空间飞行诱发水稻基因组不稳定序列特征分析
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摘要
空间辐射包括电子、质子、重离子等电离辐射。电离辐射能够引起基因组不稳定,即细胞受到辐射后,在被辐射的细胞的多世代中仍出现基因组突变率增加,引起后代延迟突变。基因组不稳定表现为基因突变、微卫星不稳定等。经神舟6号载人飞船飞行搭载后水稻种子于地面连续种植,在第三代中发现紫色突变。将紫色突变植株种植,后代出现紫色和绿色性状分离现象,表明空间飞行能够诱发水稻紫色突变表型不稳定遗传,这种表型的不稳定性可能是基因组的不稳定导致。为探索空间飞行引起水稻基因组不稳定的序列特征和分子机理,本研究利用AFLP技术,对空间环境引起水稻叶色突变植株进行了基因组突变位点扫描,研究多态性位点的序列特征,并检测多态性位点在后代中的遗传或突变情况。实验结果检测到29个多态位点,对其中20个测序后比对水稻基因组序列,发现14个位点(70%)位于水稻基因组非基因区,5个位点(25%)位于基因区,1个位点(5%)与现有水稻基因组序列不匹配,表明空间飞行引起的水稻基因组不稳定主要发生在非基因编码区。检测其中8个突变位点紫色和绿色同源植株的突变序列特征,得到34个点突变、1个微卫星序列异常和4处缺失突变。相同方法检测M4代紫色和绿色植株,发现点突变增至43个,其中10个位点(23.26%)稳定遗传,19个位点(44.19%)与颜色性状共发生。本次研究发现空间飞行能够诱发基因组不稳定,水稻的紫色突变可能是空间引起基因组不稳定的表现；空间飞行引起水稻基因组不稳定主要发生在非基因编码区,主要突变类型为点突变,其他突变类型包括微卫星序列重复数改变和小片段缺失；本次研究为从分子机理解释空间生物学效应表现提供了有价值的数据。
Space environment included a variety of ionizing radiation such as electron, proton and heavy-ion. Ionizing radiation can cause genome instability, In principle, this radiation-induced genome instability will contribute to increase mutation rates in multi-generation. Genome instability encompasses a wide range of alterations, including gene mutation, microsatellite instability. Rice seed was growth on ground after space flight by Shenzhou-6 spacecraft. After three generations of continuous cultivation, we found the offspring of 4 individuals in the second generation appeared purple leave. The offsprings of purple mutation was observed both purple leave and green leave. We discovered that the purple rice trait was the performance of genome instability. To find genome sequence characteristics and molecular mechanism of space radiation induced genome instability, AFLP molecular marker technology was used to search for polymorphisms of sequence characteristics of purple rice induced by the space environment.29 polymorphism loci were detected by AFLP. By sequencing 20 polymorphism loci, we found those 14 polymorphism loci on non-coding sequence and 5 polymorphism loci on coding sequence. This result showed that space-flight induced rice genome instability mainly occurs in the non-coding region. To investigate the genome sequence characteristics of the mutation caused by space-flight,8 polymorphism loci were used to study the purple mutation.34 point mutations,1 microsatellite sequence anomalies and 4 deletion mutants were found in purple mutation. To study the genetic of the mutation, we found 43 polymorphism loci on the next generation, including 10 polymorphism loci exhibited genetic stability and 19 polymorphism locic concurrences with purple mutation. These results show that space flight could induce rice genome instability. The point mutation was mainly occurred on non-coding sequence. The purple mutation was the performance of rice genome instability. The purple mutation also exhibited genetic instability. Our findings may also have general implications in the molecular mechanism of space biology.
Space environment included a variety of ionizing radiation such as electron, proton and heavy-ion. Ionizing radiation can cause genome instability, In principle, this radiation-induced genome instability will contribute to increase mutation rates in multi-generation. Genome instability encompasses a wide range of alterations, including gene mutation, microsatellite instability. Rice seed was growth on ground after space flight by Shenzhou-6 spacecraft. After three generations of continuous cultivation, we found the offspring of 4 individuals in the second generation appeared purple leave. The offsprings of purple mutation was observed both purple leave and green leave. We discovered that the purple rice trait was the performance of genome instability. To find genome sequence characteristics and molecular mechanism of space radiation induced genome instability, AFLP molecular marker technology was used to search for polymorphisms of sequence characteristics of purple rice induced by the space environment.29 polymorphism loci were detected by AFLP. By sequencing 20 polymorphism loci, we found those 14 polymorphism loci on non-coding sequence and 5 polymorphism loci on coding sequence. This result showed that space-flight induced rice genome instability mainly occurs in the non-coding region. To investigate the genome sequence characteristics of the mutation caused by space-flight,8 polymorphism loci were used to study the purple mutation.34 point mutations,1 microsatellite sequence anomalies and 4 deletion mutants were found in purple mutation. To study the genetic of the mutation, we found 43 polymorphism loci on the next generation, including 10 polymorphism loci exhibited genetic stability and 19 polymorphism locic concurrences with purple mutation. These results show that space flight could induce rice genome instability. The point mutation was mainly occurred on non-coding sequence. The purple mutation was the performance of rice genome instability. The purple mutation also exhibited genetic instability. Our findings may also have general implications in the molecular mechanism of space biology.
