Phenotypic Analysis and Gene Mapping of a White Stripe Mutant st13 in Rice

doi:10.16819/j.1001-7216.2017.6165 355

Abstract

Abstract:

【Objective】Rice leaf color mutation identification and cloning of related genes is helpful to research photosynthesis and further understand the mechanism of chloroplast development and pigment synthesis pathway, providing theoretical basis for the breeding of rice with high photosynthetic efficiency.【Method】A somaclonal mutant (designated as stripe leaf 13, st13) was isolated from tissue-cultured progenies of a japonica rice variety Dongjin. The main agronomic traits of wild type and st13 were determined at maturity, and the pigment contents and ultrastructure of chloroplast were observed at the seedling stage. Dongjin and st13 were reciprocally crossed to observe the phenotype of F₁ and genetic analysis was carried out. An F₂ population and F_2:3 population derived from the cross st13 × Nanjing 11 (an indica rice variety) were used for gene mapping of ST13. Quantitative RT-PCR was carried out to analyze the relative expression of genes associated with chloroplast development and chlorophyll biogenesis in the wild-type Dongjin and st13 mutant. 【Result】Compared to the wild type, the plant height, number of effective panicles, panicle length, seed-setting rate, and 1000-grain weight were all significantly reduced in the mutant. In agreement with leaf color, the pigment content of st13 mutant was reduced at the seedling stage but rose to almost the same level as the wild type at the tillering stage. With a transmission electron microscopy, defective chloroplasts with no thylakoid were observed in the st13 mutant. Genetic analysis indicated the mutant phenotype was controlled by a single recessive nuclear gene. ST13 was restricted to an interval between the InDel (insertion/deletion) markers I3-21 and I3-22 on chromosome 3. Six InDel markers were further developed and the ST13 gene was narrowed down to a 94-kb region, containing eight putative open reading frames. 【Conclusion】These eight candidate genes encode three putative proteins and five functional proteins respectively, while none of them have been reported in rice. Thus, ST13 was an unreported gene in rice. Relative to wild type, the expression levels of genes associated with chloroplast development, chlorophyll biosynthesis, and photosynthetic system were significantly altered in the mutant. So we hypothesized that ST13 might be the key gene for regulating chloroplast development.

Key words: rice, white stripe mutant, chloroplast, fine mapping

摘要：

目的叶色突变相关基因鉴定和克隆有助于研究光合作用,补充并完善叶绿体发育机理和色素合成代谢途径,为开展水稻的高光效育种提供理论依据。方法从粳稻品种Dongjin的组培后代中分离出一个白条纹突变体st13,成熟期测定野生型和st13的主要农艺性状,苗期测定色素含量并观察叶绿体的超微结构;将st13和Dongjin进行正反交,观察F₁植株表型,并对F₂表型分离进行卡方检验,对st13进行遗传分析;利用st13×南京11（籼稻品种）的F₂和F_2:3群体,对st13突变基因定位;采用qPCR分析叶绿体发育和叶绿素合成相关基因在st13与野生型相对表达量。结果与野生型Dongjin相比,该突变体的株高、单株有效穗数、穗长、结实率和千粒重等主要农艺性状显著下降。苗期的色素含量降低,分蘖期无差异。突变体的叶绿体中既有含丰富的类囊体膜结构的正常叶绿体,也存在无类囊体结构的叶绿体。遗传分析和基因定位结果表明,st13的突变表型受1对隐性核基因控制,突变基因位于第3条染色体长臂InDel（Insertion-Deletion）标记I3-21和I3-22之间。进一步在这两个标记之间设计了6对InDel标记,最终将基因定位在94 kb区间内,此区间共有8个候选基因。结论这8个候选基因中,有5个假定的蛋白,其他三个都是有功能注释的蛋白,而这三个蛋白在水稻中均未见报道,因此,st13突变是由一个新的叶色基因突变引起的;同时st13中叶绿体发育、叶绿素合成和光合系统相关基因的表达也发生了显著改变,推测ST13可能是调控叶绿体发育的关键基因。

关键词: 水稻, 白条纹突变体, 叶绿体, 精细定位

Liting SUN, Tianzi LIN, Yunlong WANG, Mei NIU, Tingting HU, Shijia LIU, Yihua WANG, Jianmin WAN. Phenotypic Analysis and Gene Mapping of a White Stripe Mutant st13 in Rice[J]. Chinese Journal OF Rice Science, 2017, 31(4): 355-363.

