水稻黄绿叶突变体ygl13的鉴定及候选基因分析

doi:10.3864/j.issn.0578-1752.2015.21.001

摘要/Abstract

摘要： 【目的】对水稻黄绿叶突变体ygl13 (yellow-green leaf 13 )进行表型鉴定和候选基因检测，以便了解水稻叶色形成和调控的分子机制。【方法】经甲基磺酸乙酯（EMS）诱变籼稻恢复系缙恢10号（Jinhui 10），从中筛选出1份遗传稳定的黄绿叶突变体命名为ygl13，对突变体的表型进行系统观察，调查其成熟期的主要农艺性状，分别测定野生型和突变体苗期和孕穗期的叶片光合色素含量，同时利用透射电镜观察野生型和突变体ygl13的叶肉细胞及叶绿体结构。将表型正常的不育系西农1A与突变体ygl13杂交，根据F₁和F₂群体的性状表现与分离情况,分析该突变性状的遗传行为，并以F₂作为基因定位群体，对突变体ygl13进行候选基因遴选和突变位点测序验证。【结果】突变体ygl13的植株叶片在整个生育期均呈现黄绿色，与野生型缙恢10号相比，突变体ygl13苗期和孕穗期叶片叶绿素a、叶绿素b和类胡萝卜素含量均极显著降低。透射电镜观察结果显示，与野生型相比，突变体ygl13叶绿体结构异常，基质片层减少退化，类囊体片层减少，不规则的散乱分布。农艺性状调查结果表明，突变体ygl13穗总粒数增加了26.06%，株高和结实率分别降低了12.33%和18.82%，但穗长、有效穗、穗实粒数和千粒重无显著差异。F₂群体正常叶色的植株数与黄绿叶植株数分离比经χ²测验符合3﹕1分离比例（χ²=2.35＜χ²_0.05=3.84），表明ygl13的黄绿叶性状由1对隐性核基因控制。YGL13被定位于第8染色体短臂InDel标记ID43和ID69之间，遗传距离分别为4.0和0.5 cM，区间物理距离约为318 kb，共有52个基因。经测序比对分析发现，ygl13突变体在OsSIG1编码区的第1 005个碱基G突变为碱基A（位于第三外显子），造成编码色氨酸（Trp或W）的密码子突变为终止密码子，导致蛋白翻译提前终止，则该基因编码520个氨基酸的蛋白质突变为334个氨基酸的截短蛋白。qRT-PCR结果表明，突变体ygl13部分光合色素代谢途径和光系统相关基因表达紊乱。【结论】水稻突变体ygl13的黄绿叶性状由1对隐性核基因控制，该基因与已报道的水稻质体σ因子OsSIG1为等位基因。