引文
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[22]King G J. Morphological development in Brassica oleracea is modulated by in vivo treatment with 52azacytidine. J Hort Sci.,1995,70(2):333-342.
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Acad. Sci. USA,1998,95:6251-6255.
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[38]Dubrov Y E, Nesterov V N, Krouchinsky N G et al. Human minisatellite mutation rate after the Chernobyl accident. Nature,1996,380:683-686.
[39]Dubrova Y E, Bersimbaev R I, Djansugurova L B et al. Nuclear weapons tests and human germline mutation rate. Science,2002,295:1037.
[40]Noda A, Kodama Y, Cullings H M et al. Radiation-Induced Genomic Instability in Tandem Repeat Sequences is not Predictive of Unique Sequence Instability. Radiat. Res,2007, 167:526-534.
[41]Bouff ler S D, Bridges B A, Cooper D N et al. Assessing Radiation-Associated Mutational Risk to the Germline:Repetitive DNA Sequences as Mutational Targets and Biomarkers. Radiat. Res,2006,165:249-268.
[42]郑先云.AFLP分子标记技术的发展.生命的化学,2003,23(1)：65-68.
[43]Vos P, Hogers R. Bleeker M et al. AFLP:a new technique for DNA fingerprinting. Nude/c Acids Res,1995,23(21):4407-4414.
[44]BECKER J, VOS P, KUIPER M et al. Combined mapping of AFLP and RFLP markers in barley. Md. Gen. Genet,1995,249:65-73.
[45]张留所.AFLP技术在水生动物遗传学研究中的应用及前景展望.高技术通讯,2003,4：95-100.
[46]Montano P, Author K, Reprint A M et al. AFLP (amplified fragment length polymorphism) and its application in aquaculture. INTERCIENCIA,2006,31(8):563-569.
[47]Corsini G, Manubens A, Lladser M et al. AFLP Analysis of the Fruit Fly Ceratitis capitata. Agricultural bio. technDIogy,1999,21(3):72-73.
[48]张金卫等.应用AFLP分子标记对6个家蚕品种的鉴定.蚕业科学,2004,30(2)：137-142.
[49]Hiiseyin G, Ztibeyir D. Determination of Genctic Variation in Populations of Bemisia tabaci in Antalya. Turk. J. Agric For,2002,26:211-216.
[50]朱伟铨,王义权.AFLP分子标记技术及其在动物学研究中的应用.动物学杂志,2003,38(2)：101-108.
[51]杜金友等.AFLP标记在玉米种质资源鉴定中的应用.西北植物学报.2006,26(5)：927-932.
[52]田清震,贾继增.AFLP分子标记在小麦种质资源研究中的应用.麦类作物学报.2002.22(1)：95-99.
[53]黄建安,黄意欢,刘仲华.AFLP技术及其在茶树种质资源研究中的应用(综述).亚热带植物科学.2003,32(2)：60-63.
[54]Wang L, Xing S Y, Yang K Q et al. Genetic relationships of ornamental cultivars of Ginkgo biloba analyzed by AFLP techniques. Acta Genetica Sinica,2006,33(11):1020-1026.
[55]蔡健,王永杰,扬剑波.AFLP分子标记在作物育种中的应用.安徽农业科学.2002,30(2)：167-169.
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[62]Kadhim M A, Marsden S J, Malcolmson A M et al. Long-term genomic instability in human lymphocytes induced by single particle irradiation. Radiat Res,2001,155:122-126.
[63]Olga K, Paula B, Andrey A et al. Genome hypermethylation in Pinus silvestris of Chernobyl—a mechanism for radiation adaptation?. Mutation Research,2003,529:13-20.
[64]Pogribny I, Raiche J, Slovack M et al. Dose-dependence, sex and tissue specificity, and persistence of radiat ion-induced genomic DNA methylation changes. Biochem Biophys Res Commun,2004,320(4):1253-1261.
