一个水稻早衰突变体基因的精细定位

doi:10.3864/j.issn.0578-1752.2014.11.001

摘要/Abstract

摘要： 【目的】对水稻叶片早衰突变体W330进行遗传分析及精细定位，获得控制突变表型的基因。【方法】用60Co-γ辐射诱变籼型水稻恢复系中恢8015，从突变体库获得一份叶片早衰突变体W330。对该突变体进行表型观察和主要农艺性状调查。利用多代自交稳定的突变体W330与粳稻品种02428杂交，观察F1和 F2的表型，并统计F2群体中早衰突变表型与正常野生型的分离情况，分析该突变表型的遗传行为。利用构建的F2群体进行精细定位和候选基因分析，然后对候选基因进行DNA测序、酶切分析、表达分析、酶活测定及进化分析。【结果】突变体W330从三叶期开始出现叶片衰老，直至抽穗期及黄熟期。与野生型相比，突变体W330株高变矮、分蘖减少、叶片变窄、抽穗期不变、每株有效穗数、每穗着粒数和结实率亦显著降低。W330与02418杂交的F1表现正常，F2群体中正常植株与早衰突变植株的分离比符合3﹕1，表明突变体W330的突变性状受1对隐性核基因控制。利用F2定位群体及SSR、Indel标记，最终将目标基因定位在第3染色体短臂上2个分子标记CD-5与CD-7之间，物理距离约为21.5 kb。基因预测表明该区域共有4个完整的ORFs。其中，LOC_Os03g0131200编码一个过氧化氢酶OsCATC，基因组序列分析表明，W330突变体中的该基因从ATG开始第109位，在第一个内含子的末位发生了一个C到G的颠换，造成第一个内含子没有剪切，最终导致翻译提前终止，酶切试验验证了这一突变位点。与野生型亲本中恢8015相比，W330突变体在三叶期叶片中的过氧化氢酶的活力下降了47.8%，而过氧化氢含量上升了2.7倍。由此，推定W330与OsCATC等位。系统进化分析发现，OsCATC与水稻中同源的过氧化氢酶不在同一进化分支上。实时荧光定量PCR发现，与其野生型相比，突变体W330叶片中的OsCATA和OsCATB的表达量显著上升，而OsCATC的表达量没有明显的变化。推测，这3个高度同源的基因在水稻体内可能存在互补机制。【结论】W330突变体基因是已报道的过氧化氢酶基因OsCATC的等位基因。W330突变体在第一个内含子上发生的一个点突变造成可变剪切的发生，使得水稻一个过氧化氢酶失活，导致突变体W330突变表型的出现。

关键词: 水稻 , 早衰 , 精细定位 , 可变剪切

Abstract: 【Objective】The objective of this study is to conduct genetic analysis and gene mapping for the rice early leaf senescence mutant W330.【Method】An early leaf senescence mutant, designated as W330, was isolated from the progeny of 60Co-γ-treated indica rice cv. Zh8015. Phenotypes of the W330 mutant were observed and its main agronomic traits were analyzed under field conditions in Hangzhou and Sanya. After the W330 mutant was crossed with 02428, leaf phenotypes of the F1 progenies，the segregation ratio of normal and early senescence plants in the F2 populations were investigated. An F2 population derived from the cross of cv. 02428 with W330 was used for genetic analysis and gene fine mapping. Candidate gene assay was conducted by gene sequencing, enzyme digestion, gene expression and phylogenetic analysis.【Result】W330 plants showed leaf senescence phenotype from 3-leaf-stage to maturity stage. Compared with wild-type parent Zhonghui8015, the plant height，tiller and blade width of W330 decreased significantly, and the number of productive panicles per plant, the number of spikelets per panicle and seed setting rate were also significantly reduced. All F1 plants generated by crossing early senescence mutant W330 with 02428 showed normal leaf. The segregation ratios of normal plants and early senescence plants in two F2 populations were both 3:1, indicating that the W330 was controlled by a single recessive nuclear gene which was mapped on the short arm of chromosome 3. With developed SSR and Indel markers, the gene was finally narrowed to an interval of 21.5 kb between markers CD-5 and CD-7. Among four ORFs in this region, the LOC_Os03g0131200 encoding a Catalase (OsCATC) was probably the candidate gene. The sequencing analysis and enzyme digestion revealed the variable splicing in the first intron was probably responsible for the early senescence phenotype, which was confirmed by assay of gene expression and enzyme activity. Compared with wild-type parent Zhonghui8015，the content of catalase activity decreased by 47.8% and the content of H2O2 increased by 2.7 times. It was speculated that the W330 mutant gene was allelic to OsCATC gene. Otherwise, phylogenetic analysis revealed that OsCATC was an independent evolution in rice. Expression analysis of catalase genes by real-time PCR showed that OsCATA and OsCATB have a significant increase in the expression of leaves, while the expression of OsCATC has no significant changes. It was a speculated that the three highly homologous genes have complementary mechanisms in rice plants. 【Conclusion】 The W330 mutant gene was allelic to OsCATC gene. A point mutation in introns of OsCATC gene in the W330 mutant makes catalase inactive, resulting in the leaf senescence phenotype.