关键词: 水稻（Oryza sativa L.）, 黄绿叶突变体, OsSIG1, 遗传分析, 候选基因分析

Abstract: 【Objective】The current study was conducted aiming at phenotypic characterization and candidate gene analysis of the yellow-green mutant ygl13, so as to add to our knowledge of the formation and regulation of the molecular mechanisms responsible for leaf-color mutations in rice.【Method】A new rice mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate (EMS)-treated restorer line Jinhui10 (Oryza sativa), tentatively named as yellow-green leaf 13 (ygl13). Morphological characteristics, the photosynthetic pigment contents and the agronomic traits were measured systematically. Transmission electron microscopy was conducted to analyze the ultrastructure of the mesophyll cells and chloroplasts in the ygl13 mutant and wide-type plants. The ygl13 was crossed with indica sterile line Xinong1A whose plant and leaves were normally green, and the morphological phenotype and segregation ratio of F₁ and F₂ were used for genetic analysis, F₂ for gene mapping, and putative genes in the fine mapped region were analyzed, and the candidate genes in the mutant and the wild type were sequenced, respectively.【Result】The ygl13 leaves displayed yellow-green compared with the wild type. And the photosynthetic pigment contents of chlorophyll a, chlorophyll b, and carotenoid decreased significantly at the seedling and the booting stages. The results from transmission electron microscope demonstrated that the structure of the chloroplast in the mutant ygl13 developed abnormally with poor thylakoids, less grana stacks and scattered distribution when compared with the wide type. According to the performance of agronomic traits, compared with the wild type Jinhui 10, the grain number per panicle increased by 26.06%, and the pant height and seed setting rate decreased by 12.33% and 18.82%. As for the panicle length, effective panicles per plant, filled grain number per panicle and 1000-grain weight, there was no significant difference between the wild type and ygl13. Genetic analysis demonstrated that the mutant trait was controlled by a single recessive gene as the number of green seedlings verses that of yellow-green seedlings approached 3:1（χ²=2.35＜χ²_0.05=3.84). Genetic mapping of the mutant gene was conducted using 602 recessive individuals from the F₂ segregation population. Finally, YGL13 was mapped on the short arm of chromosome 8 between InDel marker ID43 and ID69, and with an interval of 318 kb. There were 52 genes in this region and the sequencing analysis of these candidate genes between the mutant and its wild type revealed a single base change (G1005A) of the OsSIG1 gene (LOC_Os08g06630) in the encoded product resulted in a premature stop codon and protein truncation with 334 residues not the primary protein with 520 residues in the mutant ygl13. The qRT-PCR results showed that, the expression level of genes associated with pigment metabolism and photosynthesis is in disorder. 【Conclusion】 The mutant ygl13 was controlled by a single recessive gene. The gene YGL13 was allelic to OsSIG1 which was documented as a plastid sigma factor previously in rice.

Key words: rice (Oryza sativa L.), yellow-green leaf mutant, OsSIG1, genetic analysis, candidate gene analysis

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[J]. 中国农业科学, 2015, 48(21): 4197-4208.

WANG Ya-qin, SHI Jun-qiong, ZHANG Ting, LI Yan, ZHANG Tian-quan, ZHANG Xiao-long, SANG Xian-chun, LING Ying-hua, HE Guang-hua. Characterization and Candidate Gene Analysis of Yellow-Green Leaf Mutant ygl13 in Rice (Oryza sativa)[J]. Scientia Agricultura Sinica, 2015, 48(21): 4197-4208.

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参考文献

[1] Fromme P, Melkozernov A, Jordan P, Krauss N. Structure and function of photosystem: Ⅰ Interaction with its soluble electron carriers and external antenna systems. Febs Letters, 2003, 555: 40-44.

[2] Larkin R M, Alonso J M, Ecker J R, Chory J. GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science, 2003, 299: 902-906.

[3] Dong H, Fei G L, Wu C Y, Wu F Q, Sun Y Y, Chen M J, Ren Y L, Zhou K N, Cheng Z J, Wang J L, Jiang L, Zhang X, Guo X P, Lei C L, Su N, Wang H Y, Wan J M. A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants. The Plant Physiology, 2013, 162: 1867-1880.

[4] Yoo S C, Cho S H, Sugimoto H, Li J 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. The Plant Physiology, 2009, 150: 388-401.

[5] 李贤勇, 王楚桃, 李顺武, 何永歆, 陈世全. 一个水稻高叶绿素含量基因的发现. 西南农业学报, 2002, 15(4): 122-123.

Li X Y, Wang C T, Li S W, He Y X, Chen S Q. The discovery of a high chlorophyll content gene in rice. Southwest China Journal of Agricultural Science, 2002, 15(4): 122-123. (in Chinese)

[6] Wang F H, Wang G X, Li X Y, Huang J L, Zheng J K. Heredity, physiology and mapping of a chlorophyll content gene of rice (Oryza sativa L.). Journal of Plant Physiology, 2008, 165: 324-330.

[7] 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. The Plant Journal, 1997, 12(6): 1241-1250.

[8] 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.The Plant Journal, 2011, 68: 1039-1050.

[9] Goh C H, Satoh K, Kikuchi S, Kim S C, Ko S M, Kang H G, Jeon J S, Kim C S, Park Y I. Mitochondrial activity in illuminated leaves of chlorophyll-deficient mutant rice (OsCHLH) seedlings. Plant Biotechnology Reports, 2010, 4: 281-291.

[10] 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 Molecular Biology, 2005, 57: 805-818.

