水稻矮化并花发育异常突变体dwarf and deformed flower 2 (ddf2)的基因定位与候选基因分析

doi:10.3864/j.issn.0578-1752.2015.10.001

摘要/Abstract

摘要： 【目的】对一个同时导致营养和生殖器官发育异常的水稻突变体进行表型鉴定、基因定位和候选基因分析，为下一步的基因克隆与功能分析奠定基础。【方法】在水稻籼型恢复系602组织培养后代中，发现一个矮化并花发育异常突变体dwarf and deformed flower 2（ddf2）。抽穗期，以野生型为对照，对ddf2株高、主穗长、节间和功能叶的长宽等性状进行统计分析；同时利用冷冻切片等技术对茎、叶和花器官进行详细的形态和组织学分析。分别以西农1A和中花11为母本，以DDF2/ddf2杂合株系为父本构建2个F₂群体进行遗传分析和基因定位，并对候选基因进行实时荧光定量PCR（real-time PCR）分析。【结果】相较于野生型，突变体各节间的长和茎粗均极显著降低，叶片极显著变短、变窄，同时花序也极显著变短。组织细胞学分析发现，突变体大叶脉数目和相邻2个大叶脉之间的小叶脉数都没有明显的变化，但相邻2个大叶脉之间的宽度明显减小，进一步比较2个小叶脉之间的叶肉细胞，发现在突变体中细胞数目和尺寸均显著降低；突变体茎秆维管束的数目与野生型相比没有明显的变化，但统计发现2个大维管束之间基本组织细胞的数量和细胞的大小都显著小于野生型，表明ddf2突变体茎、叶细胞分裂和膨胀都受到了抑制；此外，ddf2突变体的花器官特征发育受到了严重干扰：第一轮外稃顶部弯曲、内稃不同程度退化，第三轮雄蕊器官严重退化，部分甚至转化为雌蕊状器官，另外部分ddf2小穗的护颖过度发育，转变成稃片状，一些小穗还表现分生组织确定性的丢失，发育出2个以上的小花。遗传分析表明该突变性状受1对隐性基因控制。利用中花11/ddf2的1 024株F₂分离群体，最终将DDF2精细定位在第11染色体短臂近着丝粒位置处，位于insertion/deletion（in/del）标记S-11和S-14之间，遗传距离分别为0.049和0.098 cM，物理距离为90.295 kb，并与标记S-24共分离。分析定位区间的基因，发现共有MSU注释基因12个，其中一个编码Sec3_C蛋白的LOC_Os11g17600内部包含共分离标记S-24，进一步对该基因进行表达分析，发现该基因在突变体的叶、茎和穗中都表现出明显的下调，初步将LOC_Os11g17600作为DDF2候选基因。【结论】DDF2是一个同时控制水稻茎/叶和花器官发育的新基因。