Key words: Oryza sativa L. , early senescence , fine mapping , variable splicing

赵春德1, 2, 张迎信1, 2, 刘群恩1, 2, 余宁1, 2, 程式华1, 2, 曹立勇1, 2. 一个水稻早衰突变体基因的精细定位[J]. 中国农业科学, 2014, 47(11): 2069-2077.

ZHAO Chun-De-1, 2 , ZHANG Ying-Xin-1, 2 , LIU Qun-恩1, 2 , YU Ning-1, 2 , CHENG Shi-Hua-1, 2 , CAO Li-Yong-1, 2 . Fine Mapping of an Early Senescence Gene in Rice[J]. Scientia Agricultura Sinica, 2014, 47(11): 2069-2077.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.11.001

https://www.chinaagrisci.com/CN/Y2014/V47/I11/2069

参考文献

[1]梁建生, 曹显祖. 杂交水稻叶片的若干生理指标与根系伤流强度关系. 江西农学院学报, 1993, 14(4): 25-30.

Liang J S, Cao X Z, Studies on the relationship between several physiological characteristics of leaf and bleeding rate of roots in hybrid rice (O. sativa L.). Journal of Jiangsu Agricultural College, 1993, 14(4): 25-30. (in Chinese)

[2]张宝来. 水稻叶片衰老的研究进展. 天津农业科学, 2013, 19(4): 19-24.

Zhang B L. Advances of research on leaf senescence in rice. Tianjin Agricultural Sciences, 2013, 19(4): 19-24. (in Chinese)

[3]吴伟明, 王一平, 赵航, 曹立勇, 占小登, 程式华. 水稻不定根的穿鞘生长现象及其与叶片衰老的关系. 中国农业科学, 2005, 38(3): 474-479.

Wu W M, Wang Y P, Zhao H, Cao L Y, Zhan X D, Cheng S H. Growth phenomenon of adventitious root penetrating through the base of sheath and its effects on leaf senescence in rice. Scientia Agricultura Sinica, 2005, 38(3): 474-479. (in Chinese)

[4]郑建敏, 张涛, 郑家奎. 水稻叶片衰老相关基因的研究进展. 基因组学与应用生物学, 2009, 28(5): 1010-1019.

Zheng J M, Zhang T, Zheng J K. Research advances on the genes related to rice leaf senescence. Genomics and Applied Biology, 2009, 28(5): 1010-1019. (in Chinese)

[5]杜青, 方立魁, 桑贤春, 凌英华, 李云峰, 杨正林, 何光华, 赵芳明. 水稻叶尖早衰突变体lad的形态、生理分析与基因定位. 作物学报, 2012, 38(1): 168-173.