[65]Yuri E D, Gemma G, Anatoliy A et al. Elevated Minisatellite Mutation Rate in the Post-Chernobyl Families from Ukraine. Genet,2002,71:801-809.
[2]江丕栋主编.空间生物学.青岛：青岛出版社,2000.
[3]李鑫磊.空间环境引起水稻基因组多态性变化的特征分析：(硕士学位论文).大连：大连海事大学,2009.
[4]冯杰.空间飞行引起水稻当代植株基因组突变位点的特征分析：(硕士学位论文).哈尔滨：哈尔滨工业大学,2008.
[5]Romney C A, Paulauskis J D, Nagasawa H et al. Multiple manifestation of X-ray induced genomic instability in Chinese hamster ovary (CHO) cells. Mol Carcinog,2001,32(3):118-127.
[6]Loeb L A, Loeb K R, Anderson J P. Multiple mutations and cancer. Proc. Natl. Acad. Sci. 2003,100:776-781.
[7]Demerec M, Latarjet R. Mutations in bacteria induced by radiations. Cold Spring Harbor Symp. Quant. Biol.,1946,11:38-50.
[8]Morga W, Day J P, Kaplan M et al. Genomic instability induced by ionizing radiation. Radiat. Res,1996,146:247-258.
[9]Wright E G. Radiation-induced genomic instability in haemopoietic cells. Int. J. Radiat. Biol,1998,74:681-687.
[10]Little J B. Radiation carcinogenesis. Carcinogenesis,2001,21:397-404.
[11]Kadhim M A, Marsden S J, Malcolmson A M et al. Long-term genomic instability in human lymphocytes induced by single, particle irradiation. Radiat Res,2001,155:122-126.
[12]Morgan W F, Day J P, Kaplan M I et al. Genomic instability induced by ionizing radiation. Radiat Res,1996,146:247-258.
[13]王仲文.电离辐射诱发的基因组不稳定性与辐射致癌.国外医学·放射医学核医学分册,2000,24(5)：226-230.
[14]Kadhim M A, Marsden S J, Malcolmson A M et al. Long-term genomic instability in human lymphocytes induced by single, particle irradiation. Radiat Res,2001,155:122-126.
[15]Bartee L, Malagnac F, Bender J. Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Gen. Dev,2001,15:1753-1757.
[16]Mittelsten-Scheid 0, Afsar K, Paszkowsk J. Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis. Proc. Natl. Acad. Sci,1998,95:632-637.
[17]John B L, Hatsumi N, Tracy P et al. Radiation-Induced Genomic Instability:Delayed Mutagenic and Cytogenetic Effects of X Rays and Alpha Particles. Radiation Research,1997, 148(4):299-307.
[18]Benjamin C. Gene Ⅷ. Pearson Education. New York:2004.
[19]Kakutani T, Jeddeloh J A, Flowera S K et al. Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. Proc Natl Acad Sci.1996, 93:12406-12411.
[20]Finnegan E J, Peacock W J, Dennis E. DNA methylation, a key regulation of plant development and other processes. Curr Opion Genet Develop.2000,10:217-223.
[21]Ronemus M J, Galbiati M, Ticknor C et al. Demethylation2induced developmental plieotropy in Arabidopsis. Science,1996,273(2):654-657.
[22]King G J. Morphological development in Brassica oleracea is modulated by in vivo treatment with 52azacytidine. J Hort Sci.,1995,70(2):333-342.
[23]Sano H, Kamada I, Youssefian S et al. A single treatment of rice seedling with 52azacytidine induces heritable dwarfism and undermethylation of genomic DNA. Mol Gener Genet,1990,220:441-447.
[24]Finnegan E J, Peacock W J, Dennis E S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc Natl Acad Sci USA,1996,93:8449-8454.
[25]Nakano Y, Steward N, Sekine M et al. A tobacco NtMETl cDNA encoding a DNA methyltransferase:molecular characterization and abnormal phenotypes of transgenic tobacco plants. Plant Cell Physiol,2000,41(4):448-457.
[26]Finnegan E J, Peacock W J, Dennis E S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc Natl Acad Sci USA,1996,93:8449-8454.
[27]Olga K, Paula B, Jill B et al. Methylation changes in muscle and liver tissues of male and female mice exposed to acute and chronic low-dose X-ray-irradiation. Mutation Research, 2004,548:75-84.
