郑58/opaque2近等基因系中opaque2基因突变位点分析与高效分子标记开发
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摘要
opaque2突变体材料是最常用的高赖氨酸玉米供体。对已获得的郑58/o2近等基因系研究表明,胚乳发育不同时期22-kDα-醇溶蛋白的积累均明显低于郑58。荧光定量PCR结果表明,α-醇溶蛋白家族基因Z1A、Z1B、Z1C和Z1D的表达均显著低于郑58。郑58/o2胚乳发育不同时期Opaque2基因均正常表达。测序分析发现,郑58/o2中o2基因ATG后713 bp处缺失10个碱基,ORF预测Opaque2蛋白翻译提前终止。针对突变缺失位点开发基因内分子标记o2-indel-1,利用该标记进行回交转育,结果表明,o2-indel-1与o2突变表型完全连锁,错选率为0。opaque2基因突变位点的解析有助于高效分子标记的开发,有效降低错选率,提高优质蛋白鲜食玉米多基因聚合育种的选择效率。
Maize Opaque2(o2) mutant gene enhances lysine content of endosperm as compared with the wild genotype and o2 mutant is the main donor for high-quality protein fresh maize breeding. When compared with Zheng58, Zheng58/o2 had reduction of the accumulation of 22-k Da α-zeins tested by SDS-PAGE. However, q RT-PCR showed that the expression level of the o2 gene in Zheng 58/o2 was normal during the whole endosperm developing period. The expression levels of α-zein genes(Z1 A, Z1 B, Z1 C and Z1 D) in the o2 mutant were significantly reduced. Sequence analysis of o2 mutant gene in zheng58/o2 showed that a 10-nucleotide deletion mutant occurred at 713 bp downstream of ATG, which resulted in frame shift and early termination of Opaque2 protein translation. Further, a functional molecular marker o2-indel-1 was developed based on the deletion mutation. The marker was completely associated with o2 mutant phenotype when used in marker-assisted backcross breeding. Analysis of o2 mutation site would contribute to the development of functional molecular marker and efficiency increase of high-quality protein fresh maize breeding through marker assisted selection.
Maize Opaque2(o2) mutant gene enhances lysine content of endosperm as compared with the wild genotype and o2 mutant is the main donor for high-quality protein fresh maize breeding. When compared with Zheng58, Zheng58/o2 had reduction of the accumulation of 22-k Da α-zeins tested by SDS-PAGE. However, q RT-PCR showed that the expression level of the o2 gene in Zheng 58/o2 was normal during the whole endosperm developing period. The expression levels of α-zein genes(Z1 A, Z1 B, Z1 C and Z1 D) in the o2 mutant were significantly reduced. Sequence analysis of o2 mutant gene in zheng58/o2 showed that a 10-nucleotide deletion mutant occurred at 713 bp downstream of ATG, which resulted in frame shift and early termination of Opaque2 protein translation. Further, a functional molecular marker o2-indel-1 was developed based on the deletion mutation. The marker was completely associated with o2 mutant phenotype when used in marker-assisted backcross breeding. Analysis of o2 mutation site would contribute to the development of functional molecular marker and efficiency increase of high-quality protein fresh maize breeding through marker assisted selection.
引文
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[18]姜伟,李新海,李明顺,等.opaque-2基因微卫星标记与玉米子粒赖氨酸含量的关系[J].作物学报,2004,30(8):739-744.Jiang W,Li X H,Li M S,et al.Microsatellite marker of opaque2gene related to the lysine content of maize[J].Acta Agronomica Sinica,2004,30(8):739-744.(in Chinese)
[19]宋敏,田清震,李新海,等.分子标记在优质蛋白玉米育种中的应用[J].新疆农业大学学报,2005,28(3):5-8.Song M,Tian Q Z,Li X H,et al.Molecular markers assisted selection in quality protein maize breeding[J].Xinjiang Agricultural Science,2005,28(3):5-8.(in Chinese)
[20]宋敏,张世煌.分子标记在优质蛋白玉米(QPM)前景选择和背景选择中的应用[J].新疆农业科学,2007,44(增刊):61-66.Song M,Zhang S H.Molecular markers assisted selection in quality protein maize breeding[J].Xinjiang Agricultural Science,2007,44(S):61-66.(in Chinese)
[21]韩小花,铁双贵,卢彩霞,等.o2与淀粉合成突变基因互作对子粒赖氨酸含量的影响[J].玉米科学,2016,24(1):14-18.Han X H,Tie S G,Lu C X,et al.Interaction of the o2 gene with starch-forming mutant genes on the lysine content in maize kernel[J].Journal of Maize Sciences,2016,24(1):14-18.(in Chinese)
[22]田清震,李新海,李明顺,等.优质蛋白玉米的分子标记辅助选择[J].玉米科学,2004,12(2):108-110.Tian Q Z,Li X H,Li M S,et al.Molecular markers assisted selection to quality protein maize[J].Journal of Maize Sciences,2004,12(2):108-110.(in Chinese)
[2]Mertz E T,Bates L S,Nelson O E.Mutant gene that changes protein composition and increases lysine content of maize endosperm[J].Science,1964,145:295-280.
[3]Schmidt R J,Ketudat M,Aukerman M J,Hoschek,G.Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-k D zein genes[J].Plant Cell,1992,4:689-700.
[4]Neto G C,Yunes J A,da Silva M J,et al.The involvement of Opaque2on beta-prolamin gene regulation in maize and Coix suggests a more general role for this transcriptional activator[J].Plant Molecular Biology,1995,27:1015-1029.