孙立亭, 林添资, 王云龙, 牛梅, 胡婷婷, 刘世家, 王益华, 万建民. 水稻白条纹突变体st13的表型分析及基因定位[J]. 中国水稻科学, 2017, 31(4): 355-363.

Figures/Tables 10

References 38

[1]	Fambrini M, Castagna A, Dalla V F, Degl’Innocenti E, Ranieri A, Vernieri P, Pardossi A, Guidi L, Rascio N, Pugliesi C. Characterization of a pigment-deficient mutant of sunflower (Helianthus annuus L.) with abnormal chloroplast biogenesis, reduced PSII activity and low endogenous level of abscisic acid. Plant Breeding, 2004, 6: 645-650.
[2]	Agrawal G K, Yamazaki M, Kobayashi M, Hirochika R, Miyao A, Hirochika H.Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion: Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene. Plant Physiol, 2001, 125(3): 1248-1257.
[3]	Parks B M, Quail P H.Phytochrome-deficient hy1 and hy2 long hypocotyl mutants of Arabidopsis are defective in phytochrome chromophore biosynthesis. Plant Cell, 1991, 3(11): 1177-1186.
[4]	Kusumi K, Sakata C, Nakamura T, Kawasaki S, Yoshimura A, Iba K.A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions.Plant J, 2011, 68(6): 1039-1050.
[5]	Lee S, Kim J H, Yoo E S, Lee C H, Hirochika H, An G.Differential regulation of chlorophyll a oxygenase genes in rice.Plant Mol Biol, 2005 57: 805-818.
[6]	Sugimoto H, Kusumi K, Noguchi K, Yano M, Yoshimura A, Iba K.The rice nuclear gene,VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria. Plant J, 2007, 52: 512-527.
[7]	Zhang H, Li J, Yoo J, Yoo S, Cho S, Koh H, Seo H S, Paek N.Rice chlorina-1 and chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol Biol, 2006, 62: 325-337.
[8]	Jung K H, Hur J, Ryu C H, Choi Y, Chung Y Y, Miyao A, Hirochika H, An G.Characterization of a rice chlorophyll- deficient mutant using the T-DNA gene-trap system.Plant Cell Physiol, 2003, 44: 463-472.
[9]	Kong W, Yu X, Chen H, Liu L, Xiao Y, Wang Y, Wang C, Lin Y, Yu Y, Wang C, Jiang L, Zhai H, Zhao Z, Wan J.The catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase forms a chloroplast complex to regulate chlorophyll biosynthesis in rice.Plant Mol Biol, 2016, 92(1-2): 177-191.
[10]	Wang P, Gao J, Wan C, Zhang F, Xu Z, Huang X, Sun X, Deng X.Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol, 2010, 153: 994-1003.
[11]	Sakuraba Y, Rahman M L, Cho S H, Kim Y S, Koh H J, Yoo S C, Paek N C.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions. Plant J, 2013, 74: 122-133.
[12]	Wu Z M, Zhang X, He B, Diao L, Sheng S, Wang J, Guo X, Su N, Wang L, Jiang L, Wang C, Zhai H, Wan J.A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis.Plant Physiol, 2007, 145: 29-40.
[13]	Yang Y, Xu J, Huang L, Leng Y, Dai L, Rao Y, Chen L, Wang Y, Tu Z, Hu J, Ren D, Zhang G, Zhu L, Guo L, Qian Q, Zeng D.PGL, encoding chlorophyllide a oxygenase 1, impacts leaf senescence and indirectly affects grain yield and quality in rice. J Exp Bot, 2016, 67(5): 1297-1310.
[14]	Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M.Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice.Plant J, 2009, 57: 120-131.
[15]	Wang L, Wang C, Wang Y, Niu M, Ren Y, Zhou K, Zhang H, Lin Q, Wu F, Cheng Z, Wang J, Zhang X, Guo X, Jiang L, Lei C, Wang J, Zhu S, Zhao Z, Wan J.WSL3, a component of the plastid-encoded plastid RNA polymerase, is essential for early chloroplast development in rice.Plant Mol Biol, 2016, 92(4-5): 581-595.
[16]	Wang Y, Wang C, Zheng M, Lyu J, Xu Y, Li X, Niu M, Long W, Wang D, Wang H Y, William T, Wang Y, Wan J.WHITE PANICLE1, a Val-tRNA synthetase regulating chloroplast ribosome biogenesis in rice, is essential for early chloroplast development.Plant Physiol, 2016, 170(4): 2110-2123.
[17]	Lopez-Juez E.Plastid biogenesis between light and shadows.J Exp Bot, 2007, 58: 11-26.
[18]	Sakamoto W, Miyagishima S Y, Jarvis P.Chloroplast biogenesis: Control of plastid development, protein import, division and inheritance.Arabidopsis Book, 2008, 6: e110.
