研究报告

水稻黄叶转绿突变体818-6-8的目标基因定位与转录组分析

展开
  • 1 农业农村部淮河下游种质创制重点实验室/江苏省农业科学院 粮食作物研究所/生物育种钟山实验室南京 210014
    2 扬州大学 江苏省粮食作物现代化产业协同创新中心江苏 扬州 225009
    3 江苏大学 生命科学学院江苏 镇江 212013

收稿日期: 2024-11-08

  修回日期: 2024-12-27

  网络出版日期: 2026-05-13

基金资助

江苏省重点研发计划资助项目(BE2022383);生物育种钟山实验室项目(ZSBBL-KY2023-04-1)

Gene Mapping and Transcriptome Analysis of a Green-revertible Yellow Leaf Mutant 818-6-8 in Rice

Expand
  • 1 Key Laboratory of Germplasm Innovation in Downstream of Huaihe River, Ministry of Agriculture and Rural Affairs/Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Zhongshan Biological Breeding Laboratory, Nanjing 210014, China
    2 Jiangsu Co-innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China
    3 Institute of Life Science, Jiangsu University, Zhenjiang 212013, China

Received date: 2024-11-08

  Revised date: 2024-12-27

  Online published: 2026-05-13

摘要

【目的】叶片是水稻最重要的光合作用器官。筛选和鉴定水稻叶色突变体,有利于探究叶绿体发育与光合作用的分子调控机制。【方法】在金粳818背景的基因编辑后代中获得一个黄叶转绿突变体818-6-8,并对其进行表型鉴定、遗传分析、基因定位和转录组分析。【结果】与野生型相比,突变体三叶期前叶片明显黄化,并伴随叶绿素含量显著降低;第4叶开始,突变体开始转绿并逐渐表现为与野生型无异。突变体中叶绿体数量较少、类囊体形态和基粒片层结构异常。818-6-8黄叶性状受1对隐性核基因控制。突变位点被定位于5号染色体2.29 Mb的区间内,分析并筛选得到20个候选基因。突变体中2090个基因表达量发生显著改变,其中1343个上调,747个下调;差异表达基因显著富集在叶绿体组织等生物学过程、叶绿体等细胞组分。突变体中多个叶绿素合成和叶绿体发育相关基因表达发生改变。【结论】本研究完成了突变体818-6-8的目标基因定位与转录组分析,为下一步目标基因克隆和叶绿体发育分子调控机理解析提供支持,为景观稻育种提供优异种质资源。

本文引用格式

许扬, 王芳权, 李文奇, 陶亚军, 范方军, 陈智慧, 蒋彦婕, 朱建平, 李霞, 杨杰 . 水稻黄叶转绿突变体818-6-8的目标基因定位与转录组分析[J]. 中国水稻科学, 2026 , 40(3) : 327 -340 . DOI: 10.16819/j.1001-7216.2026.241105

Abstract

【Objective】Leaf is the most important photosynthetic organ in rice. Screening and identification of rice leaf color mutants are beneficial for exploring the molecular regulatory mechanisms of chloroplast development and photosynthesis. 【Method】A green-revertible yellow leaf mutant 818-6-8 was obtained from the gene-editing lines of Jingeng 818. Phenotypic identification, genetic analysis, gene mapping, and transcriptome analysis for 818-6-8 were performed. 【Results】Compared with the wild type, the mutant showed an obvious yellow-leaf phenotype until the three-leaf stage, accompanied by a significant decrease in chlorophyll content. As it grew, the mutant turned green and gradually became indistinguishable from the wild type at the four-leaf stage. Fewer chloroplasts, abnormal thylakoid morphology and stromal lamellas structure were observed in the mutant. The yellow-leaf trait of 818-6-8 was controlled by a pair of recessive nuclear alleles. The target mutation site was located within a 2.29 Mb interval on chromosome 5, and 20 annotated candidate genes were listed. The expression levels of 2090 genes in the mutant were significantly changed compared to the wild type, of which 1343 genes were up-regulated and 747 genes were down-regulated. Further, the differentially expressed genes were enriched in biological processes such as chloroplast organization and cellular components such as chloroplasts. The expression of genes associated with chlorophyll biosynthesis and chloroplast development of 818-6-8 changed significantly. 【Conclusion】These results support the next studies for cloning target genes and analyzing chloroplast-development molecular mechanisms, and provide a germplasm resource for ornamental rice breeding.

