重要水稻资源分子查验技术研究
摘要
水稻是禾本科中很重要的粮食作物,养活着世界上大部分人口。水稻中含有丰富的种质资源,而目前由于人为破坏活动及缺乏相应的检验技术,致使我国水稻资源流失严重,造成巨大的损失。对稻种资源及多样性及时进行有效的保护,是保证我国水稻品质改善和持久生产的基础。为建立快速、高效的重要水稻资源的分子查验技术体系,本研究共收集了普通野生稻,药用、斑点、疣粒、高杆、宽叶、紧穗和长雄蕊8种类型的野生稻以及2种类型的栽培稻材料共计105份,采用基因条码技术及dCAPS分子标记旨在建立对野生稻、栽培稻等种的特异性进行有效的区分的资源查验技术体系。
1.采用基因条码技术,对水稻材料的matK、trnL、rpoB、rpoC、Nadl、ndhJ、atpF-atpH、psbK-psb1、Adh2、Os9971进行扩增并自行测序,对测序结果较好的trnL、rpoB、rpoC、Nad1、ndhJ、atpF-atpH、psbK-psbl7个条码基因,结合genebank已公布的基因序列信息,利用MEGA4软件进行系统进化亲缘关系分析,得出以下结论：(1)trnL基因具有较多的多态性位点,不仅能将禾本科作物中属级作物区分开,而且能定位至基因组型,将所分析的36份水稻材料共分为AA,CC, DD,CCDD四个染色体组型(2)通过对Nadl与psbK-psbl两个基因的分析,表明了宽叶和高杆野生稻与其他水稻材料相比具有较近的亲缘关系。(3)atpF-atpH、rpoB、rpoC、ndhJ等条码基因鉴于非常保守的特点,比较适合做属以上的分类结元进行鉴定。
2.探讨了基于SNP位点建立dCAPS分子标记体系。采用生物信息学软件寻找了药用野生稻和斑点野生稻上trnL、rpoB、rpoC、matK、atpF-atpH、psbK-psb1 ndhJ8个基因碱基序列上适合设计错配引物的SNP位点,设计合成适当的错配引物进行PCR扩增,选择相应的限制性内切酶进行酶切。在这2种野生稻DNA的matK基因上分别找到1个合适的SNP位点,设计了3对错配引物,斑点野生稻的trnL基因上找到了1个SNP位点,设计合成1对错配引物。PCR后4种相应的限切酶都可以特异性地切开PCR产物,将药用野生稻或斑点野生稻与栽培稻和其它野生稻区分开来。本结果证明了dCAPS分子标记可以在口岸查验上应用。
Rice is the important crops in the gramineous ones,it feeds the most of population in the world.There are abundant germplasm resource in rice,but at present,becase it lacked of corresponding inspection technology and destroyed by people, it causes our rice resources huge losses.It is necessary to protect the rice resource and diversity in order to improve the rice quality and sustainable production. For establishing a rapid and efficient rice resources molecules inspection technology system, this paper collected one hundred and five materials, Itcontains O.rufipogon, O.latiflia, O.alta, O.meyeriana.O.punctata O.officinalis, O.longistaminata, O.eichingeri,eight types of wild rices and two types of cultivated rices. It aimed to established the rice resource inspection technology system for distinguishing the species specificity of wild rice and cultivated rice.
1.Using the genetic bar code technology, amplify the rice gene of trnL matK, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psb1, Adh2, Os9971,then sequencing,it conducted the system Phylogenetic evolution relation analysi by using MEGA4 for the better sequence result of trnL, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psbl and combining with the genetic sequence of genebank published information,the following conclusion (1) trnL had the more polymorphism sites,it can not only distinguish the genetic generic crops in the gramineous ones,but also locate the genomic,it divided the analytical rice material into AA,CC, DD, CCDD four chromosome type. (2) Comparing with other rice material, O.latifolia and O.alta has relative near genetic relationship. (3) atpF-atpH, rpoB, rpoC,ndhJ gene is suit to identify the above of generic crops.
