].PLoS ONE,2012,7(7):e41274.
[31] Heslop-Harrison J S,Brandes A,Taketa S,et al.The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implication for genome evolution[J].Genetica,1997,100(1/2/3):197-204.
[32] Ostertag E M,Madison B B,Kano H.Mutagenesis in rodents using the L1 retrotransposon[J].Genome Biology,2007,8 (Supplement 1):S16.
[33] Doolittle R F,Feng D F,Johnson M S,et al.Origins and evolutionary relationships of retroviruses[J].Quarterly Review of Biology,1989,64(1):1-30.
[34] Bennetzen J L,Wang H.The contributions of transposable elements to the structure,function,and evolution of plant genomes[J].Annual Review of Plant Biology,2014,65(1):505-530.
[35] Kumar A.The evolution of plant retroviruses:moving to green pastures[J].Plant Science,1998,3(10):371-374.
[36] Diao X M,Freeling M,Lish D.Horizontal transfer of a plant transposon[J].PLoS Biology,2006,4(1):119-128.
[37] Tang Y M,Ma Y Z,Li L C,et al.Identification and characterization of reverse transcriptase domain of transcriptionally active retrotransposons in wheat genome[J].Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica),2005,47(5):604-612.
[2] Kumar A,Bennetzen J L.Plant retrotransposons:annual review of genetics[J].1999,33:479-532.
[3] Feschotte C,Jiang N,Wessler S R.Plant transposable elements:where genetics meets genomics[J].Nature Reviews Genetics,2002,3(5):329-341.
[4] Bonchev G,Parisod C.Transposable elements and microevolutionary changes in natural populations[J].Molecular Ecology Resources,2013,13(5):765-775.
[5] Voytas D F,Cummings M P,Konieczny A,et al.Copia-like retrotransposons are ubiquitous among plants[J].Proceedings of the National Academy of Sciences of the United States of America,1992,89(15):7124-7128.
[6] Kumekawa N,Ohtsubo E,Ohtsubo H.Identification and phylogenetic analysis of gypsy-type retrotransposons in the plant kingdom[J].Genes & Genetic Systems,1999,74(6):299-307.
[7] Kalendar R,Grob T,Regina M,et al.IRAP and REMAP:two new retrotransposon-based DNA fingerprinting techniques[J].Theoretical and Applied Genetics,1999,98(5):704-711.
[8] Waugh R,Mclean K,Flavell A J,et al.Genetic distribution of bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP) [J].Molecular and General Genetics,1997,253(6):687-694.
[9] Patel M,Jung S,Moore K,et al.High-oleate peanut mutants result from a MITE insertion into the FAD2 gene[J].Theoretical and Applied Genetics,2004,108(8):1492-1502.
[10] Gowda M V C,Bhat R S,Motagi B N,et al.Association of high-frequency origin of late leaf spot resistance mutants with AhMITE1 transposition in peanut[J].Plant Breeding,2010,129(5):567-569.
[11] Gowda M V C,Bhat R S,Sujay V,et al.Characterization of AhMITE1 transposition and its association with the mutational and evolutionary origin of botanical types in peanut (Arachis spp.) [J].Plant Systematics and Evolution,2011,291(3/4):153-158.
[12] Shirasawa K,Hirakawa H,Tabata S,et al.Characterization of active miniature inverted-repeat transposable elements in the peanut genome[J].Theoretical and Applied Genetics,2012,124 (8):1429-1438.
[13] Shirasawa K,Koilkonda P,Aoki K,et al.In silico polymorphism analysis for the development of simple sequence repeat and transposon markers and construction of linkage map in cultivated peanut[J].BMC Plant Biology,2012,12(1):80-92.
[14] Shirasawa K,Bertioli D J,Varshney R K,et al.Integrated consensus map of cultivated peanut and wild relatives reveals structures of the A and B genomes of arachis and divergence of the legume genomes[J].DNA Research,2013,20(2):173-184.
[15] Mondal S,Hande P,Badigannavar A M.Identification of transposable element markers for a rust (Puccinia arachidis Speg.) resistance gene in cultivated peanut[J].Journal of Phytopathology,2014,162(7/8):548-552.
