Identification and Genetic Analysis of Thermo-sensitive Mutant osv15 in Rice

doi:10.16819/j.1001-7216.2018.7131

Abstract

Abstract:

：【Objective】The objective of the research is to identify and clone a new thermo-sensitive gene of rice, and to reveal the molecular mechanism involved in the process of chloroplast development and photosynthesis, providing theoretical support for photosynthetic efficiency breeding. 【Method】A yellow leaf mutant osv15 was obtained from rice cultivar Nipponbare (abbreviated as Nip) mutagenized by irradiation. The phenotypic, agronomic traits and genetic patterns of osv15 were analyzed in detail, and F₂ population was constructed by crossing the mutant with Kasalath, and mutant individuals were selected to map the gene OsV15.【Result】The mutant osv15 exhibited etiolation at seedling stage, with only ten percent of the chlorophyll content of the wild type, declined photochemical efficiency and abnormal structure of chloroplasts under low temperature condition (22℃). As temperature rose, leaf color of the mutant regreened due to increased chlorophyll content, which recovered to sixty-eight percent of that of the wild type under higher temperature condition (30℃). In natural environment, osv15 showed leaf yellowing from seedling stage to maturity stage, and significant differences in plant height, tiller number, seed setting rate and so on. Genetic analysis revealed that the mutant phenotype of osv15 was controlled by a single recessive gene. The gene was located in the 84 kb region of chromosome 6, between molecular markers S4 and S5. Sequencing analysis in this region showed that Cpn60β1(LOC_Os06g02380) that encodes a putative chaperonin 60 beta precursor, carried a single base deletion in osv15.【Conclusion】osv15 is a new thermo-sensitive mutant, and Cpn60β1 may be the mutant gene.

Key words: rice, thermo-sensitive, chloroplast, gene mapping

摘要：

【目的】本研究旨在鉴定和克隆水稻温敏转绿新基因,揭示其参与叶绿体发生发育和光合作用的分子机制,为高光效育种提供理论支撑。【方法】利用辐射诱变的方法,从粳稻品种日本晴中筛选获得叶片黄化突变体osv15,并对其表型、农艺性状和遗传方式进行详细分析。构建了突变体与Kasalath的F₂群体,利用多态性分子标记对目的基因进行定位和测序分析。【结果】osv15幼苗期在22℃低温下叶片黄化,叶绿素含量仅为野生型的10%,光化学效率下降,叶绿体结构异常;随着温度的升高,osv15的叶色由黄转绿,30℃时叶绿素含量恢复到野生型的68%,光化学效率和叶绿体发育与野生型相近。在自然环境下,osv15突变体从苗期至成熟期均表现为叶片黄化,且株高、分蘖数和产量等农艺性状与野生型相比差异显著。遗传分析表明osv15突变体的表型由一对隐性核基因控制。将OsV15基因定位到第6染色体多态性标记S4和S5之间84 kb的区间内,定位区间测序发现突变体中编码分子伴侣蛋白的基因Cpn60β1(LOC_Os06g02380)发生单碱基缺失。【结论】osv15是一个新的水稻温敏转绿突变体,Cpn60β1可能为突变基因。

关键词: 水稻, 温敏, 叶绿体, 基因定位

CLC Number:

Yuehan QI, Ruili LI, Fang WANG, Hongjia LIU, Keke YI, Cheng ZHU. Identification and Genetic Analysis of Thermo-sensitive Mutant osv15 in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(4): 349-356.

齐岳翰, 李瑞莉, 王芳, 刘洪家, 易可可, 朱诚. 水稻温敏转绿突变体osv15的鉴定和遗传分析[J]. 中国水稻科学, 2018, 32(4): 349-356.

