控制水稻金黄色颖壳与节间基因OsCAD2的图位克隆

doi:10.16819/j.1001-7216.2017.6170 465

摘要/Abstract

摘要：

目的水稻色素不仅对其自身生长发育有重要生理作用，而且在水稻育种、农副产品改良等方面运用比较广泛。对水稻色素相关基因进行表型分析和基因定位，为进一步研究色素代谢途径及调控机理奠定基础。方法利用EMS诱变粳稻长粒粳(CLJ)，在突变体库内筛选到一个金黄色颖壳与节间突变体gh881;在成熟期，测定野生型与gh881的主要农艺性状;将gh881与野生型及中恢8015杂交，观察BC₁F₁及F₁植株表型，并对BC₁F₂及F₂表型分离进行卡方检验，对gh881进行遗传分析;利用F₂群体和图位克隆的方法对gh881突变基因进行定位;采用qPCR检测颖壳颜色相关基因在gh881与野生型不同发育时期的幼穗、节间以及剑叶叶鞘的相对表达量。结果与野生型相比，突变体的颖壳与节间均呈金黄色;除单株有效穗数外，gh881突变体的株高、每穗总粒数及实粒数、结实率和千粒重等性状均极显著降低。遗传分析和基因定位结果表明，gh881的突变表型受1对隐性核基因控制，位于第2染色体短臂，并最终将该基因精细定位于标记FH-13和RH-25之间，物理距离约33.2 kb，该区域中包含4个开放阅读框(ORFs)。结论序列分析结果表明，发现其中一个编码肉桂醇脱氢酶(CAD)的基因OsCAD2(Os02g0187800)的 3 563 bp处发生了1个单碱基突变(G转换为A)，导致该基因编码区的第297位氨基酸由甘氨酸突变为天冬氨酸，由此认为该突变体为OsCAD2基因单碱基突变的新等位基因。qRT-PCR结果表明，突变体的节间中OsCAD2相对表达量极显著下调，而在剑叶叶鞘及穗部则基本都是极显著增加，其他相关基因也发生显著变化，证实OsCAD2是木质素代谢中的重要基因，且可能与其他相关基因存在反馈调节。

关键词: 水稻, 色素, 突变体, 颖壳, 节间, 基因定位

Abstract:

【Objective】Rice pigments not only have important physiological effects on their own development, but also have been widely used in rice breeding, by-product improvement, and so on. Phenotypic analysis and gene mapping of pigment-related genes could lay the foundation for the further study of rice pigments metabolism and regulation mechanism. 【Method】A golden hull and internode rice mutant, gh881, was isolated from an elite japonica cultivar Changlijing (CLJ) using ethyl methane sulphonate(EMS) mutagenesis strategy;the main agricultural traits of gh881 and its wild type(WT) were measured at mature stage; we crossed gh881 with WT and ZH8015, respectively, followed by observation of F₁ and BC₁F₁ plants phenotype, and we made genetic analysis using Chi-square test in BC₁F₂population and gene mapping using map-based cloning in F₂ population; the related-genes expression of young panicles, internodes and flag leaf sheaths of gh881 and WT at various stages were studied by qPCR. 【Result】Compared with wild type, gh881 exhibited golden hull and internode at the mature stage. Except for no significant difference in the number of panicles per plant, there were significant reduction in plant height, the seed-setting rate, the number of spikelets per panicle, the number of grains per panicle and 1000-grain weight between WT and gh881. Genetic analysis and gene fine-mapping results suggested that gh881 was controlled by a single recessive gene and the mutant gene was mapped to a region of 33.2 kb in which four open reading flames (ORFs) existed on the short arm of chromosome 2 between markers FH-13 and RH-25. 【Conclusion】Sequencing analysis revealed that a single base mutation (G to A) occurred at the site 3563 bp of OsCAD2 (Os02g0187800) encoding a Cinnamyl-Alcohol Dehydrogenase (CAD), which led to a G297D mutation (codon GGC to GAC). This implied that gh881 might carry a novel allele of OsCAD2. The quantitative real-time PCR analysis showed that the relative expression level of OsCAD2 decreased significantly in the internode, while it almost increased significantly in the flag leaf sheath and panicle, and the related-genes also almost changed significantly. These results demonstrated that OsCAD2 is an important gene involved in lignin metabolic pathway, and may regulate other related-genes expression feedback.

Key words: rice, pigment, mutant, hull, internode, gene mapping

中图分类号:

Q343.5

王宏, 张迎信, 孙廉平, 孟帅, 徐鹏, 吴玮勋, 程式华, 曹立勇, 沈希宏. 控制水稻金黄色颖壳与节间基因OsCAD2的图位克隆[J]. 中国水稻科学, 2017, 31(5): 465-474.