2. The dCAPS molecular markers system is established on the basis of the SNP, Nucleotide sequences of eight genes in O. punctata and O.officinalis were analyzed by bio-informatics software to search for SNP loci that are suitable for designing appropriate mismatch primer(s), which allow relevant enzymes to cut on after PCR. In matK gene of two wild rice species DNA, one appropriate SNP loci were found respectively, In trnL gene of O. punctata one appropriate SNP loci were found. on the basis of which four mismatch primer pairs were designed and distinguished O. punctata or O.officinalis from other O.sativa and wild rice after restriction enzyme digestion of PCR products. This result supported that dCAPS molecular markers can be used in port inspection.
1.采用基因条码技术,对水稻材料的matK、trnL、rpoB、rpoC、Nadl、ndhJ、atpF-atpH、psbK-psb1、Adh2、Os9971进行扩增并自行测序,对测序结果较好的trnL、rpoB、rpoC、Nad1、ndhJ、atpF-atpH、psbK-psbl7个条码基因,结合genebank已公布的基因序列信息,利用MEGA4软件进行系统进化亲缘关系分析,得出以下结论：(1)trnL基因具有较多的多态性位点,不仅能将禾本科作物中属级作物区分开,而且能定位至基因组型,将所分析的36份水稻材料共分为AA,CC, DD,CCDD四个染色体组型(2)通过对Nadl与psbK-psbl两个基因的分析,表明了宽叶和高杆野生稻与其他水稻材料相比具有较近的亲缘关系。(3)atpF-atpH、rpoB、rpoC、ndhJ等条码基因鉴于非常保守的特点,比较适合做属以上的分类结元进行鉴定。
2.探讨了基于SNP位点建立dCAPS分子标记体系。采用生物信息学软件寻找了药用野生稻和斑点野生稻上trnL、rpoB、rpoC、matK、atpF-atpH、psbK-psb1 ndhJ8个基因碱基序列上适合设计错配引物的SNP位点,设计合成适当的错配引物进行PCR扩增,选择相应的限制性内切酶进行酶切。在这2种野生稻DNA的matK基因上分别找到1个合适的SNP位点,设计了3对错配引物,斑点野生稻的trnL基因上找到了1个SNP位点,设计合成1对错配引物。PCR后4种相应的限切酶都可以特异性地切开PCR产物,将药用野生稻或斑点野生稻与栽培稻和其它野生稻区分开来。本结果证明了dCAPS分子标记可以在口岸查验上应用。
Rice is the important crops in the gramineous ones,it feeds the most of population in the world.There are abundant germplasm resource in rice,but at present,becase it lacked of corresponding inspection technology and destroyed by people, it causes our rice resources huge losses.It is necessary to protect the rice resource and diversity in order to improve the rice quality and sustainable production. For establishing a rapid and efficient rice resources molecules inspection technology system, this paper collected one hundred and five materials, Itcontains O.rufipogon, O.latiflia, O.alta, O.meyeriana.O.punctata O.officinalis, O.longistaminata, O.eichingeri,eight types of wild rices and two types of cultivated rices. It aimed to established the rice resource inspection technology system for distinguishing the species specificity of wild rice and cultivated rice.
1.Using the genetic bar code technology, amplify the rice gene of trnL matK, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psb1, Adh2, Os9971,then sequencing,it conducted the system Phylogenetic evolution relation analysi by using MEGA4 for the better sequence result of trnL, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psbl and combining with the genetic sequence of genebank published information,the following conclusion (1) trnL had the more polymorphism sites,it can not only distinguish the genetic generic crops in the gramineous ones,but also locate the genomic,it divided the analytical rice material into AA,CC, DD, CCDD four chromosome type. (2) Comparing with other rice material, O.latifolia and O.alta has relative near genetic relationship. (3) atpF-atpH, rpoB, rpoC,ndhJ gene is suit to identify the above of generic crops.
