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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)

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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
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