[19]	Webber A N, Malkin R.Photosystem I reaction-centre proteins contain leucine zipper motifs. A proposed role in dimer formation.FEBS Lett, 1990, 264: 1-4.
[20]	Rutherford A W, Faller P.Photosystem II: evolutionary perspectives.Philos Trans R SocLond B Biol Sci. 2003, 358: 245-253.
[21]	Santis-Maciossek G D, Kofer W, Bock A, Schoch S, Maier R M, Wanner G, Rüdiger W, Koop Hans-Ulrich, Herrmann R G. Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology biochemistry and ultrastructure. Plant J, 1999, 18: 477-489.
[22]	Peng L W, Yamamoto H, Shikanai T.Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex.Biochim Biophys Acta, 2011, 1807: 945-953.
[23]	Andersson I, Backlund A.Structure and function of Rubisco.Plant Physiol Biochem, 2008, 46: 275-291.
[24]	Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2 Delta Delta C (T)] method.Methods, 2001, 25: 402-408.
[25]	McCouch S R, Kochert G, Yu Z H, Wang Z Y, Khush G S, Coffman W R, Tanksley S D. Molecular mapping of rice chromosome.Theor Appl Genet, 1998, 76: 815-829.
[26]	Shi Y F, Chen J, Liu W Q, Huang Q N, Shen B, Leung H, Wu J L.Genetic analysis and gene mapping of a new rolled-leaf mutant in rice (Oryza sativa L.). Sci China C: Life Sci, 2009, 52: 885-890.
[27]	Kusumi K, Chono Y, Shimada H, Shimada H, Gotoh E, Tsuyama M, Iba K.Chloroplast biogenesis during the early stage of leaf development in rice.Plant Biotechnol, 2010, 27: 85-90.
[28]	Allen J F, Wilson B M, Puthiyaveetil S, Nield J.A structural phylogenetic map for chloroplast photosynthesis.Trends Plant Sci, 2011, 16: 645-655.
[29]	Pakrasi H B.Genetic analysis of the form, function of photosystem I and photosystem II.Annu Rev Genet, 1995, 29: 755-776.
[30]	王兴春, 王敏, 季芝娟, 陈钊, 刘文真, 韩渊怀, 杨长登. 水稻糖苷水解酶基因OsBE1在叶绿体发育中的功能. 作物学报, 2014, 40(12): 2090-2097.
	Wang X C, Wang M, Ji Z J, Chen Z, Liu W Z, Han Y H, Yang C D.Functional characterization of the glycoside hydrolase encoding gene OsBE1 during chloroplast development in Oryza sativa. Acta Agron Sin, 2014, 40(12): 2090-2097. (in Chinese with English abstract)
[31]	Gothandam K M, Kim E S, Cho H, Chung Y Y.OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis.Plant Mol Biol, 2005, 58(3): 421-433.
[32]	Lin D, Jiang Q, Zheng K, Chen S, Zhou H, Gong X, Xu J, Teng S, Dong Y.Mutation of the rice ASL2 gene encoding plastid ribosomal protein L21 causes chloroplast developmental defects and seedling death. Plant Biol (Stuttg), 2015, 17(3): 599-607.
[33]	Zhang Z, Tan J, Shi Z, Xie Q, Xing Y, Liu C, Chen Q, Zhu H, Wang J, Zhang J, Zhang G.Albino Leaf1 that encodes the sole octotricopeptide repeat protein is responsible for chloroplast development. Plant Physiol, 2016, 171(2): 1182-1191.
[34]	Yoo S C, Cho S H, Sugimoto H, Li J, Kusumi K, Koh H J, Iba K, Paek N C.Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development.Plant Physiol, 2009, 150(1): 388-401.
[35]	Wang Y, Zhang J, Shi X, Peng Y, Li P, Lin D, Dong Y, Teng S.Temperature-sensitive albino geneTCD5, encoding a monooxygenase, affects chloroplast development at low temperatures. J Exp Bot, 2016, 67(17): 5187-5202.
[36]	Su N, Hu M L, Wu D X, Wu F Q, Fei G L, Lan Y, Chen X L, Shu X L, Zhang X, Guo X P, Cheng Z J, Lei C L, Qi C K, Jiang L, Wang H, Wan J M.Disruption of a rice pentatricopeptide repeat protein causes a seedling- specific albino phenotype and its utilization to enhance seed purity in hybrid rice production.Plant Physiol, 2012, 159(1): 227-238.
[37]	Klinghammer M, Tenhaken R.Genome-wide analysis of the UDP-glucose dehydrogenase gene family in Arabidopsis, a key enzyme for matrix polysaccharides in cell walls. J Exp Bot, 2007, 58(13): 3609-3621.
[38]	孙兰茜, 常平安, 曾鑫, 韩丽萍, 冉凤, 王超颖, 王玲, 黄飞飞. 甘油磷酸二酯酶家族蛋白的分子进化. 基因组学与应用生物学, 2015, 34(27): 172-178.
	Sun L X, Chang P A, Zeng X, Han L P, Ran F, Wang C Y, Wang L, Huang F F.Molecular evolution of the glycerophospho- diesterase family proteins.Genomics Appl Boil, 2015, 34(27): 172-178. (in Chinese with English abstract)