参考文献

[1] 徐娜, 徐江民, 蒋玲欢, 饶玉春. 水稻叶片早衰成因及分子机理研究进展[J]. 植物学报, 2017, 52(1): 102-112.
  Xu N, Xu J M, Jiang L H, Rao Y C. Advances in understanding leaf premature senescence and its molecular mechanism in rice[J]. Chinese Bulletin of Botany, 2017, 52(1): 102-112. (in Chinese with English abstract)
[2] 周亭亭, 饶玉春, 任德勇. 水稻卷叶细胞学与分子机制研究进展[J]. 植物学报, 2018, 53(6): 848-855.
  Zhou T T, Rao Y C, Ren D Y. Research advances in the cytological and molecular mechanisms of leaf rolling in rice[J]. Bulletin of Botany, 2018, 53(6): 848-855. (in Chinese)
[3] 张萍, 柳梦林, 叶胜海, 翟荣荣, 朱国富, 叶靖, 张小明. 水稻叶色突变体研究进展[J]. 分子植物育种, 2021, 19(17): 5712-5719.
  Zhang P, Liu M L, Ye S H, Zhai R R, Zhu G F, Ye J, Zhang X M. Research progress of rice leaf color mutants[J]. Molecular Plant Breeding, 2021, 19(17): 5712-5719. (in Chinese with English abstract)
[4] 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[J]. Plant Physiology, 2012, 159(1): 227-238.
[5] 戴红燕, 华劲松. 对观赏稻的认识和思考[J]. 作物杂志, 2020(4): 1-8.
  Dai H Y, Hua J S. Understanding and thinking about ornamental rice[J]. Crops, 2020(4): 1-8. (in Chinese with English abstract)
[6] 杨颜榕, 黄纤纤, 赵亚男, 汤佳玉, 刘喜. 水稻叶色基因克隆与分子机制研究进展[J]. 植物遗传资源学报, 2020, 21(4): 794-803.
  Yang Y R, Huang X X, Zhao Y N, Tang J Y, Liu X. Advances on gene isolation and molecular mechanism of rice leaf color genes[J]. Journal of Plant Genetic Resources, 2020, 21(4): 794-803. (in Chinese)
[7] 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]. Journal of Experimental Botany, 2016, 67(5): 1297-1310.
[8] 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[J]. The Plant Journal, 2013, 74(1): 122-133.
[9] 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[J]. Plant Physiology, 2010, 153(3): 994-1003.
[10] Wu Z, 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[J]. Plant Physiology, 2007, 145(1): 29-40.
[11] Zhang H, Li 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[J]. Plant Molecular Biology, 2006, 62(3): 325-337.
[12] Lee S, Kim J H, Yoo E S, Lee C H, Hirochika H, An G. Differential regulation of chlorophyll a oxygenase genes in rice[J]. Plant Molecular Biology, 2005, 57(6): 805-818.
[13] Webber A N, Malkin R. Photosystem I reaction-centre proteins contain leucine zipper motifs: A proposed role in dimer formation[J]. FEBS Letters, 1990, 264(1): 1-4.
[14] Li Z, Mo W, Jia L, Xu Y C, Tang W, Yang W, Guo Y L, Lin R. Rice FLUORESCENT1 is involved in the regulation of chlorophyll[J]. Plant & Cell Physiology, 2019, 60(10): 2307-2318.
[15] Sakuraba Y, Kim E Y, Han S H, Piao W, An G, Todaka D, Yamaguchi-Shinozaki K, Paek N C. Rice Phytochrome-Interacting Factor-Like1 (OsPIL1) is involved in the promotion of chlorophyll biosynthesis through feed-forward regulatory loops[J]. Journal of Experimental Botany, 2017, 68(15): 4103-4114.
[16] Andersson I, Backlund A. Structure and function of rubisco[J]. Plant Physiology and Biochemistry, 2008, 46(3): 275-291.
[17] 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, Wan J M. A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants[J]. Plant Physiology, 2013, 162(4): 1867-1880.