[5]Zhang Z Y,Yang J,Wu Y R.Transcriptional regulation of zein gene expression in maize through the additive and synergistic action of opaque2,prolamine-box binding factor,and O2 heterodimerizing proteins[J].The Plant Cell,2015,27:1162-1172.
[6]Qiao Z Y,Qi W W,Wang Q,et al.Zm MADS47 regulate zein gene transcription through interaction with opaque2[J].PLo S Genetics,2016,12(4):e1005991.
[7]Feng L H,Zhu J,Wang G,et al.Expressional profiling study revealed unique expressional patterns and dramatic expressional divergence of maizeα-zein super gene family[J].Plant Molecular Biology,2009,69:649-659.
[8]Coleman C E,Larkins B A.Prolamines of maize.In Seed Proteins.Edited by:Casey R,Shewry P R.Kluwer Academic Publishers[J].Dordrecht,The Netherlands;1998.
[9]王伟,杨文鹏,王明春.优质蛋白糯玉米自交系的选育及品质分析[J].种子,2017,36(4):105-108.Wang W,Yang W P,Wang M C.Quality analysis and breeding of maize inbred lines with high quality protein and waxy characteristics[J].Seed,2017,36(4):105-108.(in Chinese)
[10]梁国虎,江小林,邱红波,等.分子标记辅助聚合育种选育优质蛋白糯玉米创新性品系的初步研究[J].种子,2011,30(5):103-105.Liang G H,Jiang X L,Qiu H B,et al.Preliminary study on breeding innovative maize lines with quality protein and waxy characteristics by molecular marker-assisted methold[J].Seed,2011,30(5):103-105.(in Chinese)
[11]张述宽,滕辉升,苏琪,等.应用SSR辅助选择技术选育优质蛋白糯玉米自交系[J].广西农业科学,2009(10):1279-1283.Zhang S K,Teng H S,Su Q,Yang Y J.Selection of waxy-QPMmaize lines with SSR markers[J].Guangxi Agricutural Sciences,2009(10):1279-1283.(in Chinese)
[12]Chen Y,Zhou Z Q,Zhao G,et al.Transposable element rbg induces the differential expression of opaque-2 mutant gene in two maize o2 NIL derived from the same inbred line[J].Plos One,2014,9(1):e85159.
[13]Wu Y R,Wang W Q,Messing J.Balancing of sulfur storage in maize seed[J].BMC Plant Biology,2012,12:1471-1480.
[14]赵刚,吴子恺,陈亮,等.几个供体对优质蛋白玉米(QPM)近等基因系构建效果的比较[J].玉米科学,2010,18(6):11-14.Zhao G,Wu Z K,Chen L,et al.Comparison of several donor inbred line on development of quality protein maize(QPM)near-isogenic lines[J].Journal of Maize Sciences,2010,18(6):11-14.(in Chinese)
[15]Chin E C L,Shu H,Smith J S C,et al.Maize simple repetitive DNAsequence:abundance and allele variation[J].Genome,1996,39(5):866-873.
[16]Zhao G,Li M S,Zhang D G,et al.Kernel lysine content dose not increase in some maize opaque2 mutant[J].Planta,2012,235(1):205-215.
[17]谭华,郑德波,邹成林,等.利用分子标记辅助选择优质蛋白玉米近等基因系[J].中国农学通报,2015,32(15):146-150.Tan H,Zheng D B,Zou C L,et al.Near-isogenic lines(NILs)selection of quality protein maize(QPM)by marker assisted selection[J].Chinese Agricultural Science Bulletin,2015,32(15):146-150.(in Chinese)
[18]姜伟,李新海,李明顺,等.opaque-2基因微卫星标记与玉米子粒赖氨酸含量的关系[J].作物学报,2004,30(8):739-744.Jiang W,Li X H,Li M S,et al.Microsatellite marker of opaque2gene related to the lysine content of maize[J].Acta Agronomica Sinica,2004,30(8):739-744.(in Chinese)
[19]宋敏,田清震,李新海,等.分子标记在优质蛋白玉米育种中的应用[J].新疆农业大学学报,2005,28(3):5-8.Song M,Tian Q Z,Li X H,et al.Molecular markers assisted selection in quality protein maize breeding[J].Xinjiang Agricultural Science,2005,28(3):5-8.(in Chinese)
[20]宋敏,张世煌.分子标记在优质蛋白玉米(QPM)前景选择和背景选择中的应用[J].新疆农业科学,2007,44(增刊):61-66.Song M,Zhang S H.Molecular markers assisted selection in quality protein maize breeding[J].Xinjiang Agricultural Science,2007,44(S):61-66.(in Chinese)
[21]韩小花,铁双贵,卢彩霞,等.o2与淀粉合成突变基因互作对子粒赖氨酸含量的影响[J].玉米科学,2016,24(1):14-18.Han X H,Tie S G,Lu C X,et al.Interaction of the o2 gene with starch-forming mutant genes on the lysine content in maize kernel[J].Journal of Maize Sciences,2016,24(1):14-18.(in Chinese)
[22]田清震,李新海,李明顺,等.优质蛋白玉米的分子标记辅助选择[J].玉米科学,2004,12(2):108-110.Tian Q Z,Li X H,Li M S,et al.Molecular markers assisted selection to quality protein maize[J].Journal of Maize Sciences,2004,12(2):108-110.(in Chinese)