[16] Hake A A,Shirasawa K,Yadawad A,et al.Mapping of important taxonomic and productivity traits using genic and non-genic transposable element markers in peanut (Arachis hypogaea L.) [J].PLoS ONE,2017,12(10):e0186113.
[17] Gayathri M,Shirasawa K,Varshney R K,et al.Development of AhMITE1 markers through genome-wide analysis in peanut (Arachis hypogaea L.) [J].BMC Research Notes,2018,11(1):10-15.
[18] 王洁,李双铃,王辉,等.利用AhMITE1转座子分子标记鉴定花生F1代杂种[J].花生学报,2012,41(2):8-12.
[19] 王辉,李双铃,任艳,等.利用AhMITE转座子分子标记研究花生栽培种及高世代材料的亲缘关系[J].农业生物技术学报,2013,21(10):1176-1184.
[20] 尹亮,任艳,石延茂,等.利用AhMITE1转座子分子标记鉴定栽培花生杂交F1代种子真伪[J].山东农业科学,2015,47(12):1-5.
[21] 许梦琦,李双铃,任艳,等.花生作图亲本间分子标记的多态性分析[J].湖北农业科学,2015,54(11):2763-2766.
[22] 吴琪,曹广英,尹亮,等.利用AhMITE转座子分子标记鉴定花生杂交F1代真假杂种[J].花生学报,2017,46(3):48-53.
[23] 刘婷,王传堂,唐月异,等.利用近红外技术和转座子标记鉴定花生杂交F1真杂种[J].分子植物育种,2017,15(9):3592-3598.
[24] Nielen S,Campos-Fonseca F,Leal-Bertioli S,et al.FIDEL-a retrovirus-like retrotransposon and its distinct evolutionary histories in the A- and B-genome components of cultivated peanut[J].Chromosome Research,2010,18(2):227-246.
[25] Nielen S,Vidigal B S,Leal-Bertioli S C M,et al.Matita,a new retroelement from peanut:characterization and evolutionary context in the light of the Arachis A-B genome divergence[J].Molecular Genetics and Genomics,2012,287(1):21-38.
[26] 熊发前,刘俊仙,贺梁琼,等.花生LTR和MITE转座子及其分子标记开发利用研究进展[J].分子植物育种,2017,15(2):640-647.
[27] 熊发前,刘俊仙,刘菁,等.花生DNA的五种改良CTAB提取方法的比较分析及其应用[J].分子植物育种,2019,17(7):2207-2216.
[28] Steinhauer D A,Holland J J.Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA[J].Journal of Virology,1986,57(1):219-228.
[29] Gabriel A,Willems M,Mules E H,et al.Replication infidelity during a single cycle of Ty1 retrotransposition[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(15):7767-7771.
[30] Song Y,Ji D,Li S,et al.The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean[J].PLoS ONE,2012,7(7):e41274.
[31] Heslop-Harrison J S,Brandes A,Taketa S,et al.The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implication for genome evolution[J].Genetica,1997,100(1/2/3):197-204.
[32] Ostertag E M,Madison B B,Kano H.Mutagenesis in rodents using the L1 retrotransposon[J].Genome Biology,2007,8 (Supplement 1):S16.
[33] Doolittle R F,Feng D F,Johnson M S,et al.Origins and evolutionary relationships of retroviruses[J].Quarterly Review of Biology,1989,64(1):1-30.
[34] Bennetzen J L,Wang H.The contributions of transposable elements to the structure,function,and evolution of plant genomes[J].Annual Review of Plant Biology,2014,65(1):505-530.
[35] Kumar A.The evolution of plant retroviruses:moving to green pastures[J].Plant Science,1998,3(10):371-374.
[36] Diao X M,Freeling M,Lish D.Horizontal transfer of a plant transposon[J].PLoS Biology,2006,4(1):119-128.
[37] Tang Y M,Ma Y Z,Li L C,et al.Identification and characterization of reverse transcriptase domain of transcriptionally active retrotransposons in wheat genome[J].Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica),2005,47(5):604-612.