欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2018, Vol. 44 ›› Issue (9): 1301-1310.doi: 10.3724/SP.J.1006.2018.01301

• 研究论文 • 上一篇    下一篇

水稻窄叶突变体nal20的表型分析与基因定位

龙海馨(),邱海阳,MuhammadUZAIR,房静静,赵金凤,李学勇()   

  1. 中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程, 北京100081
  • 收稿日期:2018-02-07 接受日期:2018-06-12 出版日期:2018-09-10 网络出版日期:2018-07-02
  • 通讯作者: 李学勇
  • 基金资助:
    本研究由国家转基因生物新品种培育科技重大专项(2016ZX08009-003)资助

Phenotypic Analysis and Gene Mapping of the Rice Narrow-leaf Mutant nal20

Hai-Xin LONG(),Hai-Yang QIU,UZAIR Muhammad,Jing-Jing FANG,Jin-Feng ZHAO,Xue-Yong LI()   

  1. National Key Facility for Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-02-07 Accepted:2018-06-12 Published:2018-09-10 Published online:2018-07-02
  • Contact: Xue-Yong LI
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops (2016ZX08009-003)

RichHTML

14

PDF

720

PDF

61

摘要：

叶片作为植物光合作用的主要器官, 其面积的大小影响着光能利用率和最终产量。为了研究水稻叶片形态建成的分子机制, 利用 60Co-γ射线诱变粳稻品种春江06, 在M2代中得到1份窄叶突变体, 命名为narrow leaf20 (nal20)。该突变体叶片变窄、株高降低、分蘖增多、茎节间缩短、抽穗期提前。本研究重点调查了3片功能叶的形态, 发现突变体叶片宽度减少了50%, 叶片长度变化较小。细胞学观察表明, 叶片变窄主要是由于表皮细胞数目的减少, 而细胞大小变化不大。遗传分析表明, 该突变体表型受1对隐性基因控制。利用具有多态性的InDel分子标记及nal20与Dular配制的F2定位群体, 将该基因定位于第7染色体着丝粒区1.9 Mb范围内。二代测序结果表明, 在该范围内有455 kb的大片段缺失。本研究结果为窄叶基因NAL20的克隆和功能分析奠定了良好基础, 也为水稻株型改良提供了基因资源和育种材料。

关键词: 水稻, 叶片, 窄叶突变体, 抽穗期, 基因定位

Abstract:

Leaf is the major organ for photosynthesis in plant, and its area influences the light energy utilization efficiency and grain yield. To study the molecular mechanism of rice leaf morphology, we mutagenized the japonica cultivar Chunjiang 06 through 60Co-γ radiation. A stable narrow leaf mutant named as narrow leaf20 (nal20) was obtained in the M2 population. The nal20 mutant showed narrow leaf, reduced plant height, increased tiller number, shortened internodes, and earlier heading date. Here we mainly focused on the morphology of the flag leaf, the second top leaf and the third top leaf. We found that the blade width of the mutant reduced by 50%, compared with the wild type. Meanwhile, the variation on the blade length was relatively small. Cytological observation of leaf epidermal cells indicated that the reduced leaf width was mainly due to the decreased cell number, but not the cell size. Genetic analysis indicated that the narrow leaf phenotype was controlled by a recessive gene. Using the mutant plants from the F2 mapping population derived from a cross between nal20 and Dular, the candidate mutation locus was mapped to 1.9 Mb within the centromere of chromosome 7 by using polymorphic InDel markers. The next-generation sequencing result showed that a deletion of 455 kb occurred in the predicted region. Our study lays a good foundation for the cloning and functional study of the NAL20 gene and provides gene resources and breeding materials for the improvement of rice plant architecture.

Key words: rice, leaf, narrow-leaf mutant, heading date, gene mapping

图1

野生型和突变体nal20的叶片宽度 A: 野生型(左)和突变体nal20 (右)抽穗期的剑叶形态比较, 标尺为10 mm; B: 剑叶、倒二叶和倒三叶叶片宽度比较。以t测验计算P值, 柱形图中**表示突变体在P <0.01水平差异显著(n = 20)。"

图2

野生型和突变体nal20的叶片长度 A: 野生型(左)和突变体nal20 (右)抽穗期的剑叶形态比较, 标尺为5 cm; B: 剑叶、倒二叶和倒三叶叶片长度比较。t测验计算P值, 柱形图中 *表示突变体在P <0.05水平差异显著; **表示突变体在P < 0.01水平差异显著(n = 20)。"