关键词: 水稻, 矮化, 花器官, dwarf and deformed flower 2 (ddf2), 基因定位

Abstract: 【Objective】A rice mutant, dwarf and deformed flower 2 (ddf2) , was characterized in the present paper. These results will provide a foundation for map-based cloning and functional analysis of the DDF2 gene. 【Method】 The ddf2 mutant was derived from tissue culture mutation generations of indica restorer 602. At the heading stage, the plant height, panicle length, width and length of leaves and internodes of the ddf2 mutant and wild type were measured. Moreover, the phenotype and histology analyses of stem, leaf and spikelet were conducted. A population containing 1 024 F₂ mutants from the zhonghua11/ddf2 cross was used to map the DDF2 gene. The expression of candidate genes were detected by real time PCR. 【Result】 The leaves and internodes of ddf2 mutant were all much shorter and thinner than those of the wild type, and the panicleof ddf2 mutant was shorter than that of the wild type. According to the histology analysis, the width between two large veins of leaf was decreased dramatically in ddf2 mutant, while no obvious difference in the numbers of large and small veins was observed between the ddf2 mutant and the wild type. Further analysis showed that the size and number of mesophyll cells in ddf2 leaves were all reduced significantly. Furthermore, parenchyma cells were significantly reduced in size and number in ddf2 stems, while there was no difference in the number of vascular bundles in stem between the ddf2 mutant and the wild type. These results indicated both the cell division and cell expansion were inhibited in ddf2 leaves and stems. Additionally, the spikelet and floral organs in ddf2 mutant display serious defects. In ddf2 florets, the lemmas were distorted while the paleas were degraded in whorl 1. The stamens were degenerated seriously or transformed into pistil-like organs in whorl 3. Elongated sterile lemma and extra florets were often observed in some ddf2 spikelets. Genetic analysis indicated that ddf2 was a nuclear recessive gene. By using 1 024 F₂ mutants from the zhonghua11/ddf2 cross, the DDF2 gene was mapped between In/Del markers S-11 and S-14 on the short arm of chromosome 11 near the centromere, with genetic distance of 0.049 cM and 0.098 cM, respectively, and an approximate physical distance of 90.295 kb. The gene was co-segregating with another In/Del marker S-24. There were 12 MSU annotation genes within the mapping region. Based on the qRT-RCR, it was found that the expression of LOC_Os11g17600, encoding an exocyst complex component Sec3_C protein, was down regulated in the ddf2 mutant. Then, the LOC_Os11g17600 was considered as candidate gene of DDF2.【Conclusion】DDF2 is considered to be a novel gene controlling both stem, leaf and floral organ development.

Key words: rice (Oryza Sativa), dwarf, floral organ, dwarf and deformed flower 2 (ddf2), gene mapping

张玲，郭爽，汪玲，张天泉，庄慧，龙珏臣，何光华，李云峰. 水稻矮化并花发育异常突变体dwarf and deformed flower 2 (ddf2)的基因定位与候选基因分析[J]. 中国农业科学, 2015, 48(10): 1873-1881.

ZHANG Ling, GUO Shuang, WANG Ling, ZHANG Tian-quan, ZHUANG Hui, LONG Yu-chen, HE Guang-hua, LI Yun-feng. Gene Mapping and Candidate Gene Analysis of a dwarf and deformed flower 2 (ddf2) Mutant in Rice (Oryza sativa)[J]. Scientia Agricultura Sinica, 2015, 48(10): 1873-1881.

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.0578-1752.2015.10.001

https://www.chinaagrisci.com/CN/Y2015/V48/I10/1873

参考文献

[1] Coen E S, Meyerowitz E M. The war of the whorls: Genetic interactions controlling flower development. Nature, 1991, 353(6339): 31-37.

[2] Weigel D, Meyerowitz E M. The ABCs of floral homeotic genes. Cell, 1994, 78(2): 203-209.

[3] Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky M F. The SEP4 Gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology, 2004, 14(21): 1935-1940.

[4] Nagasawa N, Miyoshi M, Sano Y, Satoh H, Sakai H, Nagato Y. SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice. Development, 2003, 130(4): 705-718.

[5] Yao S G, Ohmori S, Kimizu M, Yoshida H. Unequal genetic redundancy of rice PISTILLATA orthologs, OsMADS2 and OsMADS4, in lodicule and stamen development. Plant and Cell Physiology, 2008, 49(5): 853-857.

[6] Yamaguchi T, Lee D Y, Miyao A, Hirochika H, An G H, Hirano H Y. Functional diversification of the two C-class MADS box genes OsMADS3 and OsMADS58 in Oryza sativa18(1): 15-28.. The Plant Cell, 2006,

[7] Yamaguchi T, Nagasawa N, Kawasaki S, Matsuoka M, Nagato Y, Hirano H. The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa. The Plant Cell, 2004, 16(2): 500-509.

[8] Sanchez-Corrales Y E, Alvarez-Buylla E R, Mendoza L. The Arabidopsis thaliana flower organ specification gene regulatory network determines a robust differentiation process. Journal of Theoretical Biology, 2010, 264(1): 971-983.

[9] Ikeda K, Ito M, Nagasawa N, Kyozuka J, Nagato Y. Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate. The Plant Journal, 2007, 51(6): 1030-1040.

