菊芋叶绿体分离方法比较研究
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摘要
以菊芋叶片为材料,通过比较提取其叶绿体DNA的5种方法(改良高盐-低pH法、Percoll密度梯度离心法、GENMED试剂盒物理法、GENMED试剂盒化学法及DNaseⅠ差速离心分离法),筛选出适合菊芋叶绿体NDA的提取方法。利用显微镜、核酸蛋白测定仪、琼脂糖凝胶电泳及菊芋基因特异引物RAPD检测。结果显示:改良高盐-低pH法分离得到的叶绿体在40×物镜下其数目多,浓度高,背景清晰,提取菊芋叶片cpDNA的OD_(260)/OD_(280)为1.926,质量浓度为295.63μg/μL。经10 g/L琼脂糖凝胶电泳检测发现,cpDNA条带清晰整齐,无核DNA污染;且提取的cpDNA通过4对引物均能扩增到片段。结果表明,改良高盐-低pH法能够快速获得菊芋高质量的叶绿体DNA。
In order to screen out the chloroplast DNA(cpDNA) extraction method suitable for Jerusalem artichoke chloroplast DNA.In this experiment,Jerusalem artichoke leaves were used as materials to compare the modified high salt-low pH method,Percoll density gradient centrifugation method,GENMED kit physics method,GENMED kit chemical method and DNaseⅠ differential centrifugation method. The DNA quality of Jerusalem artichoke chloroplast was determined by microscope,nucleic acid protein analyzer,agarose gel electrophoresis and Jerusalem artichoke gene-specific primer RAPD. The results showed that the chloroplasts isolated by the modified high-salt-low pH method had a large number,high concentration and clear background under the 40× objective lens. The OD_(260)/OD_(280) of cpDNA extracted from Jerusalem artichoke leaves was 1.926,and the mass concentration was 295.63 μg/μL. After 10 g/L agarose gel electrophoresis,the cpDNA band was clear and neat,and there was non udear DNA contamination.The extracted cpDNA could amplify the fragment by 4 pairs of primers.The results showed that the improved high-salt-low pH method could quickly obtain the high quality chloroplast DNA of Jerusalem artichoke,which laid a foundation for the study of the chloroplast genome of Jerusalem artichoke and even the compositae.
In order to screen out the chloroplast DNA(cpDNA) extraction method suitable for Jerusalem artichoke chloroplast DNA.In this experiment,Jerusalem artichoke leaves were used as materials to compare the modified high salt-low pH method,Percoll density gradient centrifugation method,GENMED kit physics method,GENMED kit chemical method and DNaseⅠ differential centrifugation method. The DNA quality of Jerusalem artichoke chloroplast was determined by microscope,nucleic acid protein analyzer,agarose gel electrophoresis and Jerusalem artichoke gene-specific primer RAPD. The results showed that the chloroplasts isolated by the modified high-salt-low pH method had a large number,high concentration and clear background under the 40× objective lens. The OD_(260)/OD_(280) of cpDNA extracted from Jerusalem artichoke leaves was 1.926,and the mass concentration was 295.63 μg/μL. After 10 g/L agarose gel electrophoresis,the cpDNA band was clear and neat,and there was non udear DNA contamination.The extracted cpDNA could amplify the fragment by 4 pairs of primers.The results showed that the improved high-salt-low pH method could quickly obtain the high quality chloroplast DNA of Jerusalem artichoke,which laid a foundation for the study of the chloroplast genome of Jerusalem artichoke and even the compositae.
引文
[1] 张泽生,刘亚萍,李雨蒙,等.菊粉的研究与开发[J].中国食品添加剂,2017(10):183-188. ZHANG Z SH,LIU Y P,LI Y M,et al.Research and development of inulin[J].Chinese Food Additives,2017(10):183-188.
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[3] 刘祖昕,谢光辉.菊芋作为能源植物的研究进展[J].中国农业大学学报,2012,17(6):122-132. LIU Z X,XIE G H.An overview of researches on Jerusalem artichoke as a biofuel cropb[J].Journal of China Agricultural University,2012,17(6):122-132.