[28]Olga K, Paula B, Andrey A et al. Genome hypermethylation in Pinus silvestris of Chernobyl-a mechanism for radiation adaptation?. Mutation Research,2003,529:13-20.
[29]Kalinich J F, Catravas G N, Snyder S L. The efect of gamma radiation on DNA methylation. Radiat Res,1989,117(21):185-197.
[30]Dubrova Y E, Jeffreys A J, Malashenko A M. Mouse minisatellite mutations induced by ionizing radiation. Nat. Genet,1993,5:92-94.
[31]Sadamot S, Suzuki S, Kamiya K et al. Radiation induction of germline mutation at hypervariable mouse minisatellite locus. Int. J. Radiat. Biol,1994,65:549-557.
[32]Dubrova Y E, Plum M, Brown J et al. Radiationinduced germline instability at minisatellite loci. Int. J. Radiat. Biol.
[33]Jeffreys A J, Royle N J, Wilso V et al. Spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human DNA. Nature,1988,332:278-281.
[34]Mitani K, Takahashi Y, Kominami R. A GGCAGG motif in minisatellites affecting their germline instability. Biol. Chem,1990,265:15203-15210.
[35]Dubrova Y E, Plumb M, Brown J et al. Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation. Proc. Natl.
Acad. Sci. USA,1998,95:6251-6255.
[36]Barber R, Plumb M A, Boulton E et al. Elevated mutation rates in the germ line of first-and second-generation offspring of irradiated male mice. Proc. Natl. Acad. Sci. USA,2002, 99:6877-6882.
[37]Yuri E D, Gemma G, Anatoliy A et al. Elevated Minisatellite Mutation Rate in the Post-Chernobyl Families from Ukraine. Genet,2002,71:801-809.
[38]Dubrov Y E, Nesterov V N, Krouchinsky N G et al. Human minisatellite mutation rate after the Chernobyl accident. Nature,1996,380:683-686.
[39]Dubrova Y E, Bersimbaev R I, Djansugurova L B et al. Nuclear weapons tests and human germline mutation rate. Science,2002,295:1037.
[40]Noda A, Kodama Y, Cullings H M et al. Radiation-Induced Genomic Instability in Tandem Repeat Sequences is not Predictive of Unique Sequence Instability. Radiat. Res,2007, 167:526-534.
[41]Bouff ler S D, Bridges B A, Cooper D N et al. Assessing Radiation-Associated Mutational Risk to the Germline:Repetitive DNA Sequences as Mutational Targets and Biomarkers. Radiat. Res,2006,165:249-268.
[42]郑先云.AFLP分子标记技术的发展.生命的化学,2003,23(1)：65-68.
[43]Vos P, Hogers R. Bleeker M et al. AFLP:a new technique for DNA fingerprinting. Nude/c Acids Res,1995,23(21):4407-4414.
[44]BECKER J, VOS P, KUIPER M et al. Combined mapping of AFLP and RFLP markers in barley. Md. Gen. Genet,1995,249:65-73.
[45]张留所.AFLP技术在水生动物遗传学研究中的应用及前景展望.高技术通讯,2003,4：95-100.
[46]Montano P, Author K, Reprint A M et al. AFLP (amplified fragment length polymorphism) and its application in aquaculture. INTERCIENCIA,2006,31(8):563-569.
[47]Corsini G, Manubens A, Lladser M et al. AFLP Analysis of the Fruit Fly Ceratitis capitata. Agricultural bio. technDIogy,1999,21(3):72-73.
[48]张金卫等.应用AFLP分子标记对6个家蚕品种的鉴定.蚕业科学,2004,30(2)：137-142.
[49]Hiiseyin G, Ztibeyir D. Determination of Genctic Variation in Populations of Bemisia tabaci in Antalya. Turk. J. Agric For,2002,26:211-216.
[50]朱伟铨,王义权.AFLP分子标记技术及其在动物学研究中的应用.动物学杂志,2003,38(2)：101-108.
[51]杜金友等.AFLP标记在玉米种质资源鉴定中的应用.西北植物学报.2006,26(5)：927-932.
[52]田清震,贾继增.AFLP分子标记在小麦种质资源研究中的应用.麦类作物学报.2002.22(1)：95-99.
[53]黄建安,黄意欢,刘仲华.AFLP技术及其在茶树种质资源研究中的应用(综述).亚热带植物科学.2003,32(2)：60-63.
[54]Wang L, Xing S Y, Yang K Q et al. Genetic relationships of ornamental cultivars of Ginkgo biloba analyzed by AFLP techniques. Acta Genetica Sinica,2006,33(11):1020-1026.
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