Hong WANG, Yingxin ZHANG, Lianping SUN, Shuai MENG, Peng XU, Weixun WU, Shihua CHENG, Liyong CAO, Xihong SHEN. Map-Based Cloning of OsCAD2 Regulating Golden Hull and Internode in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(5): 465-474.

图/表 7

参考文献 32

[1]	Fitzgerald M A, McCouch S R, Hall R D. Not just a grain of rice: the quest for quality.Trends Plant Sci, 2009, 14: 133-139.
[2]	Robards K, Antolovich M.Analytical chemistry of fruit bioflavonoids.Analyst, 1997, 122(2): 11-34.
[3]	Furukawa T, Maekawa M, Oki T, Suda I, Iida S, Shimada H, Takamure I, Kadowak K.The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp.Plant J, 2007, 49(1): 91-102.
[4]	Hong L, Qian Q, Tang D, Wang K, Li M, Cheng Z K.A mutation in the rice chalcone isomerase gene causes the golden hull and internode 1 phenotype.Planta, 2012, 236(1): 141-151.
[5]	Shao T, Qian Q, Tang D, Chen J, Li M, Cheng Z K, Luo Q.A novel gene IBF1 is required for the inhibition of brown pigment deposition in rice hull furrows.Theor Appl Genet, 2012, 125: 381-390.
[6]	Zhang K W, Qian Q, Huang Z J, Wang Y Q, Li M, Hong L L, Zeng D L, Gu M H, Chu C C, Cheng Z K.Gold hull and internode2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase In rice. Plant Physiol, 2006, 140(3): 972-983.
[7]	Tobias C M, Chow E K.Structure of the cinnamyl- alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification.Planta, 2005, 220(5): 678-688.
[8]	Hirano K, Aya K, Kondo M, Okuno A, Morinaka Y, Matsuoka M.OsCAD2 is the major CAD gene responsible for monolignol biosynthesis in rice culm.Plant Cell Rep, 2012, 31(1): 91-101.
[9]	Doxin R A, Paiva N L.Stress-induced pheny lpropanoid metabolism.Plant Cell, 1995, 7: 1085-1097.
[10]	Humphreys J M, Chapple C.Rewriting the lignin roadmap.Curr Opin Plant Biol, 2002, 5(3): 224-229.
[11]	Iwata N, Omura T.Linkage studies in rice plants (Oryza sativa L.) on some mutants derived from chronic gamma irradiation.J Fac Agric Kyushu Univ, 1977, 21: 117-127.
[12]	李亮杰, 周海鹏, 占小登, 楚宗丽, 程式华, 曹立勇. 一个水稻金黄色颖壳和节间基因的遗传定位. 中国水稻科学, 2008, 22(4): 432-434.
	Li L J, Zhou H P, Zhan X D, Chu Z L, Cheng S H, Cao L Y.Genetic mapping of a gold hull and internode gene in rice (Oryza sativa).Chin J Rice Sci, 2008, 22(4): 432-434.
[13]	李秋圆, 叶胜海, 张迎迎, 马晓静, 梅杰, 金庆生, 何祖华, 董彦君, 张小明. 一个水稻金黄色颖壳与节间基因的定位. 核农学报, 2012, 26(7): 983-987.
	Li Q Y, Ye S H, Zhang Y Y, Ma X J, Mei J, Jin Q S, He Z H, Dong Y J, Zhang X M.Molecular mapping of a gold hull and internode gene in Oryza sativa L.J Nucl Agric Sci, 2012, 26(7): 983-987.
[14]	Gui J, Shen J, Li L.Functional characterization of evolutionarily divergent 4-coumarate: Coenzyme A ligases in rice. Plant Physiol, 2011, 157(2): 574-586.
[15]	Tonnessen B W, Manosalva P, Lang J M, Baraoidan M, Bordeos A, Mauleon R, Oard J, Hulbert S, Leung H, Leach J E.Rice phenylalanine ammonia-lyase gene OsPAL4 is associated with broad spectrum disease resistance.Plant Mol Biol, 2015, 87(3): 273-286.
[16]	石坚. OsSHN1和OsMYB48参与水稻木质素合成调控的研究. 武汉: 华中农业大学, 2014.
	Shi J.Regulation of lignin biosynthesis by OsSHNl and OsMYB48 in rice. Wuhan: Huazhong Agricultural University, 2014.
[17]	Zeng D L, Qian Q, Dong G J. Zhu X D, Dong F G, Teng S, Gong L B, Cao L Y, Cheng S H, Xiong Z M.Development of isogenic lines of morphological markers in indica rice.Acta Bot Sin, 2003, 45: 1116-1120.
[18]	SAS Institute Inc, SAS / STAT User's Guide. Cary, NC, USA: SAS Institute, 1999.
[19]	Rogers S O, Bendich A J.Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues.Plant Mol Biol, 1985, 5: 69-76.
[20]	程式华, 曹立勇, 占小登. 杂交水稻制种技术. 北京: 金盾出版社, 2005.
	Cheng S H, Cao L Y, Zhan X D.Seed Production Technology of Hybrid Rice. Beijing: Jindun Press, 2005.
[21]	Orjuela J, Garavito A, Bouniol M, Arbelaez J D, Moreno L, Kimball J, Wilson G, Rami J F, Tohme J, McCouch S R, Lorieux M. A universal core genetic map for rice.Theor Appl Genet, 2010, 120: 563-572.
[22]	Li Z, Zhang Y, Liu L, Liu Q, Bi Z, Yu N, Cheng S, Cao L.Fine mapping of the lesion mimic and early senescence 1 (lmes 1) in rice (Oryza sativa L.).Plant Physiol Biochem, 2014, 80: 300-307.
[23]	Bökel C.EMS screens: From mutagenesis to screening and mapping.Meth Mol Biol, 2008, 420: 119-138.
[24]	Mulder NJ, Apweiler R, Attwood T K, Bairoch A, Bateman A, Binns D, Bradley P, Bork P, Bucher P, Cerutti L, Copley R, Courcelle E, Das U, Durbin R, Fleischmann W, Gough J, Haft D, Harte N, Hulo N, Kahn D, Kanapin A, Krestyaninova M, Lonsdale D, Lopez R, Letunic I, Madera M, Maslen J, McDowall J, Mitchell A, Nikolskaya A N, Orchard S, Pagni M, Ponting C P, Quevillon E, Selengut J, Sigrist C J A, Silventoinen V, Studholme D J, Vaughan R, Wu C H. InterPro, progress and status in 2005.Nucleic Acids Res, 2005, 33: D201-D205.
[25]	Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer E, Studholme D J Y, Eddy S. The Pfam protein families database.Nucleic Acids Res, 2004, 32: D138-D141.
[26]	Madera M, Vogel C, Kummerfeld SK, Chothia C, Gough J.The SUPERFAMILY database in 2004: Additions and improvements.Nucleic Acids Res, 2004, 32: D235-D239.
[27]	Gough J, Karplus K, Hughey R & Chothia C. Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure.J Mol Biol, 2001, 313(4): 903-919.
[28]	Liu L, Shang-Guan K, Zhang B, Liu X, Yan M, Zhang L, Shi Y, Zhang M, Qian Q, Li J, Zhou Y.Brittle Culm1, a COBRA-Like protein, functions in cellulose assembly through binding cellulose microfibrils. PLoS Genet, 2013, 9: e1003704.
[29]	Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Takahashi H, Umemura K, Umezawa T, Shimamoto K.Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice.PNAS, 2006, 103(1): 230-235.
[30]	Lee D K, Jung H, Jang G, Jeong J S, Kim Y S, Do Choi Y, Kim J K.Overexpression of the OsERF71 transcription factor alters rice root structure and drought resistance.Plant Physiol, 2016, 172(1): 575-588.
[31]	Kong X, Xie J, Wu X, Huang Y, Bao J.Rapid prediction of acid detergent fiber, neutral detergent fiber, and acid detergent lignin of rice materials by near-infrared spectroscopy.J Agric Food Chem, 2005, 53: 2843-2848.
[32]	Reddy N, Yang Y.Biofibers from agricultural byproducts for industrial applications.Trends Biotechnol, 2005, 23: 22-27.