[4] 詹文悦,李辉,康健,等.盐胁迫对菊芋糖组分含量和分配的影响[J].草业学报,2017,26(5):127-134. ZHAN W Y,LI H,KANG J,et al.Effect of salt stress on the accumulation on and distribution of sugars in Jerusalem artichoke(Helianthus tuberosus) plant [J].Acta Prataculturae Sinica,2017,26(5):127-134.
[5] 吕世奇,寇一翾,曾军,等.菊芋新品种兰芋1号的选育[J].中国蔬菜,2018(1):76-79. Lü SH Q,KOU Y X,ZENG J,et al.A new Jerusalem artichoke variety -‘Lanyu No.1’ [J].Chinese Vegetables,2018(1):76-79.
[6] 薛志忠,杨雅华,李海山,等.58份菊芋种质资源遗传多样性SRAP分析[J].北方园艺,2017(21):31-36. XUE ZH ZH,YANG Y H,LI H SH,et al.Genetic diversity of fifty-eight Jerusalem artichoke germplasm resources revealed by sequence related amplified polymorphism( SRAP)[J].Northern Horticulture,2017(21):31-36.
[7] 钟启文.基于转录组测序菊芋果聚糖代谢生物合成相关基因的挖掘[J].园艺学报,2017,44(S1):2539. ZHONG Q W.Mining of genes related to metabolic biosynthesis of Jerusalem artichoke fructan based on transcriptome sequencing[J].Acta Horticulturae Sinica,2017,44(S1):2539.
[8] 罗安凯.洋姜茎叶等对重金属离子吸附的应用基础研究[D].四川绵阳:西南科技大学,2012. LUO A K.The applied basic research on the leaf and stem of Jerusalem artichokes for treating heavy metal wastewater[D].Mianyang Sichuan:Southwest University of Science and Technology,2012.
[9] 孙鹏程.菊芋叶片中高纯度绿原酸的规模化制备工艺研究[D].沈阳:辽宁大学,2014. SUN P CH.Study on large-scale preparation of high purity chlorogenic acid from Helianthus tuberrosus L.leaves.[D].Shenyang:Liaoning University,2014.
[10] 郑晓涛.菊芋叶总黄酮含量变化及其提取、纯化、抗氧化性研究[D].南京:南京农业大学,2012. ZHENG X T.Study on extrction and purification technology of fiavonids contene varition in Jerusalem artichoke (Helianthus tuberrosus L)leaves and antioxidative activeitiy[D].Nanjing:Nanjing Agricultural University,2012.
[11] 吕萌荔,王梦醒,贾晓鸥,等.紫茎泽兰叶绿体基因RNA编辑位点的鉴定及分析[J].西北农业学报,2015,24(2):127-133. Lü M L,WANG M X,JIA X O,et al.Identification and analysis of RNA editing sites in the chloroplast transcripts of Ageratina adenophora [J].Acta Agriculturae Boreali-occidentalis Sinica,2015,24(2):127-133.
[12] 李保珠,赵孝亮,彭雷.植物叶绿体发育及调控研究进展[J].植物学报,2014,49(3):337-345. LI B ZH,ZHAO X L,PENG L.Research advances in the development and regulation of plant chloroplasts [J].Chinese Bulletin Botany,2014,49(3):337-345.
[13] MILLIGAN B G.Purification of chloroplast DNA using hexadecyltrimethylammonium bromide[J].Plant Molecular Biology Reporter,1989,7(2):144-149.
[14] 温贝贝,苏会,李美凤,等.叶绿体基因工程发展现状[J].中国园艺文摘,2016,32(6):16-18,36. WEN B B,SU H,LI M F,et al.Development of chloroplast gene engineering[J].Chinese Horticulture Abstracts,2016,32(6):16-18,36.