[11] Sugimoto H, Kusumi K, Tozawa Y, Yazaki J, Kishimoto N, Kikuchi S, Iba K. The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation. Plant Cell Physiology, 2004, 45(8): 985-996.

[12] 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. The Plant Journal, 2007, 52: 512-527.

[13] Zhang H T, Li J J, Yoo J H, Yoo S C, Cho S H, Koh H J, Seo H S, Paek N C. Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Molecular Biology, 2006, 62: 325-337.

[14] Wu Z M, Zhang X, He B, Diao L P, Sheng S L, Wang J L, Guo X P, Su N, Wang L F, Jiang L, Wang C M, Zhai H Q, Wan J M. A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. The Plant Physiology, 2007, 145: 29-40.

[15] Wang P R, Gao J X, Wan C M, Zhang F T, Xu Z J, Huang X Q, Sun X Q, Deng X J. Divinyl chlorophyll(ide) a can be converted to monovinyl chlorophyll(ide) a by a divinyl reductase in rice. The Plant Physiology, 2010, 153: 994-1003.

[16] 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. The Plant Journal, 2013, 74: 122-133.

[17] Koussevitzky S, Nott A, Mockler T C, Hong F X, Sachetto-Martins G, Surpin M, Lim J, Mittler R, Chory J. Signals from chloroplasts converge to regulate nuclear gene expression. Science, 2007, 316: 715-719.

[18] Terry M J, Kendrick R E. Feedback inhibition of chlorophyll synthesis in the phyochrome chromophore-deficient aurea and yellow-green-2 mutants of tomato.The Plant Physiology, 1999, 119(1): 143-152.

[19] Huq E, Al-Sady B, Hudson M, Kim C, Apel K, Quail P H. Phytochrome-interacting factor l is a critical bHLH regulator of chlorophyll biosynthesis. Science, 2004, 305: 1937-1942.

[20] Reinbothe S, Pollmann S, Springer A, James R J, Tichtinsky G, Reinbothe C. A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH: Protochlorophyllide oxidoteductase(POR) A⁺. The Plant Journal, 2005, 42: 1-12.

[21] Krushnir S, Babiychuk E, Storozhenko S, Davey M W, Papenbrock J, Rycke R D, Engler G, Stephan U W, Lange H, Kispal G, Lill R, Montagu M V. A mutation of the mitochondrial ABC transporter sta1 leads to dwarfism and chlorosis in the Arabidopsis mutant starik. The Plant Cell, 2001, 13: 89-100.

[22] Lichtenthaler H K. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods in Enzymology, 1987, 148: 350-382.

[23] 何瑞峰, 丁毅, 余金洪, 祖明生. 水稻温敏叶绿素突变体叶片超微结构的研究. 武汉植物学研究, 2001, 19(1): 1-5.

He R F, Ding Y, Yu J H, Zu M S. Study on leaf ultrastructure of the thermo-sensitive chlorophyll deficient mutant in rice. Journal of Wuhan Botanical Research, 2001, 19(1): 1-5. (in Chinese)

[24] Michelmore R W, Paran I, Kesseli R V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88: 9828-9832.

[25] Panaud O, Chen X, McCouch S R. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L.). Molecular and General Genetics, 1996, 252: 597-607.

[26] 桑贤春, 何光华, 张毅, 杨正林, 裴炎. 水稻PCR扩增模板的快速制备. 遗传, 2003, 25(6): 705-707.

Sang X C, He G H, Zhang Y, Yang Z L, Pei Y. The simple gain of templates of rice genomes DNA for PCR. Hereditas, 2003, 25(6): 705-707. (in Chinese)

[27] Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8: 4321-4325.

[28] Gothandarn K M, Kim E S, Cho H, Chung Y Y. OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis. Plant Molecular Biology, 2005, 58(3): 421-433.

[29] Kusumi K, Yara A, Mitsui N, Tozawa Y, Iba K. Characterization of a rice nuclear-encoded plastid RNA polymerase gene OsRpoTp. Plant Cell Physiology, 2004, 45(9): 1194-1201.