[18] Peng L, Yamamoto H, Shikanai T. Structure and biogenesis of the chloroplast NAD(P)H dehydrogenase complex[J]. Biochimica et Biophysica Acta, 2011, 1807(8): 945-953.
[19] Zhu X, Guo S, Wang Z, Du Q, Xing Y, Zhang T, Shen W, Sang X, Ling Y, He G. Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice (Oryza sativa)[J]. BMC Plant Biology, 2016, 16(1): 134.
[20] Zhu X, Mou C, Zhang F, Huang Y, Yang C, Ji J, Liu X, Cao P, Nguyen T, Lan J, Zhou C, Liu S, Jiang L, Wan J. WSL9 encodes an HNH endonuclease domain-containing protein that is essential for early chloroplast development in rice[J]. Rice, 2020, 13(1): 45.
[21] Zhou K, Zhang C, Xia J, Yun P, Wang Y, Ma T, Li Z. Albino seedling lethality 4; chloroplast 30S ribosomal protein S1 is required for chloroplast ribosome biogenesis and early chloroplast development in rice[J]. Rice, 2021, 14(1): 47.
[22] Yin C C, Ma B, Collinge D P, Pogson B J, He S J, Xiong Q, Duan K X, Chen H, Yang C, Lu X, Wang Y Q, Zhang W K, Chu C C, Sun X H, Fang S, Chu J F, Lu T G, Chen S Y, Zhang J S. Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase-mediated abscisic acid pathway[J]. The Plant Cell, 2015, 27(4): 1061-1081.
[23] Zheng H, Wang Z, Tian Y, Liu L, Lv F, Kong W, Bai W, Wang P, Wang C, Yu X, Liu X, Jiang L, Zhao Z, Wan J. Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice[J]. Plant Physiology and Biochemistry, 2019, 139: 495-503.
[24] Fang G, Yang S, Ruan B, Liu C, Zhang A, Jiang H, Ding S, Tian B, Zhang Y, Jahan N, Zhu L, Zhang G, Dong G, Zhang Q, Zeng D, Guo L, Gao Z, Qian Q. Isolation of TSCD11 gene for early chloroplast development under high temperature in rice[J]. Rice, 2020, 13(1): 49.
[25] Xu Y, Lin Q, Li X, Wang F, Chen Z, Wang J, Li W, Fan F, Tao Y, Jiang Y, Wei X, Zhang R, Zhu Q H, Bu Q, Yang J, Gao C. Fine-tuning the amylose content of rice by precise base editing of the Wx gene[J]. Plant Biotechnology Journal, 2021, 19(1): 11-13.
[26] Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. The sequence alignment/map format and SAMtools[J]. Bioinformatics, 2009, 25(16): 2078-2079.
[27] McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo M A. The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data[J]. Genome Research, 2010, 20(9): 1297-1303.
[28] Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, Sharma S, Sharma S, Kanzaki H, Matsumura H, Saitoh H, Mitsuoka C, Utsushi H, Uemura A, Kanzaki E, Kosugi S, Yoshida K, Cano L, Kamoun S, Terauchi R. MutMap+: Genetic mapping and mutant identification without crossing in rice[J]. PLoS One, 2013, 8(7): e68529.
[29] Takagi H, Uemura A, Yaegashi H, Tamiru M, Abe A, Mitsuoka C, Utsushi H, Natsume S, Kanzaki H, Matsumura H, Saitoh H, Yoshida K, Cano L M, Kamoun S, Terauchi R. MutMap-Gap: Whole-genome resequencing of mutant F2 progeny bulk combined with de novo assembly of gap regions identifies the rice blast resistance gene Pii[J]. The New Phytologist, 2013, 200(1): 276-283.