引物名称 Primer name	上游引物 Forward primer(5′-3′)	下游引物 Reverse primer(5′-3′)	用途 Purpose
FH-1	TTACCATATAGTATAGCTGGTTGG	AACGATTTAGAAGTCAAATTAAAA	精细定位 Fine mapping
FH-5	ATTTTGATAATGGATAGAGATATTG	CACTTATAAATATAGAGAACGGATCT	精细定位 Fine mapping
FH-13	ATTTGGTTACATCCGCCTTT	CAGTTCAGCCGTCCAATAGA	精细定位 Fine mapping
RH-1	AGGAGCCTTGGATTACTACGA	GTTCACTGCCATGAACACATG	精细定位 Fine mapping
RH-4	GAGAAGAGCACAAACAATCACA	GTTTTTCGGTGTCTCTCAACTA	精细定位 Fine mapping
RH-7	TGCCAAACATATCGTGTCAC	ATTCCAAGCCCACTTCTTAA	精细定位 Fine mapping
RH-10	TGAAACTGTTTTAAACTAGATTCG	TGCTGAAAAGACTACAAAGATATG	精细定位 Fine mapping
RH-21	TTGATTGATTTTTTTCGATACAC	GGATTGTTAACTTTTAAAACTTCTG	精细定位 Fine mapping
RH-25	TATGTGACTTGTATGTGTCCAGC	TTCCTATCATTTGATTCATTGTT	精细定位 Fine mapping
SE-5	GGTGTAGACGTCGTTGTAGG	AGATGGATCATGGCTCATG	测序 Sequencing
Os4CL3	CCAGCAGGTGGATGGAGAG	AGCACGGAGTTGAGCGAGTA	qRT-PCR
OsMYB48	TGTATGGGTGGTGTGTGGCT	CAGGAATGCTCTTGGCGA	qRT-PCR