2. The dCAPS molecular markers system is established on the basis of the SNP, Nucleotide sequences of eight genes in O. punctata and O.officinalis were analyzed by bio-informatics software to search for SNP loci that are suitable for designing appropriate mismatch primer(s), which allow relevant enzymes to cut on after PCR. In matK gene of two wild rice species DNA, one appropriate SNP loci were found respectively, In trnL gene of O. punctata one appropriate SNP loci were found. on the basis of which four mismatch primer pairs were designed and distinguished O. punctata or O.officinalis from other O.sativa and wild rice after restriction enzyme digestion of PCR products. This result supported that dCAPS molecular markers can be used in port inspection.
引文
[1]江川,王金英,李清华.野生稻的优异特性及其在水稻育种中的利用[J].福建稻麦科技.2003,21(3)：8-9.
[2]赵颖,黄凤宽,童晓.药用野生稻与栽培稻抗褐飞虱HPLC指纹图谱的比较[J].植物保护,2005,31(5)：36-37.
[3]鄂志国,郑传举,王磊.中国野生稻资源资源及其在水稻抗性育种上的应用[J].2008,(4)：3-6
[4]钟代彬,罗利军,应存山.野生稻有利基因转移研究进展.中国水稻科[J].2000,14(2)：103-106.
[5]傅立国主编.中国植物红皮书(第一册)[M].北京：科学出版社,1992,314-316.
[6]Song Ge, Tao Sang, Bao Rong Lu, et al. Phylogeny of rice genomes with emphasis on origins of allotetraploid species.www.pnas.org,1999,96(25):14400-14405.
[7]卢宝荣,葛颂,桑涛,等.稻属分类的现状及存在的问题[J].植物分类学报,2001,39(4)：373-388.
[8]高立志,张寿洲,周毅,等.中国野生稻的现状调查[J].生物多样性,1996,4(3)：160-166.
[9]范树国,张再君,刘林,等.中国野生稻资源的保护及其在育种中的应用[J].2000,(2)：102-107
[10]刘克德,张启发.云南地方稻种的遗传变异和籼粳分化.植物学报[J].1995,37(9)：718-724.
[11]包颖,葛颂.根据DNA的ITS序列的分析鉴定稻属的CD染色体组物种,植物学报[J].2003,45(7)：762-765.
[12]孙传清,王象坤,才宏伟,等.中国普通野生稻和亚洲栽培稻核基因组的遗传分化[J].中国农业大学学报,1997,2(5)：65-71.
[13]周宇爝.分子标记发展史,现代农业科技[J].2009,11：264-270.
[14]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J].果树学报.2003,20(4)：270-274.
[15]万亚涛,阿新祥,樊传章,等.云南疣粒野生稻34个居群遗传多样性的ISSR分析[J].中国水稻科学,2006,20(6)：596-602.
[16]程毅,朱水芳,陈红运,等.三系杂交水稻真伪及纯度实时PCR技术的鉴定[J].农业生物技术学报,2007,15(2)：295-300.
[17]王印肖,徐秀琴,韩宏伟.分子标记在品种鉴定中的应用[J].河北林业科学,2006,9：46-48.
[18]陈良兵,朱汝财,吴海斌,等.海南普通野生稻和山栏稻的SSR比较[M].2005年植物分子育种国际学术研讨会论文集,2005：70-73.
[19]王艳红,王辉,高立志.普通野生稻的SSR遗传多样性研究[J].西北植物学报,2003,23(10)：1750-1754.
[20]任羽,王德元,张银东.相关序列扩增多态性(SRAP)一种性新的分子标记技术[J].中国农业通报,2004,20(6)：11-13.
[21]柳李旺,龚义勤,黄浩,等.新型分子标记SRA与TRAP及其应用[J].遗传,2004,26(5)：777-781.
[22]张金梅,王建秀,夏涛,等.基于系统发育分析的DNA条形码技术在澄清芍药属牡丹组物种问题中的应用[J].生命科学,2008,38(12)：1166-1168.