Figures/Tables 8

References 34

[1]	Mullet J E.Dynamic regulation of chloroplast transcription.Plant Physiol, 1993, 103(2): 309-313.
[2]	Abdallah F, Salamini F, Leister D.A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis. Trends Plant Sci, 2000, 5(4): 141-142.
[3]	何冰, 刘玲珑, 张文伟, 万建民. 植物叶色突变体. 植物生理学报, 2006, 42(1): 1-9.
	He B, Liu L L, Zhang W W, Wan J M.Plant leaf color mutants.Plant Physiol J, 2006, 42(1): 1-9. (in Chinese with English abstract)
[4]	刘艳霞, 林冬枝, 董彦君. 水稻温敏感叶色突变体研究进展. 中国水稻科学, 2015, 29(4): 439-446.
	Liu Y X, Lin D Z, Dong Y J.Research advances in thermo-sensitive leaf coloration mutants in rice.Chin J Rice Sci, 2015, 29(4): 439-446. (in Chinese with English abstract)
[5]	初志战, 刘小林, 陈远玲, 刘耀光. 一个水稻白化致死突变基因的精细定位和遗传研究. 中国水稻科学, 2016, 30(2): 136-142.
	Chu Z Z, Liu X L, Chen Y L, Liu Y G.Genetic analysis and gene mapping of an albino lethal mutant in rice. Chin J Rice Sci, 2016, 30(2): 136-142. (in Chinese with English abstract)
[6]	李自壮, 徐乾坤, 余海平, 周亭亭, 薛大伟, 曾大力, 郭龙彪, 钱前, 任德勇. 水稻淡黄叶矮化突变体yld的遗传分析及基因定位. 作物学报, 2017, 43(4): 522-529.
	Li Z Z, Xu Q K, Yu H P, Zhou T T, Xue D W, Zeng D L, Guo L B, Qian Q, Ren D Y.Genetic analysis and gene mapping of Yellow leaf and dwarf ( yld ) mutant in rice. Act Agron Sin, 2017, 43(4): 522-529. (in Chinese with English abstract)
[7]	刘胜, 魏祥进, 邵高能, 唐绍清, 胡培松. 一个水稻“斑马叶”叶色突变体基因zebra leaf2(zl2)的图位克隆. 中国水稻科学, 2013, 27(3): 231-239.
	Liu S, Wei J X, Shao G N, Tang S Q, Hu P S.Map-based cloning of a zebra leaf mutant gene zl2 in rice.Chin J Rice Sci, 2013, 27(3): 231-239. (in Chinese with English abstract)
[8]	王亚琴, 施军琼, 张婷, 李燕, 张天泉, 张小龙, 桑贤春, 凌英华, 何光华. 水稻黄绿叶突变体ygl13的鉴定及候选基因分析. 中国农业科学, 2015, 48(21): 4197-4208.
	Wang Y Q, Shi J Q, Zhang T, Li Y, Zhang T Q, Zhang X L, Sang X C, Ling Y H, He G H .Characterization and candidate gene analysis of Yellow-Green Leaf mutant ygl13 in rice(Oryza sativa). Sci Agric Sin, 2015, 48(21): 4197-4208. (in Chinese with English abstract)
[9]	涂政军, 邹国兴, 黄李超, 陈龙, 代丽萍, 高易宏, 冷语佳, 朱丽, 张光恒, 胡江, 任德勇, 高振宇, 董国军, 陈光, 郭龙彪, 钱前, 曾大力. 水稻淡绿叶基因PGL11的鉴定与精细定位. 中国水稻科学, 2017, 31(5): 489-499.
	Tu Z J, Zou G X, Huang L C, Chen L, Dai L P, Gao Y H, Leng Y J, Zhu L, Zhang G H, Hu J, Ren D Y, Gao Z Y, Dong G J, Chen G, Guo L B, Qian Q, Zeng D L.Identification and fine mapping of pale green leaf PGL11 in rice. Chin J Rice Sci, 2017, 31(5): 489-499. (in Chinese with English abstract)
[10]	邹金财, 张维林, 夏明辉, 邱洋松, 王长春, 杨玲, 张小明. 水稻阶段性温敏白化转绿突变体stgra254的特征和基因定位. 华北农学报, 2017, 32(3): 1-6.
	Zhou J C, Zhang W L, Xia M H, Qiu Y S, Wang C C, Yang L, Zhang X M.Characterization and gene mapping of stage thermo-sensitive green-revertible albino mutant stgra254 in rice. Acta Agric Boreali-Sin, 2017, 32(3): 1-6. (in Chinese with English abstract)
[11]	朱环环, 刘艳霞, 潘倩文, 郑凯伦, 林冬枝, 董彦君. 一个水稻白化致死突变体abl25鉴定及其基因定位. 上海师范大学学报: 自然科学版, 2014, 43(3): 238-244.
	Zhu H H, Liu Y X, Pan Q W, Zheng K W, Lin D Z, Dong Y J.Identification and gene mapping of a novel rice albino lethal abl25 mutant. J Shanghai Normal Univ: Nat Sci, 2014, 43(3): 238-244. (in Chinese with English abstract)
[12]	高小丽, 李素娟, 邵健丰, 刘洪家, 陶跃之. 水稻POR基因的分离、定位与功能的初步研究. 植物生理学报, 2015, 51(6): 860-868.
	Gao X L, LI S J, Shao J F, Liu H J, Tao Y Z.Preliminary study on isolation, mapping, and function of NADPH: Proto-chlorophyllide oxidoreductase( POR) gene in rice(Oryza sativa). Plant Physiol J, 2015, 51(6): 860-868. (in Chinese with English abstract)
[13]	Li Q, Zhu F Y, Gao X, Sun Y, Li S, Tao Y, Lo C, Liu H.Young Leaf Chlorosis 2 encodes the stroma-localized heme oxygenase 2 which is required for normal tetrapyrrole biosynthesis in rice. Planta, 2014, 240(4): 701-712.
[14]	Zhou K, Ren Y, Lv J, Wang Y, Liu F, Zhou F, Zhao S, Chen S, Peng C, Zhang X.Young Leaf Chlorosis 1 , a chloroplast-localized gene required for chlorophyll and lutein accumulation during early leaf development in rice. Planta, 2013, 237(1): 279.
[15]	Chen H, Cheng Z, Ma X, Wu H, Liu Y, Zhou K, Chen Y, Ma W, Bi J, Zhang X.A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep, 2013, 32(12): 1855.