Du Q, Fang L K, Sang X C, Ling Y H, Li Y F, Yang Z L, He G H, Zhao F M. Analysis of phenotype and physiology of leaf apex dead mutant (lad) in rice and mapping of mutant gene. Acta Agronomica Sinica, 2012, 38(1): 168-173. (in Chinese)

[6]Doelling J H, Walker J M, Friedman E M, Thompson A R, Vierstra R D. The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. Journal of Biological Chemistry, 2002, 277(36): 33105-33114.

[7]Hanaka H, Takashi N, Yumiko S, Tomohiko K, Hiroaki H, Daisuke S, Satoshi T, Yoshinori O. Leaf senescence and starva-tion-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiology, 2002, 129: 1181-1193.

[8]Yoshida S, Ito M, Nishida I, Ikuo N, Akira W. Identification of a novel gene HYS1/CPR5 that has a repressive role in the induction of leaf senescence and pathogen-defence responses in Arabidopsis thaliana. The Plant Journal, 2002, 29(4): 427-437.

[9]Jing H C, Sturre M J G, Hille J, Dijkwel P P. Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence. The Plant Journal, 2002, 32(1): 51-63.

[10]Grbi? V, Bleecker A B. Ethylene regulates the timing of leaf senescence in Arabidopsis. The Plant Journal, 1995, 8(4): 595-602.

[11]Oh S A, Park J H, Lee G I, Paek K H, Park S K, Nam H G. Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana. The Plant Journal, 1997, 12(3): 527-535.

[12]Woo H R, Goh C H, Park J H, de la Serve B T, Kim J H, Park Y I, Nam H G. Extended leaf longevity in the ore4-1 mutant of Arabidopsis with a reduced expression of a plastid ribosomal protein gene. The Plant Journal, 2002, 31(3): 331-340.

[13]Woo H R, Chung K M, Park J H, Oh S A, Hong S H, Jang S K, Nam H G. ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. The Plant Cell Online, 2001, 13(8): 1779-1790.

[14]Ansari M I, Lee R H, Chen S C G. A novel senescence-associated gene encoding γ-aminobutyric acid (GABA): Pyruvate transaminase is upregulated during rice leaf senescence. Physiologia Plantarum, 2005, 123(1): 1-8.

[15]Lee R H, Lin M C, Chen S C G. A novel alkaline α-galactosidase gene is involved in rice leaf senescence. Plant Molecular Biology, 2004, 55(2): 281-295.

[16]Cha K W, Lee Y J, Koh H J, Lee B M, Nam Y W, Paek N C. Isolation, characterization, and mapping of the stay green mutant in rice. Theoretical and Applied Genetics, 2002, 104(4): 526-532.

[17]Jiang H W, Li M R, Liang N T, Yan H B, Wei Y B, Xu X L, Liu J, Xu Z F, Chen F, Wu G J. Molecular cloning and function analysis of the stay green gene in rice. The Plant Journal, 2007, 52(2): 197-209.

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

[19]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 Online, 2007, 19(4): 1362-1375.

[20]Kong Z, Li M N, Yang W Q, Xu W Y, Xue Y B. A novel nuclear- localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant Physiology, 2006, 141(4): 1376-1388.

[21]Wu Z M, Zhang X, He B, 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. Plant Physiology, 2007, 145(1): 29-40.

[22]Qiao Y L, Jiang W Z, Lee J H, Park M S, Piao R H, Woo M O, Roh J H, Han L Z, Paek N C, Seo H S, Koh H J. SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit μ1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa). New Phytologist, 2010, 185(1): 258-274.

[23]Wang J, Wu S, Zhou Y, Zhou L H, Xu J F, Jing H, Fang Y X, Gu M H, Liang G H. Genetic analysis and molecular mapping of a presenescing leaf gene psl1 in rice (Oryza sativa L.). Chinese Science Bulletin, 2006, 51(24): 2986-2992.