[23]H eb ert PDN, Cywin ska A, Bal 1 SL et al. Biological iden tificat ions th rou gh DNA bar codes[J]. Pr oceedings:Biological Scien ces,2003,270 (1512):3132321.
[24]杜启艳,常重杰.DNA条形码在鉴定物种中的应用[J].生物学教学,2010,35(12)：60-61.
[24]傅美兰,彭建军,王莹,等.DNA条形码技术的应用与分析[J].河南师范大学学报(自然科学版),2010,38(4)：118-122.
[25]Hebert PDN, Stoeckl e M Y, Zemlak T S, et al. Ident ificat ion of birds through DNA barcodes[J]. Plos Biology,2004,2(10):312-313.
[26]Ward R D, H ann er R, Hebert PDN.The campaign to DNA barcode all fishes[J]. Journal of Fish Biology,2009,74:329-356.
[27]HajibabaeiM, JanzenD H, Burns J M, et al.DNA barcodes distinguish species of tropical Lepidoptera[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103 (4):968-971.
[28]BallS L, Armstrong.DNA barcodes for inseetpest identification:a test case with tussock moths(Lepidoptera:Lymantriidae)[J].Canadian Journal of Forest Research, 2006,36:337-350.
[29]Smith M A, Poyark ov N A, Hebert PDN.CO1 DNA barcoding amphibians:take the chance meet the challenge[J].Molecular Ecology Resources,2008,8:235-236.
[30]Toshiyuki Komori, Naoto Notta.Utilization of the CAPS/dCAPS Method to Convert Rice SNPs into PCR based Markers[J].Breeding Science,2005, (55):93-98.
[31]隋标峰,张朝贤,崔海兰,等.杂草对AHAS抑制剂的抗药性分子机理研究进展[J].农药学学报,2009,211(4)：399-406.
[32]Paul K. B, Mark R. T. Association of dwarfism and floral induction with a grape green revolution mutation[J]. Nature,2002,4(416):847-850.
[33]Liu S.P., Wang J.R., Wang L, et al.Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family[J].Cell Research,2009,19 (9):1110-1119.
[34]Salah E, Din E.A. A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2[J]Nature Genetics,2001,29 (4):435-440.
[35]Olivier L., Vera S. Christine C, Fanny C., et al.Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2[J].Nature Genetics, 2007,39 (7):896-900.
[36]冯国忠,万树青,潘大建.斑点野生稻拒食活性组分的分离及其对斜纹夜蛾消化酶活性的影响[J].昆虫学报,2006,49(4)：556-561.
[37]王洪水,侯相山.基因芯片技术研究进展[J].安徽农业科学,2007,35(8)：2241-2245.
[38]高志勇.应用基因芯片检测水稻基因表达的研究[J].安徽农业科学,2007,35(10)：3007-3008.
[39]王曼玲,Rocha Pedro,李落叶,等.应用基因表达芯片分析水稻高温胁迫相关基因[J].生物技术通报,2009,(10)：92-97.
[40]Renaud L. Vincent S. Sylvie D.Olivier M and Michelle B. A test of psbK-psbI and atpF-atpH as potential plant DNA barcodes using the flora of the Kruger National Park as a model system[J].2008,1896(1):1-21
[41]Qihui Zhu, Song Ge. Phylogenetic relationships among A genome species of the genus Oryza revealed by intron sequences of four nuclear genes[J].New phytologist, 2005,167,249-263.
[42]郑艳萍.一种基于聚合酶链式反应检测SNP的方法[J].中国农业大学学报,2006,11(3)：51-55.
[43]张玲,李伟,谢崇华.水稻单核苷酸多态性研究进展[J].安徽农业科学,2007,35(34)：1095510957.
[44]刘学军.植物SNP数据库及转化CAPS的方法[J].分子植物育种,2006,4(3)：443-447.