表1

叶片宽度和抽穗天数在不同日照条件下的比较"

性状
Trait		 日照条件
Daylength condition		 野生型春江06
Wild-type Chunjiang 06		 nal20
Mutant nal20
剑叶宽度 长日照(北京昌平) Long-day (Changping, Beijing) 15.10±0.67 8.60±0.49**
Flag leaf width (mm) 短日照(海南三亚) Short-day (Sanya, Hainan) 15.62±0.85 8.27±0.46**
抽穗天数 长日照(北京昌平) Long-day (Changping, Beijing) 131.65±2.04 94.92±1.57**
Heading date (d) 短日照(海南三亚) Short-day (Sanya, Hainan) 89.96±1.82 92.31±1.79

图3

野生型和突变体nal20叶脉数目比较 A, C, E: 野生型剑叶、倒二叶和倒三叶的叶脉, 标尺为1 mm; B, D, F: 突变体剑叶、倒二叶和倒三叶的叶脉, 标尺为1 mm; 图中*表示大叶脉, 2个大叶脉中的圆点表示小叶脉; G, H: 野生型和突变体大叶脉数目(G)和小叶脉数目(H)的比较。t测验计算P值, 柱形图中**表示突变体在P < 0.01水平差异显著(n = 20)。"

图4

野生型和突变体nal20叶片细胞大小和数目 A, B: 野生型(A)和突变体nal20 (B)成熟期剑叶下表皮细胞形态, 标尺为50 μm; C, D: 野生型和突变体nal20抽穗期的叶片下表皮细胞大小和细胞数目的比较。t测验计算P值, **表示突变体在P < 0.01水平差异显著(n = 20)。"

图5

野生型和突变体nal20的整体株型 A: 野生型(左)和突变体nal20 (右)抽穗期的株型比较, 标尺为5 cm; B: 植株高度比较; C: 分蘖数目比较。t测验计算P值, 柱形图中**表示突变体在P < 0.01水平差异显著(n = 20)。"

图6

野生型和突变体nal20穗型和茎节长度 A: 野生型(左)和突变体nal20 (右)抽穗期的主茎穗形态; B: 主茎穗粒数比较, t测验计算P值, 柱形图中**表示突变体在P < 0.01水平差异显著(n = 20); C: 野生型(左)和突变体nal20 (右)抽穗期的节间形态; D: 节间长度比较(由上而下分别是穗长、穗茎节、第1茎节、第2茎节、第3茎节、第4茎节、第5茎节、第6茎节)。"

表2

InDel分子标记"

分子标记
Marker		 正向引物序列
Forward primer sequence (5°-3°)		 反向引物序列
Reverse primer sequence (5°-3°)
R7-3 GGCAAGTTAAAACCGAGCAG CCATGGAAGGCTGTAACCAT
R7-4 GATAGCTTGACAACGGTGGCAC CCATACATTGTTGCACTTGTGAC
R7-6 CCCCATGAGGCCTACACTT AGCAGCATAATCAGATGAGACG
R7-7 ATCGGTGCCGCTCCTAGAT CACTCCACAGACATGCAATTT
R7-8 TTCCAGGCTGCATCTTATTC GCAGGACCCATGCTGAAAAG
C7-1 ATCTAGCGGCTAGCACACTGG ATCACCTCATGTCTCCGGACG
C7-2 ACTGTGTGCTGCCTGACATAC AGACGAATGGTCAAACATGTG
C7-3 GATGGTAGGAGGCCGGACTGG GCCTCCTTTACTACCGACCGC
C7-4 GTGGTGACAATGTGGTACAAT CCACTTATACGTGCGTAACAC
C7-5 CGCCGTTCGCATAAAGTCCTG TTGGACTTGTTTGGGCATACG
C7-6 AAGGACTTGCCGTTTGATCTC CATGATCGGTACTAGCAATTC

图7

突变体nal20的基因定位图谱 A: 初步定位结果; B: 精细定位结果; C: 目的基因所在区域, 虚线表示455 kb片段缺失。A, B图中横线上方为定位所用标记, 下方数字为交换单株个数。"

表3

455 kb缺失区间内的基因"

