DONG Bangning,LONG Wuhua,LIU Xi,et al.Phenotypic analysis and gene cloning of a dwarf mutant htr in rice[J].Journal of Nanjing Agricultural University,2017,(4):573-583.[doi:10.7685/jnau.201612014]





Phenotypic analysis and gene cloning of a dwarf mutant htr in rice
董邦宁 龙武华 刘喜 刘世家 田云录 陈亮明 江玲 王益华 万建民
南京农业大学作物遗传与种质创新国家重点实验室, 江苏 南京 210095
DONG Bangning LONG Wuhua LIU Xi LIU Shijia TIAN Yunlu CHEN Liangming JIANG Ling WANG Yihua WAN Jianmin
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
rice(Oryza sativa L.)dwarfgene cloningtranscription factorAP2
[目的] 对水稻矮秆突变体htr进行表型分析与基因克隆,为水稻育种提供新的矮秆基因资源。[方法]对矮秆突变体htr茎秆进行细胞学观察,并将htr与‘N22’和‘9311’杂交构建F2群体,提取隐性极端个体定位基因HTR[结果]htr是一个稳定遗传的矮秆突变体,其株高、穗长、千粒质量、分蘖数等性状均降低,但其叶片变宽,叶色深绿,茎秆增粗。成熟期对htr倒二节间进行切片观察发现:相比‘9311’,htr在茎秆的纵轴方向细胞未能正常伸长,横向细胞数目增多,细胞变小。遗传分析表明突变体htr的矮秆性状受1对隐性单基因控制。利用F2群体中隐性极端个体将HTR定位在第5染色体长臂ttr-1和ttr-2两个分子标记之间,物理距离大约在144 kb范围内。测序结果发现htr中编码AP2转录因子(AP2-like ethylene-responsive transcription factor)基因发生点突变,导致保守氨基酸发生替换,亚细胞定位显示HTR定位于细胞核中。HTR呈现组成型表达,但以在茎秆中表达量最高。[结论]矮秆突变体htr突变性状受一对编码AP2转录因子的隐性单基因控制。HTR基因的克隆对培育厚壁粗秆抗倒伏水稻新品种提供了一定的理论参考。
[Objectives]The aim is to identify rice dwarf mutant htr,localize the target gene and provide new dwarf gene resources for rice breeding. [Methods]Sections of the stem were prepared for cytological observation. The dwarf mutant htr was crossed with ‘N22’ and ‘9311’ to construct F2 populations for genetic analysis and gene mapping,respectively. We selected the recessive extreme individuals to localize the gene HTR. [Results]htr was a stably inherited dwarf mutant. The plant height,panicle length,1 000-grain weight,and tiller number in mature plants all decreased compared with wild type,while the leaf width and culm diameter were increased and the leaf colour of htr was much darker green than wild type. By observing sections of the second internodes at the mature stage,we found in the longitudinal direction of the stem,the cells failed to elongate normally and became small,but the cell number increased. Genetic analysis showed that the dwarf trait of mutant htr was controlled by a pair of recessive gene. The HTR locus was mapped to a region between the markers ttr-1 and ttr-2 on chromosome 5,which was further narrowed down to a 144 kb region. Sequence analysis revealed that the coding region of the AP2-like ethylene-responsive transcription factor had a single nucleotide substitution,resulting in a change in the conserved amino acid sequence in the htr mutant. Subcellular localization assay showed that HTR was located in the nucleus,and the HTR was constitutively expressed,but it had the highest expression in the stem. [Conclusions]The phenotype of htr was controlled by a single recessive gene,which encodes the AP2-like ethylene-responsive transcription factor. The cloning of HTR gene could provide some theoretical reference for cultivating thick-walled and lodging resistant new rice varieties.


[1] Sasaki A,Ashikari M,Ueguchi-Tanaka M,et al. A mutant gibberellin-synthesis gene in rice[J]. Nature,2002,416(6882):701-702.