[15] 李孟竹.植物叶绿体提取方法比较和两种药用植物叶绿体基因组分析[D].杭州:浙江大学,2016. LI M ZH.Comparative studies on the extraction methods of cpDNA and analysis of two medical plants [D].Hangzhou:Zhejiang University,2016.
[16] CROUSE E J,SCHMITT J M,BOHNERT H J.Chloroplast and cyanobacterial genomes,genes,and RNAs:a compilation[J].Plant Molecular Biology Reporter,1985,3(1/2):43-89.
[17] WOLTERS A M A,SCHOENMAKERS H C H,KOORNNEEF M.Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum[J].TAG Theoretical and Applied Genetics,1995,90(2):285-293.
[18] 姚丹,闫伟,关淑艳,等.高盐低pH值法提取大豆不同组织DNA的效果[J].河南农业科学,2009(12):50-54. YAO D,YAN W,GUAN SH Y,et al.Extraction effect of genomic DNA from different tissues of soybean with high-salt low-pH methods[J].Journal of Henan Agricultural Sciences,2009(12):50-54.
[19] 龙兴,曾继吾,黄秉智,等.香蕉叶绿体分离及叶绿体DNA提取方法[J].江西农业大学学报,2008(3):534-537,542. LONG X,ZENG J W,HUANG B ZH,et al.The method for isolation of banana chloroplast and cpDNA[J].Journal of Jiangxi Agricultural University,2008(3):534-537,542.
[20] 谢海坤,焦健,樊秀彩,等.中国野生葡萄叶绿体分离及叶绿体DNA提取的研究[J].西北植物学报,2016,36(7):1464-1469. XIE H K,JIAO J,FAN X C,et al.An optimized chloroplaste isolation and chloroplast DNA extraction protocol for Chinese wild grapes [J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1464-1469.
[21] 刘少林,靖深蓉,郭立平,等.用改进的高盐低pH法分离和纯化棉花叶绿体及叶绿体DNA[J].棉花学报,1997(5):16-18,24. LIU SH L,JING SH R,GUO L P,et al.A preliminary study on genetic diversity of upland cotton cultivars in China [J].Acta Gossypil Sinica,1997(5):16-18,24.
[22] 陆丹,牛楠,李玥莹.高粱基因组DNA提取方法的比较与优化[J].生物技术,2010,20(3):49-52. LU D,NIU N,LI Y Y.Sorghum genomic DNA extraction methods and optimization of process [J].Biotechnology,2010,20(3):49-52.
[23] 梁凤萍,文祥宁,高赫一,等.菊科植物叶绿体基因组特征分析[J].基因组学与应用生物学,2018,37(12):5437-5447. LIANG F P,WEN X N,GAO H Y,et al.Analysis of chloroplast genomes features of Asteraceae species[J].Genomics and Applied Biology,2018,37(12):5437-5447.
[24] 黎金燕,王春晖,陈天带等.高盐低pH法提取尾叶桉叶绿体DNA方法建立及质量分析[J].分子植物育种,2017,15(3):890-894. LI J Y,WANG CH H,CHEN T D,et al.Establishment and quality analysis of chloroplast DNA extraction from Eucalyptus urophylla with high salt and low pH method[J].Molecular Plant Breeding,2017,15(3):890-894.
[25] MICHAUD H,LUMARET R,RIPOLL J P,et al.A procedure for the extraction of chloroplast DNA from broad-leaved tree species[J].Plant Molecular Biology Reporter,1995,13(2):131-137.
[26] 姜少俊,周海辰,金樑,等.紫花苜蓿线粒体和叶绿体DNA高效提取体系的建立[J].云南农业大学学报(自然科学),2015,30(5):747-754. JIANG SH J,ZHOU H CH,JIN L,et al.The efficient extraction system of alfalfa mitochondrial and chloroplast DNA[J].Journal of Yunnan Agricultural University(Natural Science),2015,30(5):747-754.