引物名称 Primer name	上游引物 Forward primer(5′-3′)	下游引物 Reverse primer(5′-3′)	用途 Purpose
FH-1	TTACCATATAGTATAGCTGGTTGG	AACGATTTAGAAGTCAAATTAAAA	精细定位 Fine mapping
FH-5	ATTTTGATAATGGATAGAGATATTG	CACTTATAAATATAGAGAACGGATCT	精细定位 Fine mapping
FH-13	ATTTGGTTACATCCGCCTTT	CAGTTCAGCCGTCCAATAGA	精细定位 Fine mapping
RH-1	AGGAGCCTTGGATTACTACGA	GTTCACTGCCATGAACACATG	精细定位 Fine mapping
RH-4	GAGAAGAGCACAAACAATCACA	GTTTTTCGGTGTCTCTCAACTA	精细定位 Fine mapping
RH-7	TGCCAAACATATCGTGTCAC	ATTCCAAGCCCACTTCTTAA	精细定位 Fine mapping
RH-10	TGAAACTGTTTTAAACTAGATTCG	TGCTGAAAAGACTACAAAGATATG	精细定位 Fine mapping
RH-21	TTGATTGATTTTTTTCGATACAC	GGATTGTTAACTTTTAAAACTTCTG	精细定位 Fine mapping
RH-25	TATGTGACTTGTATGTGTCCAGC	TTCCTATCATTTGATTCATTGTT	精细定位 Fine mapping
SE-5	GGTGTAGACGTCGTTGTAGG	AGATGGATCATGGCTCATG	测序 Sequencing
Os4CL3	CCAGCAGGTGGATGGAGAG	AGCACGGAGTTGAGCGAGTA	qRT-PCR
OsMYB48	TGTATGGGTGGTGTGTGGCT	CAGGAATGCTCTTGGCGA	qRT-PCR

性状 Trait		gh881	P
株高 Plant height/cm	74.75±0.96	72.80±1.62	**P<0.01
单株有效穗数 No. of panicles per plant	11.20±1.69	12.40±1.43	P=0.07
每穗总粒数 No. of spikelets per panicle	132.31±6.19	104.03±8.08	**P<0.01
每穗实粒数 No. of grains per panicle	117.46±4.94	65.31±10.99	**P<0.01
结实率 Seed-setting rate/%	88.82±2.15	62.87±9.71	**P<0.01
千粒重 1000-grain weight/g	28.92±0.51	27.13±1.12	**P<0.01

性状 Trait		gh881	P
株高 Plant height/cm	74.75±0.96	72.80±1.62	**P<0.01
单株有效穗数 No. of panicles per plant	11.20±1.69	12.40±1.43	P=0.07
每穗总粒数 No. of spikelets per panicle	132.31±6.19	104.03±8.08	**P<0.01
每穗实粒数 No. of grains per panicle	117.46±4.94	65.31±10.99	**P<0.01
结实率 Seed-setting rate/%	88.82±2.15	62.87±9.71	**P<0.01
千粒重 1000-grain weight/g	28.92±0.51	27.13±1.12	**P<0.01

杂交组合 Cross combination	F₁表型 F₁ phenotype	F₂分离比 Segregation in F₂		总数 Total	χ²_3：1
杂交组合 Cross combination	F₁表型 F₁ phenotype	野生型表型 Wild-type	突变体表型 Mutant-type	总数 Total	χ²_3：1
gh881×中恢8015 gh881×Zhonghui 8015	正常Normal	764	236	1000	1.05
gh881×长粒粳 gh881×Changlijing	正常Normal	756	244	1000	0.19