基因
Gene		 功能注释
Function annotation
LOC_Os07g15640 CRR4, putative, expressed
LOC_Os07g15650 Expressed protein
LOC_Os07g15670 Peroxiredoxin, putative, expressed
LOC_Os07g15680 Phospholipase D, putative, expressed
LOC_Os07g15720 Hypothetical protein
LOC_Os07g15770 CCT motif family protein, expressed
LOC_Os07g15820 Expressed protein
LOC_Os07g15860 Expressed protein
LOC_Os07g15870 Expressed protein
LOC_Os07g15880 Mitochondrial prohibitin complex protein 2, putative, expressed
LOC_Os07g15910 Expressed protein
LOC_Os07g15920 Expressed protein
LOC_Os07g15930 Legume lectins beta domain containing protein, expressed
LOC_Os07g15940 Legume lectins beta domain containing protein, expressed
LOC_Os07g15950 Expressed protein
LOC_Os07g15959 Expressed protein
LOC_Os07g15970 Erythronate-4-phosphate dehydrogenase domain containing protein, expressed
LOC_Os07g15980 Expressed protein
LOC_Os07g16030 Expressed protein
LOC_Os07g16040 Erythronate-4-phosphate dehydrogenase domain containing protein, expressed
LOC_Os07g16054 Hypothetical protein
LOC_Os07g16130 Acetyltransferase, GNAT family, putative, expressed
LOC_Os07g16140 FAD binding protein, putative, expressed
LOC_Os07g16150 Expressed protein
LOC_Os07g16180 Hypothetical protein
LOC_Os07g16210 Expressed protein
LOC_Os07g16224 Piwi domain containing protein, putative, expressed
LOC_Os07g16260 Expressed protein
LOC_Os07g16270 Hypothetical protein
[1] 朱雄涛, 汪真 . 水稻高光效生理育种初探. 福建稻麦科技, 2003,21(2):14-17
Zhu X T, Wang Z . Research on high photosynthetic efficiency in the physiological breeding. Fujian Sci & Technol Rice & Wheat, 2003,21(2):14-17 (in Chinese)
[2] Bowman J L, Eshed Y, Baum S F . Establishment of polarity in angiosperm lateral organs. Trends Genet, 2002,18:134-141
doi: 10.1016/S0168-9525(01)02601-4 pmid: 11858837
[3] Schraer H, Hunter S J . The development of plant leaves. Plant Physiol, 2003,131:389
doi: 10.1104/pp.015347 pmid: 12586863
[4] Murray M G, Thompson W F . Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980,8:4321-4325
doi: 10.1093/nar/8.19.4321 pmid: 324241
[5] David-Schwartz R, Koenig D, Sinha N R . LYRATE is a key regulator of leaflet initiation and lamina outgrowth in tomato. Plant Cell, 2009,21:3093-3104
doi: 10.1105/tpc.109.069948 pmid: 2782284
[6] 陈代波, 程式华, 曹立勇 . 水稻窄叶性状的研究进展. 中国稻米, 2010,16(3):1-4
Chen D B, Cheng S H, Cao L Y . Research progress of narrow leaf characters of rice. China Rice, 2010,16(3):1-4 (in Chinese)
[7] Iri A, Perera I, Ujiie K, Ishimaru K, Hirotsu N, Kashiwagi T . Partial loss-of-function of NAL1 alters canopy photosynthesis by changing the contribution of upper and lower canopy leaves in rice. Sci Rep, 2017,7(1):15958
[8] Qi J, Qian Q, Bu Q, Li S, Chen Q, Sun J, Liang W, Zhou Y, Chu C . Mutation of the rice narrow leaf1 gene, which encodes a novel protein, affects vein patterning and polar auxin transport. Plant Physiol, 2008,147:1947