[27] 寇一翾.菊芋种质资源多样性及高产量形成机理研究[D].兰州:兰州大学,2013. KOU Y X.Germplasm diversity and high-yield formation of Helianthus tuberosus L[D].Lanzhou:Lanzhou University,2013.
[28] 赵孟良,韩睿,李莉.菊芋不同部位DNA提取的比较研究[J].浙江农业学报,2013,25(5):994-996. ZHAO M L,HAN R,LI L.Comparative study on extraction of genomic DNA from different parts of Helianthus tuberosus L. [J].Journal of Zhejiang Agricultural Sciences,2013,25(5):994-996.
[29] 杨晓婷,黄建,张春梅,等.枣叶绿体基因组DNA提取方法研究[J].西北林学院学报,2015,30(2):105-110. YANG X T,HUANG J,ZHANG CH M,et al.An optimized chloroplast DNA extraction protocol for Chinese jujube[J].Journal of Northwest Forestry University,2015,30(2):105-110.
[30] 田春艳,李富生,何丽莲,等.割手密叶绿体DNA的高效提取方法[J].分子植物育种,2016,14(1):86-91. TIAN CH Y,LI F SH,HE L L,et al.An efficient method for isolation of chloroplast DNA in Saccharum spontaneum[J].Molecular Plant Breeding,2016,14(1):86-91.
[31] DALLY A M,GéRAPD SECOND.Chloroplast DNA isolation from higher plants:an improved non-aqueous method[J].Plant Molecular Biology Reporter,1989,7:135.
[32] 赵孟良,韩睿,马胜超,等.菊芋基因组DNA提取方法的比较[J].中国蔬菜,2012(14):41-44. ZHAO M L,HAN R,MA SH CH,et al.Comparison of genomic DNA extraction from Helianthus tuberosus L[J].Chinese Vegetables,2012(14):41-4.
[2] 高健,彭斌,徐虹.菊芋中菊糖的提取分离研究[J].安徽农业科学,2009,37(1):184-185,214. GAO J,PENG B,XU H.Study on extraction and separation of inulin from Jerusalem artichoke [J].Journal of Anhui Agricultural Sciences,2009,37(1):184-185,214.
[3] 刘祖昕,谢光辉.菊芋作为能源植物的研究进展[J].中国农业大学学报,2012,17(6):122-132. LIU Z X,XIE G H.An overview of researches on Jerusalem artichoke as a biofuel cropb[J].Journal of China Agricultural University,2012,17(6):122-132.
[4] 詹文悦,李辉,康健,等.盐胁迫对菊芋糖组分含量和分配的影响[J].草业学报,2017,26(5):127-134. ZHAN W Y,LI H,KANG J,et al.Effect of salt stress on the accumulation on and distribution of sugars in Jerusalem artichoke(Helianthus tuberosus) plant [J].Acta Prataculturae Sinica,2017,26(5):127-134.
[5] 吕世奇,寇一翾,曾军,等.菊芋新品种兰芋1号的选育[J].中国蔬菜,2018(1):76-79. Lü SH Q,KOU Y X,ZENG J,et al.A new Jerusalem artichoke variety -‘Lanyu No.1’ [J].Chinese Vegetables,2018(1):76-79.
[6] 薛志忠,杨雅华,李海山,等.58份菊芋种质资源遗传多样性SRAP分析[J].北方园艺,2017(21):31-36. XUE ZH ZH,YANG Y H,LI H SH,et al.Genetic diversity of fifty-eight Jerusalem artichoke germplasm resources revealed by sequence related amplified polymorphism( SRAP)[J].Northern Horticulture,2017(21):31-36.
[7] 钟启文.基于转录组测序菊芋果聚糖代谢生物合成相关基因的挖掘[J].园艺学报,2017,44(S1):2539. ZHONG Q W.Mining of genes related to metabolic biosynthesis of Jerusalem artichoke fructan based on transcriptome sequencing[J].Acta Horticulturae Sinica,2017,44(S1):2539.