[30] Park S Y, Yu J W, Park J S, Li J, Yoo S C, Lee N Y, Lee S K, Jeong S W, Seo H S, Koh H J, Jeon J S, Park Y I, Paek N C. The senescence- induced staygreen protein regulates chloropgyll degradation. The Plant Cell, 2007, 19(5): 1649-1664.

[31] Kusaba M, Ito H, Morita R, Iida S, Sato Y, Fujimoto M, Kawasaki S, Tanaka R, Hirochika H, Nishimura M, Tanaka A. Rice NON- YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence. The Plant Cell, 2007, 19(4): 1362-1375.

[32] 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. The Plant Journal, 2009, 57(1): 120-131.

[33] 李燕群, 高家旭, 肖云华, 李秀兰, 蒲翔, 孙昌辉, 王平荣, 邓晓建. 水稻ygl80黄绿叶突变体的遗传分析与目标基因精细定位. 作物学报, 2014, 40(4): 644-649.

Li Y Q, Gao J X, Xiao Y H, Li X L, Pu X, Sun C H, Wang P R, Deng X J. Genetic analysis and gene fine mapping of yellow-green leaf mutant ygl80 in rice. Acta Agronomic Sinica(in Chinese), 2014,40(4): 644-649.

[34] 孙小秋, 王兵, 肖云华, 万春美, 邓晓建, 王平荣. 水稻ygl98黄绿叶突变基因的精细定位与遗传分析. 作物学报, 2011, 37(6): 991-997.

Sun X Q, Wang B, Xiao Y H, Wan C M, Deng X J, Wang P R. Genetic analysis and fine-mapping of ygl98 yellow-green leaf gene in rice. Acta Agronomic Sinica, 2011, 37(6): 991-997. (in Chinese)

[35] 吴书俊, 杨杰, 闫影, 张丽霞, 范方军, 朱金燕, 李文奇, 仲维功, 曹黎明, 王军. 水稻黄绿叶突变体ygl11(t)的生理特性和基因克隆. 中国水稻科学, 2015, 29(2): 111-118.

Wu S J, Yang J, Yan Y, Zhang L X, Fan F J, Zhu J Y, Li W Q, Zhong W G, Cao L M, Wang J. Physiological characterization and gene identification of a yellow green leaf mutant ygl11(t) in rice. Chinese Journal of Rice Science, 2015, 29(2): 111-118. (in Chinese)

[36] Chen H, Cheng Z J, Ma X D, Wu H, Liu Y L, Zhou K N, Chen Y L, Ma W W, Bi J C, Zhang X, Guo X P, Wang J L, Lei C L, Wu F Q, Lin Q B, Liu Y Q, Liu L L, Jiang L. A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Reports, 2013, 32: 1855-1867.

[37] 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.

Lü 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. Acta Agronomic Sinica, 2009, 35(12): 2304 -2308. (in Chinese)

[38] Ishizaki Y, Tsunoyama Y, Hatano K, Ando K, Kato K, Shinmyo A, Kobori M, Takeba G, Nakahira Y, Shiina T. A nuclear-encoded sigma factor, Arabidopsis SIG6, recognizes sigma-70 type chloroplast promoters and regulates early chloroplast development in cotyledons. The Plant Journal, 2005, 42(2): 133-144.

[39] Tozawa Y, Teraishi M, Sasaki T, Sonoike K, Nishiyama Y, Itaya M, Miyao A, Hirochika H. The plastid sigma factor SIG1maintains photosystem I activity via regulated expression of the psaA operon in rice chloroplasts. The Plant Journal, 2007, 52(1): 124-132.

[40] 李小林, 邓安凤, 徐雨然, 邓君浪, 谷安宇, 年伟, 史胜利, 吴殿星, 董阳军, 李健强, 张锦文. 水稻叶缘白化叶色标记三系不育系云丰88 A的选育. 种子, 2011, 12(30): 109-111.

Li X L, Deng A F, Xu Y R, Deng J L, Gu A Y, Nian W, Shi S L, Wu D X, Dong Y J, Li J Q, Zhang J W. Breeding of rice leaf margin albino leaf-color marker three sterile line Yunfeng 88A. Seed, 2011, 12(30): 109-111. (in Chinese)