[9] Woo Y M, Park H J , Su’Udi M, Yang J I, Park J J, Back K. Constitutively wilted 1, a member of the rice YUCCA gene family, is required for maintaining water homeostasis and an appropriate root to shoot ratio. Plant Mol Biol, 2007,65:125-136
[10] Fujino K, Matsuda Y, Ozawa K, Nishimura T, Koshiba T, Fraaije M W, Sekiguchi H . NARROW LEAF 7 controls leaf shape mediated by auxin in rice. Mol Genet Genomics, 2008,279:499-507
[11] Sakamoto T, Morinaka Y, Inukai Y, Kitano H, Fujioka S . Auxin signal transcription factor regulates expression of the brassinosteroid receptor gene in rice. Plant J, 2013,73:676-688
[12] Li M, Xiong G, Li R, Cui J, Tang D, Zhang B, Ding T, Zhang B C, Pauly M, Cheng Z K, Zhou Y H . Rice cellulose synthase-like D4 is essential for normal cell-wall biosynthesis and plant growth. Plant J, 2009,60:1055-1069
doi: 10.4161/psb.5.2.10369 pmid: 19765235
[13] Wu C, Fu Y, Hu G, Si H, Cheng S, Liu W . Isolation and characterization of a rice mutant with narrow and rolled leaves. Planta, 2010,232:313-324
doi: 10.1007/s00425-010-1180-3 pmid: 20443024
[14] Hu J, Zhu L, Zeng D, Gao Z, Guo L, Fang Y, Zhang G, Dong G, Yan M, Liu J . Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice. Plant Mol Biol, 2010,73:283-292
[15] Cho S H, Yoo S C, Zhang H, Pandeya D, Koh H J, Hwang J Y, Kim G T, Paek N C . The rice narrow leaf2 and narrow leaf3 loci encode WUSCHEL-related homeobox 3A (OsWOX3A) and function in leaf, spikelet, tiller and lateral root development. New Phytol, 2013,198:1071-1084
[16] Lin H, Niu L, Mchale N A, Ohme-Takagi M, Mysore K S, Tadege M . Evolutionarily conserved repressive activity of WOX proteins mediates leaf blade outgrowth and floral organ development in plants. Proc Natl Acad Sci USA, 2013,110:366-371
[17] Ishiwata A, Ozawa M, Nagasaki H, Kato M, Noda Y, Yamaguchi T, Nosaka M, Shimizu-Sato S, Nagasaki A, Maekawa M . Two WUSCHEL-related homeobox genes, narrow leaf2 and narrow leaf3, control leaf width in rice. Plant Cell Physiol, 2013,54:779-792
doi: 10.1093/pcp/pct032 pmid: 23420902
[18] Zheng M, Wang Y, Liu X, Sun J, Wang Y, Xu Y, Xu J L, Long W, Zhu X, Guo X . The RICE MINUTE-LIKE1 (RML1) gene, encoding a ribosomal large subunit protein L3B, regulates leaf morphology and plant architecture in rice. J Exp Bot, 2016,67:3457-3469
[19] Yang W, Guo Z, Huang C, Wang K, Jiang N, Feng H, Chen G, Liu Q, Xiong L . Genome-wide association study of rice (Oryza sativa L.) leaf traits with a high-throughput leaf scorer. J Exp Bot, 2015,66:5605-5615
[20] Yoshikawa T, Eiguchi M, Hibara K I, Ito J I, Nagato Y . Rice SLENDER LEAF 1 gene encodes cellulose synthase-like D4 and is specifically expressed in M-phase cells to regulate cell proliferation. J Exp Bot, 2013,64:2049
[21] Jiang D, Fang J, Lou L, Zhao J, Yuan S, Yin L, Sun W, Peng L, Guo B, Li X . Characterization of a null allelic mutant of the rice NAL1 gene reveals its role in regulating cell division. PLoS One, 2015,10:e0118169