[16]	Archer E K, Bonnett H T.Characterization of a virescent chloroplast mutant of tobacco.Plant Physiol, 1987, 83(4): 920-925.
[17]	谭炎宁, 孙学武, 袁定阳, 孙志忠, 余东, 何强, 段美娟, 邓华凤, 袁隆平. 水稻单叶独立转绿型黄化突变体grc2的鉴定与基因精细定位. 作物学报, 2015, 41(6): 831-837.
	Tan Y N, Sun X W, Yuan D Y, Sun Z Z, Yu D, He Q, Duan M J, Deng H F, Yuan L P.Identification and fine mapping of green-revertible chlorina gene grc2 in rice(Oryza sativa L.) Act Agron Sin, 2015, 41(6): 831-837. (in Chinese with English abstract)
[18]	Iba K, Takamiya K I, Toh Y, Satoh H, Nishimura M.Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice. Genesis, 2010, 12(5): 342-348.
[19]	Kusumi K, Mizutani A, Nishimura M, Iba K.A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice. Plant J, 1997, 12(6): 1241-1250.
[20]	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(3): 512-527.
[21]	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.
[22]	Gong X, Su Q, Lin D, Jiang Q, Xu J, Zhang J, Teng S, Dong Y.The rice OsV4 encoding a novel pentatricopeptide repeat protein is required for chloroplast development during the early leaf stage under cold stress. J Integr Plant Biol, 2014, 56(4): 400-410.
[23]	Liu H, Li Q, Yang F, Zhu F, Sun Y, Tao Y, Lo C.Differential regulation of protochlorophyllide oxidoreductase abundances by VIRESCENT 5A (OsV5A) and VIRESCENT 5B (OsV5B) in rice seedlings. Plant Cell Physiol, 2016, 57(11): 2392-2402.
[24]	Jiang Q, Mei J, Gong X D, Xu J L, Zhang J H, Teng S, Lin D Z, Dong Y J.Importance of the riceTCD9 encoding α subunit of chaperonin protein 60(Cpn60α) for the chloroplast development during the early leaf stage. Plant Sci, 2014(215/216): 172-179.
[25]	Liu W, Fu Y, Hu G, Si H, Zhu L, Wu C, Sun Z.Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice ( Oryza sativa L.). Planta, 2007, 226(3): 785-795.
[26]	Murray M G, Thompson W F.Rapid isolation of high weight plant DNA.Nucleic Acids Res, 1980, 8(19): 4321-4325.
[27]	张其德. 测定叶绿素的几种方法. 植物学报, 1985, 3(5): 60-64.
	Zhang Z D.Methods of determination of chlorophyll.Chin Bull Bot, 1985, 3(5): 60-64. (in Chinese)
[28]	吕典华, 宗学凤, 王三根, 凌英华, 桑贤春, 何光华. 两个水稻叶色突变体的光合特性研究. 作物学报, 2009, 35(12): 2304-2308.
	Lv D H, Zong X F, Wang S G, Ling Y H, Sang X C, He G H.Characteristics of photosynthesis in two leaf color mutants of rice. Act Agron Sin, 2009, 35(12): 2304-2308. (in Chinese with English abstract)
[29]	郭鹏. 水稻温敏黄转绿突变体v5的鉴定和基因克隆. 金华: 浙江师范大学, 2011.
	Guo P.Identification and gene mapping of a rice thermo-sensitivevirescent mutant v5. Jinhua: Zhejiang Normal University, 2011. (in Chinese with English abstract)
[30]	王文娟, 吴兰兰, 雷晓庆, 赵怀玉, 林冬枝, 董彦君. 一个新水稻低温敏感叶色突变体tcm11的鉴定及基因定位. 上海师范大学学报: 自然科学版, 2017, 46(2): 319-325.
	Wang W J, Wu L L, Lei X Q, Zhao H Y, Lin D Z, Dong Y J.Identification and gene mapping of a novel low temperature sensitive leaf color mutant tcm11 in rice(Oryza sativa L.). J Shanghai Normal Univ: Nat Sci, 2017, 46(2): 319-325. (in Chinese with English abstract)
[31]	张天雨, 周春雷, 刘喜, 孙爱伶, 曹鹏辉, Nguyen T, 田云录, 翟虎渠, 江玲. 一个水稻温敏黄化突变体的表型分析和基因定位. 作物学报, 2017, 43(10): 1426-1433.
	Zhang T Y, Zhou C L, Liu X, Sun A L, Cao P H, Nguyen T, Tian Y L, Zhai H Q, Jiang L.Phenotypes and gene mapping of a thermo-sensitive yellow leaf mutant of rice. Act Agron Sin, 2017, 43(10): 1426-1433. (in Chinese with English abstract)
[32]	Peng Y, Zhang Y, Lv J, Zhang J H, Li P, Shi X L, Wang Y F, Zhang H L, He Z H, Teng S.Characterization and fine mapping of a novel rice albino mutant low temperature albino 1. J Gene Genom, 2012, 39(8): 385-396.
[33]	Peng L W, Fukao Y, Myouga F, Motohashi R, Shinozaki K, Shikanai T.A chaperonin subunit with unique structures is essential for folding of a specific substrate.Plos Biol, 2011, 9(4): e1001040.
[34]	Suzuki K, Nakanishi H, Bower J, Yoder D W, Osteryoung K W, Miyagishima S Y.Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana. BMC Plant Biol, 2009, 9(1): 38.