[2] Peng J,Richards D E,Hartley N M,et al. ’Green revolution’genes encode mutant gibberellin response modulators[J]. Nature,1999,400(6741):256-261.
[3] 朱立宏. 关于我国水稻高产育种的我见[J]. 南京农业大学学报,2007,30(1):129-135. DOI:10.7685/j.issn.1000-2030.2007.01.026.Zhu L H. Some critical consideration on rice high-yielding breeding in china[J]. Journal of Nanjing Agricultural University,2007,30(1):129-135(in Chinese with English abstract).
[4] 曹建,孙学辉,路铁刚. 一份水稻矮秆小粒突变体的形态特征和基因定位[J]. 生物技术进展,2014,4(3):186-191.Cao J,Sun X H,Lu T G,et al. Morphological analysis and gene mapping of a rice small grain and dwarf mutant[J]. Current Biotechnology,2014,4(3):186-191(in Chinese with English abstract).
[5] Sunohara H,Satoh H,Nagato Y. Mutations in panicle development affect culm elongation in rice[J]. Breeding Science,2003,53(2):109-117.
[6] 马良勇,包劲松,李西明,等. 水稻矮生基因的克隆和功能研究进展[J]. 中国水稻科学,2009,23(1):1-11.Ma L Y,Bao J S,Li X M,et al. Progress on cloning and functional analysis of dwarfism related genes in rice[J]. Chin J Rice Sci,2009,23(1):1-11(in Chinese with English abstract).
[7] Sasaki A,Itoh H,Gomi K,et al. Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant[J]. Science,2003,299(5614):1896-1898.
[8] Komorisono M. Analysis of the rice mutant dwarf and gladius leaf 1:aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling[J]. Plant Physiology,2005,138(4):1982-1993.
[9] Yin H,Gao P,Liu C,et al. SUI-family genes encode phosphatidylserine synthases and regulate stem development in rice[J]. Planta,2013,237(1):15-27.
[10] Tanabe S. A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant,dwarf11,with reduced seed length[J]. Plant Cell,2005,17(3):776-790.
[11] Hong Z,Ueguchi-Tanaka M,Shimizu-Sato S,et al. Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem[J]. Plant Journal,2002,32(4):495-508.
[12] Hu X,Qian Q,Xu T,et al. The U-box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G α subunit to regulate brassinosteroid-mediated growth in rice[J]. PLoS Genet,2013,9(3):e1003391.
[13] Song Y,You J,Xiong L. Characterization of OsIAA1 gene,a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis[J]. Plant Molecular Biology,2009,70(3):297-309.
[14] Song Y,Xu Z F. Ectopic overexpression of an AUXIN/INDOLE-3-ACETIC ACID(Aux/IAA)gene OsIAA4 in rice induces morphological changes and reduces responsiveness to auxin[J]. International Journal of Molecular Sciences,2013,14(7):13645-13656.
[15] Kant S,Bi Y M,Zhu T,et al. SAUR39,a small auxin-up RNA gene,acts as a negative regulator of auxin synthesis and transport in rice[J]. Plant Physiology,2009,151(2):691-701.
[16] 陈峰,林红珍,周起先,等. 水稻矮秆基因的研究进展[J]. 山东农业科学,2013,45(9):127-133.Chen F,Lin H Z,Zhou Q X,et al. Research progress of dwarf genes in rice[J]. Shandong Agricultural Sciences,2013,45(9):127-133(in Chinese with English abstract).
[17] Dietz K J,Vogel M O,Viehhauser A. AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic,hormonal and environmental signals in stress acclimation and retrograde signalling[J]. Protoplasma,2010,245(1/2/3/4):3-14.
[18] Sakuma Y,Liu Q,Dubouzet J G,et al. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration- and cold-inducible gene expression[J]. Biochemical and Biophysical Research Communications,2002,290(3):998-1009.