材料 Material	株高 Plant height/cm	有效穗数 No. of effective panicles	剑叶长 Flag leaf length /cm		穗长 Panicle length /cm	结实率 Seed setting rate/%	千粒重 1000-grain weight /g
WT	112.2±2.9	9.6±0.6	31.5±2.4	23.8±0.2		95.5±1.5	27.9±1.6
st13	70.8±4.7^**	5.8±0.5^**	31.9±1.5	17.9±0.5^**		74.0±1.8^**	20.3±0.9^**

材料 Material	株高 Plant height/cm	有效穗数 No. of effective panicles	剑叶长 Flag leaf length /cm		穗长 Panicle length /cm	结实率 Seed setting rate/%	千粒重 1000-grain weight /g
WT	112.2±2.9	9.6±0.6	31.5±2.4	23.8±0.2		95.5±1.5	27.9±1.6
st13	70.8±4.7^**	5.8±0.5^**	31.9±1.5	17.9±0.5^**		74.0±1.8^**	20.3±0.9^**

组合 Combination	正常株数 No. of normal plants	白条纹株数 No. of plants with white stripes	实际分离比 Segration ratio	χ²_(3:1)	χ²_0.05
Dongjin×st13	286	98	2.92:1	0.03	3.84
st13×Dongjin	281	89	3.16:1	0.13

组合 Combination	正常株数 No. of normal plants	白条纹株数 No. of plants with white stripes	实际分离比 Segration ratio	χ²_(3:1)	χ²_0.05
Dongjin×st13	286	98	2.92:1	0.03	3.84
st13×Dongjin	281	89	3.16:1	0.13

引物名称 Primer name		后引物 Reverse primer	BAC克隆 BAC clone
I3-21	GCGAGATGGGCAGCTACTAC	ACACAATGTCCAGCTTGCAG	OSJNBa0010D22
P5	CCTCCTCATAGCCCCAATCC	TCACTCTCCCATAGGTGGTC	OSJNBb0042N11
P3-13	CCTCCGCACGAACCCT	GTGGGAAACTGGAGACGAG	OSJNBa0087M10
P3-14	GGTTACATCTCCTTTTCGTTT	TCTTTGTTTGCTGCCATCT	OJ1519_A12
S-57	AAAACCAAAACTAGAAGC	ATGTCCCTGGAAATGTA	OSJNBa0004G03
S-24	CTCCGAGCGGCTGATTA	GGATAGTACTTCGTTTCGTA	OSJNBa0004G03
P3-29	CAAACGCAATCTCACATCACA	AAGCGAGTGGGAGGGTG	OSJNBa0091E13
I3-22	AGGTCTCGTGTCGTTCATCC	TGGAGGGAGCATGTCTATCA	OSJNBa0063J18