[45]Reynato UMALI, Ikuo NAKAMURA. Identification of dCAPS Marker that Discriminate A and B Cytoplasms in Banana(Musa spp) [J].Plant Biotechnology, 2003,20(2):150-164.
[46]Komori T, N Nitta.High frequency of sequence polymorphism in rice MITEs and application to efficient development of PCR based marker[J].Breed Sci,2003, (53):85-92.
[2]赵颖,黄凤宽,童晓.药用野生稻与栽培稻抗褐飞虱HPLC指纹图谱的比较[J].植物保护,2005,31(5)：36-37.
[3]鄂志国,郑传举,王磊.中国野生稻资源资源及其在水稻抗性育种上的应用[J].2008,(4)：3-6
[4]钟代彬,罗利军,应存山.野生稻有利基因转移研究进展.中国水稻科[J].2000,14(2)：103-106.
[5]傅立国主编.中国植物红皮书(第一册)[M].北京：科学出版社,1992,314-316.
[6]Song Ge, Tao Sang, Bao Rong Lu, et al. Phylogeny of rice genomes with emphasis on origins of allotetraploid species.www.pnas.org,1999,96(25):14400-14405.
[7]卢宝荣,葛颂,桑涛,等.稻属分类的现状及存在的问题[J].植物分类学报,2001,39(4)：373-388.
[8]高立志,张寿洲,周毅,等.中国野生稻的现状调查[J].生物多样性,1996,4(3)：160-166.
[9]范树国,张再君,刘林,等.中国野生稻资源的保护及其在育种中的应用[J].2000,(2)：102-107
[10]刘克德,张启发.云南地方稻种的遗传变异和籼粳分化.植物学报[J].1995,37(9)：718-724.
[11]包颖,葛颂.根据DNA的ITS序列的分析鉴定稻属的CD染色体组物种,植物学报[J].2003,45(7)：762-765.
[12]孙传清,王象坤,才宏伟,等.中国普通野生稻和亚洲栽培稻核基因组的遗传分化[J].中国农业大学学报,1997,2(5)：65-71.
[13]周宇爝.分子标记发展史,现代农业科技[J].2009,11：264-270.
[14]乔玉山,章镇,房经贵,等.李种质资源ISSR反应体系的建立[J].果树学报.2003,20(4)：270-274.
[15]万亚涛,阿新祥,樊传章,等.云南疣粒野生稻34个居群遗传多样性的ISSR分析[J].中国水稻科学,2006,20(6)：596-602.
[16]程毅,朱水芳,陈红运,等.三系杂交水稻真伪及纯度实时PCR技术的鉴定[J].农业生物技术学报,2007,15(2)：295-300.
[17]王印肖,徐秀琴,韩宏伟.分子标记在品种鉴定中的应用[J].河北林业科学,2006,9：46-48.
[18]陈良兵,朱汝财,吴海斌,等.海南普通野生稻和山栏稻的SSR比较[M].2005年植物分子育种国际学术研讨会论文集,2005：70-73.
[19]王艳红,王辉,高立志.普通野生稻的SSR遗传多样性研究[J].西北植物学报,2003,23(10)：1750-1754.
[20]任羽,王德元,张银东.相关序列扩增多态性(SRAP)一种性新的分子标记技术[J].中国农业通报,2004,20(6)：11-13.
[21]柳李旺,龚义勤,黄浩,等.新型分子标记SRA与TRAP及其应用[J].遗传,2004,26(5)：777-781.
[22]张金梅,王建秀,夏涛,等.基于系统发育分析的DNA条形码技术在澄清芍药属牡丹组物种问题中的应用[J].生命科学,2008,38(12)：1166-1168.
[23]H eb ert PDN, Cywin ska A, Bal 1 SL et al. Biological iden tificat ions th rou gh DNA bar codes[J]. Pr oceedings:Biological Scien ces,2003,270 (1512):3132321.
[24]杜启艳,常重杰.DNA条形码在鉴定物种中的应用[J].生物学教学,2010,35(12)：60-61.