[19] Nakano T,Suzuki K,Fujimura T,et al. Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J]. Plant Physiol,2006,140(2):411-432.
[20] Kim S,Soltis P S,Wall K,et al. Phylogeny and domain evolution in the APETALA2-like gene family[J]. Mol Biol Evol,2006,23(1):107-120.
[21] Shigyo M,Hasebe M,Ito M. Molecular evolution of the AP2 subfamily[J]. Gene,2006,366(2):256-265.
[22] 张计育,王庆菊,郭忠仁. 植物AP2/ERF 类转录因子研究进展[J]. 遗传,2012,34(7):835-847.Zhang J Y,Wang Q J,Guo Z R. Progresses on plant AP2/ERF transcription factors[J]. Hereditas,2012,34(7):835-847(in Chinese with English abstract).
[23] Qi W,Sun F,Wang Q,et al. Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene[J]. Plant Physiol,2011,157(1):216-228.
[24] Yaish M W,El-Kereamy A,Zhu T,et al. The APETALA-2-like transcription factor OsAP2-39 controls key interactions between abscisic acid and gibberellin in rice[J]. PLoS Genet,2010,6(9):e1001098.
[25] Kitomi Y,Ito H,Hobo T,et al. The auxin responsive AP2/ERF transcription factor CROWN ROOTLESS5 is involved in crown root initiation in rice through the induction of OsRR1,a type-A response regulator of cytokinin signaling[J]. Plant Journal,2011,67(3):472-484.
[26] Lichtenthaler H K. Chlorophylls and carotenoids:pigments of photosynthetic biomembranes[J]. Methods in Enzymology,1987,148(1):350-382.
[27] 蒋悦,孙娟,韩思迪,等. 水稻黄叶突变体yl的遗传分析与基因定位[J]. 南京农业大学学报,2016,39(6):889-897. DOI:10.7685/jnau.201604004.Jiang Y,Sun J,Han S D,et al. Genetic analysis and gene mapping of a yellow leaf mutant yl in rice[J]. Journal of Nanjing Agricultural University,2016,39(6):889-897(in Chinese with English abstract).
[28] Wang Y,Li J. Molecular basis of plant architecture[J]. Annual Review of Plant Biology,2008,59(59):253-279.
[29] Aya K,Hobo T,Sato-Izawa K,et al. A novel AP2-type transcription factor,SMALL ORGAN SIZE1,controls organ size downstream of an auxin signaling pathway[J]. Plant Cell Physiol,2014,55(5):897-912.
[30] Huang D,Wang S,Zhang B,et al. A gibberellin-mediated DELLA-NAC signaling cascade regulates cellulose synthesis in rice[J]. Plant Cell,2015,27(6):1681-1696.
[31] Itoh H,Tatsumi T,Sakamoto T,et al. A rice semi-dwarf gene,Tan-Ginbozu(D35),encodes the gibberellin biosynthesis enzyme,ent-kaurene oxidase[J]. Plant Mol Biol,2004,54(4):533-547.
[32] Elliott R C,Betzner A S,Huttner E,et al. AINTEGUMENTA,an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth[J]. Plant Cell,1996,8(2):155-168.
[33] Chuck G,Meeley R B,Hake S. The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet1[J]. Genes Dev,1998,12(8):1145-1154.
[34] Krizek B A. Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs[J]. Dev Genet,1999,25(3):224-236.
[35] Allen M D,Yamasaki K,Ohme-Takagi M,et al. A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J]. EMBO J,1998,17(18):5484-5496.
[36] Wilson K,Long D,Swinburne J,et al. A dissociation insertion causes a semidominant mutation that increases expression of TINY,an Arabidopsis gene related to APETALA2[J]. Plant Cell,1996,8(4):659-671.