[30] Trapnell C, Williams B A, Pertea G, Mortazavi A, Kwan G, van Baren M J, Salzberg S L, Wold B J, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J]. Nature Biotechnology, 2010, 28(5): 511-515.
[31] Love M I, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biology, 2014, 15(12): 550.
[32] Moriya Y, Itoh M, Okuda S, Yoshizawa A C, Kanehisa M. KAAS: An automatic genome annotation and pathway reconstruction server[J]. Nucleic Acids Research, 2007, 35: W182-W185.
[33] Wong J L, Leydon A R, Johnson M A. HAP2(GCS1)-dependent gamete fusion requires a positively charged carboxy-terminal domain[J]. PLoS Genetics, 2010, 6(3): e1000882.
[34] Zhang F, Ma L, Zhang C, Du G, Shen Y, Tang D, Li Y, Yu H, Ma B, Cheng Z. The SUN domain proteins OsSUN1 and OsSUN2 play critical but partially redundant roles in meiosis[J]. Plant Physiology, 2020, 183(4): 1517-1530.
[35] Jadoon S, Qin Q, Shi W, Yan L, Hou S. Rice protein phosphatase 1 regulatory subunits OsINH2 and OsINH3 participate actively in growth and adaptive responses under abscisic acid[J]. Frontiers in Plant Science, 2022, 13: 990575.
[36] Kapoor M, Arora R, Lama T, Nijhawan A, Khurana J P, Tyagi A K, Kapoor S. Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice[J]. BMC Genomics, 2008, 9: 451.
[37] Zheng P, Liu Y, Liu X, Huang Y, Sun F, Wang W, Chen H, Jan M, Zhang C, Yuan Y, Tan B C, Du H, Tu J. OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice[J]. Theoretical and Applied Genetics, 2021, 134(3): 923-940.
[38] Qiu S, Ma N, Che S, Wang Y, Peng X, Zhang G, Wang G, Huang J. Repression of OsEXPA3 expression leads to root system growth suppression in rice[J]. Crop Science, 2014, 54(5): 2201-2213.
[39] Smith R A, Beebe E T, Bingman C A, Vander Meulen K, Eugene A, Steiner A J, Karlen S D, Ralph J, Fox B G. Identification and characterization of a set of monocot BAHD monolignol transferases[J]. Plant Physiology, 2022, 189(1): 37-48.
[40] You Q, Zhai K, Yang D, Yang W, Wu J, Liu J, Pan W, Wang J, Zhu X, Jian Y, Liu J, Zhang Y, Deng Y, Li Q, Lou Y, Xie Q, He Z. An E3 ubiquitin ligase-BAG protein module controls plant innate immunity and broad-spectrum disease resistance[J]. Cell Host & Microbe, 2016, 20(6): 758-769.
[41] Youssefian S, Nakamura M, Sano H. Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice[J]. Molecular & General Genetics, 1993, 237(1/2): 187-192.
[42] Kim Y J, Kim M H, Hong W J, Moon S, Kim S T, Park S K, Jung K H. OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice[J]. The Plant Journal, 2021, 107(4): 1131-1147.
[43] Kang Z, Qin T, Zhao Z. Overexpression of the zinc finger protein gene OsZFP350 improves root development by increasing resistance to abiotic stress in rice[J]. Acta Biochimica Polonica, 2019, 66(2): 183-190.
[44] Wang Y, Wang C, Zheng M, Lyu J, Xu Y, Li X, Niu M, Long W, Wang D, Wang H, Terzaghi W, Wang Y, Wan J. WHITE PANICLE1, a val-tRNA synthetase regulating chloroplast ribosome biogenesis in rice, is essential for early chloroplast development[J]. Plant Physiology, 2016, 170(4): 2110-2123.