[24]傅美兰,彭建军,王莹,等.DNA条形码技术的应用与分析[J].河南师范大学学报(自然科学版),2010,38(4)：118-122.
[25]Hebert PDN, Stoeckl e M Y, Zemlak T S, et al. Ident ificat ion of birds through DNA barcodes[J]. Plos Biology,2004,2(10):312-313.
[26]Ward R D, H ann er R, Hebert PDN.The campaign to DNA barcode all fishes[J]. Journal of Fish Biology,2009,74:329-356.
[27]HajibabaeiM, JanzenD H, Burns J M, et al.DNA barcodes distinguish species of tropical Lepidoptera[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103 (4):968-971.
[28]BallS L, Armstrong.DNA barcodes for inseetpest identification:a test case with tussock moths(Lepidoptera:Lymantriidae)[J].Canadian Journal of Forest Research, 2006,36:337-350.
[29]Smith M A, Poyark ov N A, Hebert PDN.CO1 DNA barcoding amphibians:take the chance meet the challenge[J].Molecular Ecology Resources,2008,8:235-236.
[30]Toshiyuki Komori, Naoto Notta.Utilization of the CAPS/dCAPS Method to Convert Rice SNPs into PCR based Markers[J].Breeding Science,2005, (55):93-98.
[31]隋标峰,张朝贤,崔海兰,等.杂草对AHAS抑制剂的抗药性分子机理研究进展[J].农药学学报,2009,211(4)：399-406.
[32]Paul K. B, Mark R. T. Association of dwarfism and floral induction with a grape green revolution mutation[J]. Nature,2002,4(416):847-850.
[33]Liu S.P., Wang J.R., Wang L, et al.Adventitious root formation in rice requires OsGNOM1 and is mediated by the OsPINs family[J].Cell Research,2009,19 (9):1110-1119.
[34]Salah E, Din E.A. A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2[J]Nature Genetics,2001,29 (4):435-440.
[35]Olivier L., Vera S. Christine C, Fanny C., et al.Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2[J].Nature Genetics, 2007,39 (7):896-900.
[36]冯国忠,万树青,潘大建.斑点野生稻拒食活性组分的分离及其对斜纹夜蛾消化酶活性的影响[J].昆虫学报,2006,49(4)：556-561.
[37]王洪水,侯相山.基因芯片技术研究进展[J].安徽农业科学,2007,35(8)：2241-2245.
[38]高志勇.应用基因芯片检测水稻基因表达的研究[J].安徽农业科学,2007,35(10)：3007-3008.
[39]王曼玲,Rocha Pedro,李落叶,等.应用基因表达芯片分析水稻高温胁迫相关基因[J].生物技术通报,2009,(10)：92-97.
[40]Renaud L. Vincent S. Sylvie D.Olivier M and Michelle B. A test of psbK-psbI and atpF-atpH as potential plant DNA barcodes using the flora of the Kruger National Park as a model system[J].2008,1896(1):1-21
[41]Qihui Zhu, Song Ge. Phylogenetic relationships among A genome species of the genus Oryza revealed by intron sequences of four nuclear genes[J].New phytologist, 2005,167,249-263.
[42]郑艳萍.一种基于聚合酶链式反应检测SNP的方法[J].中国农业大学学报,2006,11(3)：51-55.
[43]张玲,李伟,谢崇华.水稻单核苷酸多态性研究进展[J].安徽农业科学,2007,35(34)：1095510957.
[44]刘学军.植物SNP数据库及转化CAPS的方法[J].分子植物育种,2006,4(3)：443-447.
[45]Reynato UMALI, Ikuo NAKAMURA. Identification of dCAPS Marker that Discriminate A and B Cytoplasms in Banana(Musa spp) [J].Plant Biotechnology, 2003,20(2):150-164.
[46]Komori T, N Nitta.High frequency of sequence polymorphism in rice MITEs and application to efficient development of PCR based marker[J].Breed Sci,2003, (53):85-92.