标记名称 Marker name	正向引物(5′-3′) Forward primer(5′-3′)	反向引物(5′-3′) Reverse primer(5′-3′)	所在BAC BAC location
S1	TAACACGCACTCAAAACGTG	TGCCGACTCTCATTGATCTG	AP001552
S2	CAACCCCTAACGACCATAGC	CACATGCATGACACGATCAA	AP001552
S3	CCAACTCTACATGCTCCTTCTG	CCCCAGAGAAGATCTCCTAGC	AP001389
S4	TCCTCACCTCCTCCTCCTTT	CACAAGCTTCTCTCTTCCCTTC	AP001389
S5	AGAGGCAATGTCGCAGTATG	GGTGGTTCTGCTCCCATTTA	AP002837
S6	ACAACTTCGCTTCTCGGTTG	GTACCTGACAGAGGCGATCC	AP002838

标记名称 Marker name	正向引物(5′-3′) Forward primer(5′-3′)	反向引物(5′-3′) Reverse primer(5′-3′)	所在BAC BAC location
S1	TAACACGCACTCAAAACGTG	TGCCGACTCTCATTGATCTG	AP001552
S2	CAACCCCTAACGACCATAGC	CACATGCATGACACGATCAA	AP001552
S3	CCAACTCTACATGCTCCTTCTG	CCCCAGAGAAGATCTCCTAGC	AP001389
S4	TCCTCACCTCCTCCTCCTTT	CACAAGCTTCTCTCTTCCCTTC	AP001389
S5	AGAGGCAATGTCGCAGTATG	GGTGGTTCTGCTCCCATTTA	AP002837
S6	ACAACTTCGCTTCTCGGTTG	GTACCTGACAGAGGCGATCC	AP002838

材料 Material	株高 Plant height / cm	分蘖数 Tiller number	有效穗数 No. of productive panicles	穗长 Panicle length / cm	叶长 Leaf length / cm	叶宽 Leaf width / cm	结实率 Seed setting rate / %	千粒重 1000-grain weight / g
WT	87.3±2.0	15.8±2.2	14.3±2.1	21.5±0.6	31.5±1.3	1.3±0.1	91.4±2.1	25.8±0.6
osv15	80.2±1.9^**	8.6±1.4^**	6.8±1.0^**	20.1±1.0	29.9±1.5	1.2±0.1	80.1±4.0^**	22.8±1.8^**

材料 Material	株高 Plant height / cm	分蘖数 Tiller number	有效穗数 No. of productive panicles	穗长 Panicle length / cm	叶长 Leaf length / cm	叶宽 Leaf width / cm	结实率 Seed setting rate / %	千粒重 1000-grain weight / g
WT	87.3±2.0	15.8±2.2	14.3±2.1	21.5±0.6	31.5±1.3	1.3±0.1	91.4±2.1	25.8±0.6
osv15	80.2±1.9^**	8.6±1.4^**	6.8±1.0^**	20.1±1.0	29.9±1.5	1.2±0.1	80.1±4.0^**	22.8±1.8^**