[10] Ikeda-Kawakatsu K, Yasuno N, Oikawa T, Iida S, Nagato Y, Maekawa M, Kyozuka J. Expression level of ABERRANT PANICLE ORGANIZATION1 determines rice inflorescence form through control of cell proliferation in the meristem. Plant Physiology, 2009, 150(2): 736-747.

[11] Xiao H, Tang J F, Li Y F, Wang W M, Li X B, Jin L, Xie R, Luo H F, Zhao X F, Meng Z, He G H, Zhu L H. STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice. The Plant Journal, 2009, 59(5): 789-801.

[12] Sang X C, Li Y F, Luo Z K, Ren D Y, Fang L K, Wang N, Zhao F M, Ling Y H, Yang Z L, Liu Y S, He G H. CHIMERIC FLORAL ORGANS1, encoding a monocot-specific MADS box protein, regulates floral organ identity in rice. Plant Physiology, 2012, 160(2): 788-807.

[13] Grennan A K. Gibberellin metabolism enzymes in rice. Plant Physiology, 2006, 141(2): 524-526.

[14] Fan L M, Feng X Y, Wang Y, Deng X W. Gibberellin signal transduction in rice. Journal of Integrative Plant Biology, 2007, 49(6): 731-741.

[15] Kim T W, Wang Z Y. Brassinosteroid signal transduction from receptor kinases to transcription factors. Annual Reviews Compilations: Plant Hormones, 2010, 61: 681-704.

[16] Tong H N, Chu C C. Brassinosteroid signaling and application in rice. Journal of Genetics and Genomics, 2012, 39(1): 3-9.

[17] Sato Y, Miura Y, Hirochika H, Kitano H, Matsuoka M, Sentoku N. Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants. The EMBO Journal, 1999, 18(4): 992-1002.

[18] Wei X J, Xu J F, Guo H N, Jiang L, Chen S H, Yu C Y, Zhou Z L, Hu P S, Zhai H Q, Wan J M. DTH8 suppresses flowering in rice, influencing plant height and yield potential simultaneously. Plant Physiology, 2010, 153(4): 1747-1758.

[19] Yang W B, Ren S L, Zhang X M, Gao M J, Qi Y B, Wang J, Li Q, He Z H. BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development. The Plant Cell, 2011, 23(2): 661-680.

[20] Zhang Z, Zhang Y, Tan H X, Li G, Yuan Z, Zhang D B. RICE MORPHOLOGY DETERMINANT encodes the type II formin FH5 and regulates rice morphogenesis. The Plant Cell, 2011, 23(2): 681-700.

[21] Duan Y L, Li S P, Chen Z W, Zheng L L, Diao Z J, Zhou Y C, Lan T, Guan H Z, Pan R S, Xue Y B, Wu W R. Dwarf and deformed flower 1, encoding an F-box protein, is critical for vegetative and floral development in rice (Oryza sativa L.). The Plant Journal, 2012, 72(5): 829-842.

[22] Michelmore R W, Paran I, Kesseli R V. Identification of markers linked to disease-resistance genes by bulked segregation 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(21): 9828-9832.

[23] Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321-4325.

[24] Munson M, Novick P. The exocyst defrocked, a framework of rods revealed. Nature Structural & Molecular Biology, 2006, 13: 577-581.

[25] Wen T J, Schnable P S. Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable. American Journal of Botany, 1994, 81(7): 833-842.

[26] Wen T J, Hochholdinger F, Sauer M, Bruce W, Schnable P. The roothairless1 gene of maize encodes a homolog of sec3, which is involved in polar exocytosis. Plant Physiology, 2005, 138(3): 1637-1643.

[27] Brown P T H, Kyozuka J, Sukekiyo Y, Kimura Y, Shimamoto K, Lorz H. Molecular changes in protoplast-derived rice plants. Molecular and General Genetics, 1990, 223(2): 324-328.

[28] Kaeppler S M, Phillips R L. Tissue culture-induced DNA methylation variation in maize. Proceedings of the National Academy of Sciences of the United States of America, 1993, 90(19): 8773-8776.

[29] Krizova K, Fojtova M, Depicker A, Kovarik A. Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles. Plant Physiology, 2009, 149(3): 1493-1504.