[8] 罗安凯.洋姜茎叶等对重金属离子吸附的应用基础研究[D].四川绵阳:西南科技大学,2012. LUO A K.The applied basic research on the leaf and stem of Jerusalem artichokes for treating heavy metal wastewater[D].Mianyang Sichuan:Southwest University of Science and Technology,2012.
[9] 孙鹏程.菊芋叶片中高纯度绿原酸的规模化制备工艺研究[D].沈阳:辽宁大学,2014. SUN P CH.Study on large-scale preparation of high purity chlorogenic acid from Helianthus tuberrosus L.leaves.[D].Shenyang:Liaoning University,2014.
[10] 郑晓涛.菊芋叶总黄酮含量变化及其提取、纯化、抗氧化性研究[D].南京:南京农业大学,2012. ZHENG X T.Study on extrction and purification technology of fiavonids contene varition in Jerusalem artichoke (Helianthus tuberrosus L)leaves and antioxidative activeitiy[D].Nanjing:Nanjing Agricultural University,2012.
[11] 吕萌荔,王梦醒,贾晓鸥,等.紫茎泽兰叶绿体基因RNA编辑位点的鉴定及分析[J].西北农业学报,2015,24(2):127-133. Lü M L,WANG M X,JIA X O,et al.Identification and analysis of RNA editing sites in the chloroplast transcripts of Ageratina adenophora [J].Acta Agriculturae Boreali-occidentalis Sinica,2015,24(2):127-133.
[12] 李保珠,赵孝亮,彭雷.植物叶绿体发育及调控研究进展[J].植物学报,2014,49(3):337-345. LI B ZH,ZHAO X L,PENG L.Research advances in the development and regulation of plant chloroplasts [J].Chinese Bulletin Botany,2014,49(3):337-345.
[13] MILLIGAN B G.Purification of chloroplast DNA using hexadecyltrimethylammonium bromide[J].Plant Molecular Biology Reporter,1989,7(2):144-149.
[14] 温贝贝,苏会,李美凤,等.叶绿体基因工程发展现状[J].中国园艺文摘,2016,32(6):16-18,36. WEN B B,SU H,LI M F,et al.Development of chloroplast gene engineering[J].Chinese Horticulture Abstracts,2016,32(6):16-18,36.
[15] 李孟竹.植物叶绿体提取方法比较和两种药用植物叶绿体基因组分析[D].杭州:浙江大学,2016. LI M ZH.Comparative studies on the extraction methods of cpDNA and analysis of two medical plants [D].Hangzhou:Zhejiang University,2016.
[16] CROUSE E J,SCHMITT J M,BOHNERT H J.Chloroplast and cyanobacterial genomes,genes,and RNAs:a compilation[J].Plant Molecular Biology Reporter,1985,3(1/2):43-89.
[17] WOLTERS A M A,SCHOENMAKERS H C H,KOORNNEEF M.Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum[J].TAG Theoretical and Applied Genetics,1995,90(2):285-293.
[18] 姚丹,闫伟,关淑艳,等.高盐低pH值法提取大豆不同组织DNA的效果[J].河南农业科学,2009(12):50-54. YAO D,YAN W,GUAN SH Y,et al.Extraction effect of genomic DNA from different tissues of soybean with high-salt low-pH methods[J].Journal of Henan Agricultural Sciences,2009(12):50-54.
[19] 龙兴,曾继吾,黄秉智,等.香蕉叶绿体分离及叶绿体DNA提取方法[J].江西农业大学学报,2008(3):534-537,542. LONG X,ZENG J W,HUANG B ZH,et al.The method for isolation of banana chloroplast and cpDNA[J].Journal of Jiangxi Agricultural University,2008(3):534-537,542.
[20] 谢海坤,焦健,樊秀彩,等.中国野生葡萄叶绿体分离及叶绿体DNA提取的研究[J].西北植物学报,2016,36(7):1464-1469. XIE H K,JIAO J,FAN X C,et al.An optimized chloroplaste isolation and chloroplast DNA extraction protocol for Chinese wild grapes [J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1464-1469.