[22] 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. Proc Natl Acad Sci USA, 1991,88:9828
[23] Xue W, Xing Y, Weng X, Zhao Y, Tang W, Wang L, Zhou H, Yu S, Xu C, Li X . Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet, 2009,40:761-767
doi: 10.1038/ng.143 pmid: 18454147
[24] 谈聪, 翁小煜, 鄢文豪, 白旭峰, 邢永忠 . 多效性基因Ghd7调控水稻剑叶面积. 遗传, 2012,34:901-906
Tan C, Weng X Y, Yan W H, Bai X F, Xing Y Z . Ghd7, a pleiotropic gene controlling flag leaf erea in rice. Hereditas(Beijing), 2012,34:901-906 (in Chinese with English abstract)
[25] Li L, Yan W, Xue W, Shao D, Xing Y . Evolution and association analysis of Ghd7 in rice. PLoS One, 2012,7:e34021
[26] Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A . Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1 . Genes Dev, 2004,18:926-936
[27] Hayama R, Yokoi S, Tamaki S, Yano M, Shimamoto K . Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature, 2003,422:719-722
doi: 10.1038/nature01549 pmid: 12700762
[28] 肖珂, 左海龙, 巩迎军, 张俊芝, 张永娟, 董彦君 . 控制水稻剑叶形态相关性状的数量基因位点(QTL)的定位. 上海师范大学学报(自然科学版), 2007,36(2):66-70
Xiao K, Zuo H L, Gong Y J, Zhang J Z, Zhang Y J, Dong Y J . Locating quantitative trait loci affecting flag-leaf shape traits in rice (Oryza sativa L.).J Shanghai Norm Univ(Nat Sci), 2007,36(2):66-70 (in Chinese with English abstract)
[1] 田甜, 陈丽娟, 何华勤. 基于Meta-QTL和RNA-seq的整合分析挖掘水稻抗稻瘟病候选基因[J]. 作物学报, 2022, 48(6): 1372-1388.
[2] 郑崇珂, 周冠华, 牛淑琳, 和亚男, 孙伟, 谢先芝. 水稻早衰突变体esl-H5的表型鉴定与基因定位[J]. 作物学报, 2022, 48(6): 1389-1400.
[3] 周文期, 强晓霞, 王森, 江静雯, 卫万荣. 水稻OsLPL2/PIR基因抗旱耐盐机制研究[J]. 作物学报, 2022, 48(6): 1401-1415.
[4] 郑小龙, 周菁清, 白杨, 邵雅芳, 章林平, 胡培松, 魏祥进. 粳稻不同穗部籽粒的淀粉与垩白品质差异及分子机制[J]. 作物学报, 2022, 48(6): 1425-1436.
[5] 颜佳倩, 顾逸彪, 薛张逸, 周天阳, 葛芊芊, 张耗, 刘立军, 王志琴, 顾骏飞, 杨建昌, 周振玲, 徐大勇. 耐盐性不同水稻品种对盐胁迫的响应差异及其机制[J]. 作物学报, 2022, 48(6): 1463-1475.
[6] 杨建昌, 李超卿, 江贻. 稻米氨基酸含量和组分及其调控[J]. 作物学报, 2022, 48(5): 1037-1050.
[7] 杨德卫, 王勋, 郑星星, 项信权, 崔海涛, 李生平, 唐定中. OsSAMS1在水稻稻瘟病抗性中的功能研究[J]. 作物学报, 2022, 48(5): 1119-1128.
[8] 朱峥, 王田幸子, 陈悦, 刘玉晴, 燕高伟, 徐珊, 马金姣, 窦世娟, 李莉云, 刘国振. 水稻转录因子WRKY68在Xa21介导的抗白叶枯病反应中发挥正调控作用[J]. 作物学报, 2022, 48(5): 1129-1140.
[9] 王小雷, 李炜星, 欧阳林娟, 徐杰, 陈小荣, 边建民, 胡丽芳, 彭小松, 贺晓鹏, 傅军如, 周大虎, 贺浩华, 孙晓棠, 朱昌兰. 基于染色体片段置换系群体检测水稻株型性状QTL[J]. 作物学报, 2022, 48(5): 1141-1151.
[10] 王泽, 周钦阳, 刘聪, 穆悦, 郭威, 丁艳锋, 二宫正士. 基于无人机和地面图像的田间水稻冠层参数估测与评价[J]. 作物学报, 2022, 48(5): 1248-1261.
[11] 陈悦, 孙明哲, 贾博为, 冷月, 孙晓丽. 水稻AP2/ERF转录因子参与逆境胁迫应答的分子机制研究进展[J]. 作物学报, 2022, 48(4): 781-790.
[12] 刘磊, 詹为民, 丁武思, 刘通, 崔连花, 姜良良, 张艳培, 杨建平. 玉米矮化突变体gad39的遗传分析与分子鉴定[J]. 作物学报, 2022, 48(4): 886-895.
[13] 王吕, 崔月贞, 吴玉红, 郝兴顺, 张春辉, 王俊义, 刘怡欣, 李小刚, 秦宇航. 绿肥稻秆协同还田下氮肥减量的增产和培肥短期效应[J]. 作物学报, 2022, 48(4): 952-961.
[14] 巫燕飞, 胡琴, 周棋, 杜雪竹, 盛锋. 水稻延伸因子复合体家族基因鉴定及非生物胁迫诱导表达模式分析[J]. 作物学报, 2022, 48(3): 644-655.
[15] 陈云, 李思宇, 朱安, 刘昆, 张亚军, 张耗, 顾骏飞, 张伟杨, 刘立军, 杨建昌. 播种量和穗肥施氮量对优质食味直播水稻产量和品质的影响[J]. 作物学报, 2022, 48(3): 656-666.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2