[24]Li F Z, Hu G C, Fu Y P, Bai X M, Sun Z X. Genetic analysis and high-resolution mapping of a premature senescence gene Pse(t) in rice (Oryza sativa L.). Genome, 2005, 48(4): 738-746.

[25]Yan W Y, Ye S H, Jin Q S, Zeng L J, Peng Y, Yan D W, Yang W B, Yang D l, He Z H, Dong Y J, Zhang X M. Characterization and mapping of a novel mutant sms1 (senescence and male sterility 1) in rice. Genet Genomics, 2010, 37: 47-55

[26]Fang L K, Li Y F, Gong X P, Sang X C, Ling Y H, Wang X W, Gong Y F, He G H. Genetic analysis and gene mapping of a dominant presenescing leaf gene PSL3 in rice (Oryza sativa L.). Chinese Science Bulletin, 2010, 55(23): 2517-2521.

[27]Yang Y L, Rao Y C, Liu H J, Fang Y X, Dong G J, Huang L C, Leng Y J, Guo L B, Zhang G H, Hu J, Gao Z Y, Qian Q, Zeng D L. Characterization and fine mapping of an early senescence mutant (es-t) in Oryza sativa L.. Chinese Science Bulletin, 2011, 56(23): 2437-2443.

[28]苗润隆, 蒋钰东, 廖红香, 徐芳芳, 何光华, 杨正林, 赵芳明, 桑贤春. 水稻早衰突变提esl3的鉴定与基因定位. 作物学报, 2013, 39(5): 862-867.

Miao R L, Jiang Y D, Liao H X, Xu F F, He G H, Yang Z L, Zhao F M, Sang X C. Identification and gene mapping of rice early senescent leaf(esl3) mutant. Acta Agronomica Sinica, 2013, 39(5): 862-867. (in Chinese)

[29]卢扬江, 郑康乐. 提取水稻 DNA 的一种简易方法. 中国水稻科学, 1992, 6(1): 47-48.

Lu Y J, Zheng K L. A simple method for isolation of rice DNA. Chinese Journal of Rice Science, 1992, 6(1): 47-48. (in Chinese)

[30]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. Theoretical and Applied Genetics, 2010, 120(3): 563-572.

[31]Ferguson I B, Watkins C B, Harman J E. Inhibition by calcium of senescence of detached cucumber cotyledons effect on ethylene and hydroperoxide production. Plant Physiology, 1983, 71(1): 182-186.

[32]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[33]Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 1987, 4(4): 406-425.

[34]曹孟良. 水稻叶片早衰性的遗传分析. 湖南农业科学, 2001, 1: 13-14.

Cao M L. Genetic analysis on leaf senescence of rice. Hunan Agricultural Sciences, 2001, 1: 13-14. (in Chinese)

[35]Lin A, Wang Y, Tang J, Xue P, Li C, Liu L C, Hu B, Yang F Q, Gary J L, Chu C C. Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice. Plant Physiology, 2012, 158(1): 451-464.

[36]陈县明. 内含子的识别和选择性剪切. 安徽农学通报, 2010, 16(10): 29-30.

Chen X M. Summary on recognition and alternative splicing of intron. Anhui Agricultural Science Bulletin, 2010, 16(10): 29-30. (in Chinese)

[37]Jurica M S, Moore M J. Pre-mRNA splicing: Awash in a sea of proteins. Molecular Cell, 2003, 12(1): 5-14.

[38]Mullineaux P, Karpinski S. Signal transduction in response to excess light: Getting out of the chloroplast. Current Opinion in Plant Biology, 2002, 5(1): 43-48.

[39]Vandenabeele S, Van Der Kelen K, Dat J, Gadjev I, Boonefaes T, Morsa S, Rottiers P, Slooten L, Montagu M V, Zabeau M, Inze D,Van Breusegem F. A comprehensive analysis of hydrogen peroxide- induced gene expression in tobacco. Proceedings of the National Academy of Sciences of the USA, 2003, 100(26): 16113-16118.