[37] 王燚,魏中伟,邵平,等. 水稻茎秆特性与抗倒伏关系的研究现状[J]. 湖北农业科学,2015,54(9):2054-2057.Wang Y,Wei Z W,Shao P,et al. Research status on relationship between culm characteristics and lodging resistance of rice[J]. Hubei Agricultural Sciences,2015,54(9):2054-2057(in Chinese with English abstract).
[38] Duan C R,Wang B C,Wang P Q,et al. Relationship between the minute structure and the lodging resistance of rice stems[J]. Colloids and Surfaces B Biointerfaces,2004,35(3/4):155-158.
[39] Hirano K,Okuno A,Hobo T,et al. Utilization of stiff culm trait of rice smos1 mutant for increased lodging resistance[J]. PLoS ONE,2014,9(7):e96009.


 ZHANG Chen-ming,XU Ye-hong,ZHAO Hai-juan,et al.Effects of different nitrogen forms on nitrogen uptake and root growth of rice at the seedling stage[J].Journal of Nanjing Agricultural University,2011,34(4):72.[doi:10.7685/j.issn.1000-2030.2011.03.013]
 HAO Wen-ya,SHEN Qi-rong,RAN Wei,et al.The effects of sugars and amino acids in watermelon and rice root exudates on the growth of Fusarium oxysporum f.sp. niveum[J].Journal of Nanjing Agricultural University,2011,34(4):77.[doi:10.7685/j.issn.1000-2030.2011.03.014]
 XU Xiao-sa,LIU Xi,ZHAO Zhi-gang,et al.Construction of genetic linkage map based on a RILs population derived from the hybrid rice Peiai 64S/93-11 and detection of QTL for 1000-grain weight[J].Journal of Nanjing Agricultural University,2011,34(4):8.[doi:10.7685/j.issn.1000-2030.2011.01.002]
 WEI Guang-bin,XU Hai-gang,DING Yan-feng,et al.Development and realization of the rice design cultivation system[J].Journal of Nanjing Agricultural University,2011,34(4):14.[doi:10.7685/j.issn.1000-2030.2011.01.003]
 LI Gang-hua,WANG Hui-zhi,WANG Shao-hua,et al.Effect of nitrogen applied at rice panicle initiation stage on carbon and nitrogen metabolism and spikelets per panicle[J].Journal of Nanjing Agricultural University,2010,33(4):1.[doi:10.7685/j.issn.1000-2030.2010.01.001]
 WANG Bi-qian,FAN Xiao-rong,XU Guo-hua,et al.Characteristics of flag leaf senescence among three rice cultivars with different nitrogen use efficiency[J].Journal of Nanjing Agricultural University,2010,33(4):8.[doi:10.7685/j.issn.1000-2030.2010.02.002]
 ZHAO Cheng-guo,XU Hai-gang,LI Gang-hua,et al.Studies on population composition of super-high-yielding single-cropping japonica rice in heading stage[J].Journal of Nanjing Agricultural University,2011,34(4):23.[doi:10.7685/j.issn.1000-2030.2011.02.005]
 CHEN Zhi-de,ZHONG Wei-gong,WANG Jun,et al.Mapping of QTL of tolerance to Cd^{2+} stress at seedling stage in rice(Oryza sativa L.)[J].Journal of Nanjing Agricultural University,2010,33(4):1.[doi:10.7685/j.issn.1000-2030.2010.03.001]
 YE Li-ting,FAN Jian-bo,XU Ye-hong,et al.Characteristics of growth in rice genotypes with different nitrogen use efficiency[J].Journal of Nanjing Agricultural University,2010,33(4):77.[doi:10.7685/j.issn.1000-2030.2010.03.015]
 JIN Yu-kuan,YANG Shi-hu,YU Li,et al.Expression and resistance analysis of the Pib gene in transgenic rice under different promoters[J].Journal of Nanjing Agricultural University,2010,33(4):1.[doi:10.7685/j.issn.1000-2030.2010.04.001]


更新日期/Last Update: 2017-07-07