[21] 刘少林,靖深蓉,郭立平,等.用改进的高盐低pH法分离和纯化棉花叶绿体及叶绿体DNA[J].棉花学报,1997(5):16-18,24. LIU SH L,JING SH R,GUO L P,et al.A preliminary study on genetic diversity of upland cotton cultivars in China [J].Acta Gossypil Sinica,1997(5):16-18,24.
[22] 陆丹,牛楠,李玥莹.高粱基因组DNA提取方法的比较与优化[J].生物技术,2010,20(3):49-52. LU D,NIU N,LI Y Y.Sorghum genomic DNA extraction methods and optimization of process [J].Biotechnology,2010,20(3):49-52.
[23] 梁凤萍,文祥宁,高赫一,等.菊科植物叶绿体基因组特征分析[J].基因组学与应用生物学,2018,37(12):5437-5447. LIANG F P,WEN X N,GAO H Y,et al.Analysis of chloroplast genomes features of Asteraceae species[J].Genomics and Applied Biology,2018,37(12):5437-5447.
[24] 黎金燕,王春晖,陈天带等.高盐低pH法提取尾叶桉叶绿体DNA方法建立及质量分析[J].分子植物育种,2017,15(3):890-894. LI J Y,WANG CH H,CHEN T D,et al.Establishment and quality analysis of chloroplast DNA extraction from Eucalyptus urophylla with high salt and low pH method[J].Molecular Plant Breeding,2017,15(3):890-894.
[25] MICHAUD H,LUMARET R,RIPOLL J P,et al.A procedure for the extraction of chloroplast DNA from broad-leaved tree species[J].Plant Molecular Biology Reporter,1995,13(2):131-137.
[26] 姜少俊,周海辰,金樑,等.紫花苜蓿线粒体和叶绿体DNA高效提取体系的建立[J].云南农业大学学报(自然科学),2015,30(5):747-754. JIANG SH J,ZHOU H CH,JIN L,et al.The efficient extraction system of alfalfa mitochondrial and chloroplast DNA[J].Journal of Yunnan Agricultural University(Natural Science),2015,30(5):747-754.
[27] 寇一翾.菊芋种质资源多样性及高产量形成机理研究[D].兰州:兰州大学,2013. KOU Y X.Germplasm diversity and high-yield formation of Helianthus tuberosus L[D].Lanzhou:Lanzhou University,2013.
[28] 赵孟良,韩睿,李莉.菊芋不同部位DNA提取的比较研究[J].浙江农业学报,2013,25(5):994-996. ZHAO M L,HAN R,LI L.Comparative study on extraction of genomic DNA from different parts of Helianthus tuberosus L. [J].Journal of Zhejiang Agricultural Sciences,2013,25(5):994-996.
[29] 杨晓婷,黄建,张春梅,等.枣叶绿体基因组DNA提取方法研究[J].西北林学院学报,2015,30(2):105-110. YANG X T,HUANG J,ZHANG CH M,et al.An optimized chloroplast DNA extraction protocol for Chinese jujube[J].Journal of Northwest Forestry University,2015,30(2):105-110.
[30] 田春艳,李富生,何丽莲,等.割手密叶绿体DNA的高效提取方法[J].分子植物育种,2016,14(1):86-91. TIAN CH Y,LI F SH,HE L L,et al.An efficient method for isolation of chloroplast DNA in Saccharum spontaneum[J].Molecular Plant Breeding,2016,14(1):86-91.
[31] DALLY A M,GéRAPD SECOND.Chloroplast DNA isolation from higher plants:an improved non-aqueous method[J].Plant Molecular Biology Reporter,1989,7:135.
[32] 赵孟良,韩睿,马胜超,等.菊芋基因组DNA提取方法的比较[J].中国蔬菜,2012(14):41-44. ZHAO M L,HAN R,MA SH CH,et al.Comparison of genomic DNA extraction from Helianthus tuberosus L[J].Chinese Vegetables,2012(14):41-4.