[45] Yang M, Sakruaba Y, Ishikawa T, Ohtsuki N, Kawai-Yamada M, Yanagisawa S. Chloroplastic Sec14-like proteins modulate growth and phosphate deficiency responses in Arabidopsis and rice[J]. Plant Physiology, 2023, 192(4): 3030-3048.
[46] Wang Q, Chen J, Wang X, Sun J, Sha W. Molecular cloning and expression analysis of the rice triose phosphate/phosphate translocator gene[J]. Plant Science, 2002, 162(5): 785-790.
[47] Fan X, Wu J, Chen T, Tie W, Chen H, Zhou F, Lin Y. Loss-of-function mutation of rice SLAC7 decreases chloroplast stability and induces a photoprotection mechanism in rice[J]. Journal of Integrative Plant Biology, 2015, 57(12): 1063-1077.
[48] Kusumi K, Hirotsuka S, Kumamaru T, Iba K. Increased leaf photosynthesis caused by elevated stomatal conductance in a rice mutant deficient in SLAC1, a guard cell anion channel protein[J]. Journal of Experimental Botany, 2012, 63(15): 5635-5644.
[49] Zhang Y, Chen X, Du D, Ma L, He G. Identification and cloning of early senescence leaf mutant esl13 in rice (Oryza sativa L.)[J]. Crop Science, 2023, 63(3): 1102-1113.
[50] Itoh J I, Kitano H, Matsuoka M, Nagato Y. SHOOT ORGANIZATION genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice[J]. The Plant Cell, 2000, 12(11): 2161.
[51] Nagasaki H, Itoh J, Hayashi K, Hibara K, Satoh-Nagasawa N, Nosaka M, Mukouhata M, Ashikari M, Kitano H, Matsuoka M, Nagato Y, Sato Y. The small interfering RNA production pathway is required for shoot meristem initiation in rice[J]. Proceedings of the National Academy of Sciences, 2007, 104(37): 14867-14871.
[52] Song X, Li P, Zhai J, Zhou M, Ma L, Liu B, Jeong D, Nakano M, Cao S, Liu C, Chu C, Wang X, Green P J, Meyers B C, Cao X. Roles of DCL4 and DCL3b in rice phased small RNA biogenesis[J]. The Plant Journal, 2012, 69(3): 462-474.
[53] Wei L, Gu L, Song X, Cui X, Lu Z, Zhou M, Wang L, Hu F, Zhai J, Meyers B C, Cao X. Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(10): 3877-3882.
[54] Wang Y, Ren Y, Zhou K, Liu L, Wang J, Xu Y, Zhang H, Zhang L, Feng Z, Wang L, Ma W, Wang Y, Guo X, Zhang X, Lei C, Cheng Z, Wan J. WHITE STRIPE LEAF4 encodes a novel P-type PPR protein required for chloroplast biogenesis during early leaf development[J]. Frontiers in Plant Science, 2017, 8: 1116.
[55] Yamamoto K, Shida S, Honda Y, Shono M, Miyake H, Oguri S, Sakamoto H, Momonoki Y S. Overexpression of acetylcholinesterase gene in rice results in enhancement of shoot gravitropism[J]. Biochemical and Biophysical Research Communications, 2015, 465(3): 488-493.
[56] Ribeiro C W, Korbes A P, Garighan J A, Jardim-Messeder D, Carvalho F E L, Sousa R H V, Caverzan A, Teixeira F K, Silveira J A G, Margis-Pinheiro M. Rice peroxisomal ascorbate peroxidase knockdown affects ROS signaling and triggers early leaf senescence[J]. Plant Science, 2017, 263: 55-65.
文章导航

/

浙ICP备05004719号-5
公安备案号:33010302003356
地址:浙江省杭州市富阳区水稻所路28号 邮编:311400 电话:0571-63370278 E-mail:cjrs@263.net
本系统由北京玛格泰克科技发展有限公司设计开发
总访问量: 今日访问: 在线人数: