水稻窄叶突变体nal12的鉴定与基因精细定位

doi:10.16819/j.1001-7216.2019.8125

中国水稻科学 ›› 2019, Vol. 33 ›› Issue (3): 219-226.DOI: 10.16819/j.1001-7216.2019.8125

水稻窄叶突变体nal12的鉴定与基因精细定位

文艺^1,², 方云霞³, 胡鹏², 王月影², 侯琳琳², 谭义青², 朱黎欣², 邓雪梅², 曾大力², 张光恒², 郭龙彪², 朱丽², 陈光², 任德勇², 饶玉春¹, 薛大伟³, 钱前^1,^2,^*(), 胡江^2,^*()

1浙江师范大学化学与生命科学学院, 浙江金华 321000
2中国水稻研究所水稻生物学国家重点实验室, 杭州 310006
3杭州师范大学生命与环境科学学院, 杭州 310036;

收稿日期:2018-11-20 修回日期:2018-12-18 出版日期:2019-05-10 发布日期:2019-05-10
通讯作者: 钱前,胡江
作者简介:
^#共同第一作者;
基金资助:
国家自然科学基金资助项目(31671666;31871594;91335105);浙江省自然科学基金杰出青年项目(LR19C130001)

Identification and Fine Mapping of a Narrow Leaf Mutant nal12 in Rice

Yi WEN^1,², Yunxia FANG³, Peng HU², Yueying WANG², Linlin HOU², Yiqing TAN², Lixin ZHU², Xuemei DENG², Dali ZENG², Guangheng ZHANG², Longbiao GUO², Li ZHU², Guang CHEN², Deyong REN², Yuchun RAO¹, Dawei XUE³, Qian QIAN^1,^2,^*(), Jiang HU^2,^*()

1College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321000, China
2State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China
3College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China;

Received:2018-11-20 Revised:2018-12-18 Online:2019-05-10 Published:2019-05-10
Contact: Qian QIAN, Jiang HU
About author:
^#These authors contributed equally to this work;

摘要/Abstract

摘要：

【目的】水稻叶片是理想株型的主要组成部分。筛选和鉴定新的叶形突变材料,可为研究叶发育调控机制和塑造叶片理想形态打下基础。【方法】由粳稻品种武运粳31经EMS(ethyl methane sulphonate)诱变获得窄叶突变体nal12(narrow leaf 12);通过表型测量分析剑叶至倒4叶的叶片长度、宽度、大脉数和小脉数,并进行组织切片和显微观察;将突变体nal12与籼稻品种台中本地1号(TN1)杂交,在F₂分离群体中选取了1709个具窄叶突变表型的单株,通过已有的SSR、STS和新开发的分子标记进行精细定位。【结果】nal12在幼苗期就表现出了窄叶性状,在成熟期植株较野生型矮小,分蘖增多,茎秆变细。经组织切片观察分析,nal12叶片变窄是由于大脉和小脉数量减少造成。遗传分析表明该窄叶表型受单一隐性核基因调控;最终将该基因定位在第10染色体上LC2-RF37与LC4R-RF39标记之间约64.7 kb的范围内。该区间内共有10个开放阅读框,其中并无已报道的窄叶相关基因。qRT-PCR结果表明,NAL12基因的突变会影响生长素合成与运输相关基因的表达。【结论】NAL12为一个新的叶形调控基因。这为进一步研究水稻叶片发育,丰富其分子调控网络打下了基础。

关键词: 水稻, 窄叶, NAL12, 基因定位

Abstract:

【Objective】Leaf is a vital component of ideal plant type in rice. Screening and identification of new leaf mutants can lay the foundation for studying leaf development and constructing ideal leaf morphology. 【Method】A novel rice mutant named narrow leaf 12 (nal12) was identified from an EMS mutagenized population of Wuyunjing 31. The leaf length, leaf width, numbers of large and small veins from flag leaf to the 4th leaf from the top were measured, and the histological section and microscopic observation were conducted at the maturity stage. The F₂ segregating population was constructed from a cross between nal12 and indica variety TN1, and 1709 narrow-leaf individuals were selected for fine mapping of NAL12 with existing SSR, STS and developed molecular markers. 【Result】The nal12 showed narrow leaves at seedling stage, and exhibited dwarf, increased tillers and thinner stems at maturity stage. According to the anatomy observation of tissue sections, the narrow leaf phenotype of nal12 was caused by the decrease in the number of large veins and small veins compared with the wild type. Genetic analysis indicated that the narrow leaf trait was regulated by a single recessive nuclear gene, and NAL12 was finally mapped to a range of 64.7 kb between LC2-RF37 and LC4R-RF39 markers on chromosome 10. A total of 10 ORFs (open reading frames) were found and there were no narrow-leaf related genes reported in this region. qRT-PCR analysis revealed that the mutation of NAL12 was involved in the expression level of auxin synthesis and transport-related genes. 【Conclusion】NAL12 is a new leaf-shape regulatory gene, which lay a foundation for further research on rice leaf development and enrich its molecular regulatory network.

Key words: rice, narrow leaf, NAL12, gene mapping

中图分类号:

文艺, 方云霞, 胡鹏, 王月影, 侯琳琳, 谭义青, 朱黎欣, 邓雪梅, 曾大力, 张光恒, 郭龙彪, 朱丽, 陈光, 任德勇, 饶玉春, 薛大伟, 钱前, 胡江. 水稻窄叶突变体nal12的鉴定与基因精细定位[J]. 中国水稻科学, 2019, 33(3): 219-226.

Yi WEN, Yunxia FANG, Peng HU, Yueying WANG, Linlin HOU, Yiqing TAN, Lixin ZHU, Xuemei DENG, Dali ZENG, Guangheng ZHANG, Longbiao GUO, Li ZHU, Guang CHEN, Deyong REN, Yuchun RAO, Dawei XUE, Qian QIAN, Jiang HU. Identification and Fine Mapping of a Narrow Leaf Mutant nal12 in Rice[J]. Chinese Journal OF Rice Science, 2019, 33(3): 219-226.

图/表 6

图1 野生型武运粳31与突变体nal12的表型 A-10 d秧龄的幼苗,标尺=3 cm;B-分蘖期植株,标尺=5 cm;C-穗,标尺=1 cm;D和E-分别为茎秆倒Ⅰ节到倒Ⅵ节,标尺=1 cm;F图中,误差线表示标准差（n=20）,**表示差异达0.01显著水平。

Fig. 1. Phenotype of wild-type Wuyunjing 31 and mutant nal12. A, Seedlings(10-day-old), scale bar=3 cm; B, Plant at the tillering stage, scale bar=5 cm; C, Panicle, scale bar =1 cm; D and E, Six sections of the culm, scale bar=1 cm. Bars represent standard deviations (n=20), ** significant difference between WT and nal12 by t-test (P<0.01).

图2 野生型武运粳31(WT)与突变体nal12叶片的显微结构 A-野生型武运粳31和突变体nal12剑叶至倒4叶的形态, 标尺=1 cm;B-叶片的主脉、大脉和小脉。MV为主脉,LV为大脉,SV为小脉,标尺=1 cm;C和D-主脉的石蜡切片,标尺=200 μm;E和F-大脉和小脉的石蜡切片。标尺=200 μm;G和H-野生型武运粳31和突变体nal12的叶宽和叶长;I和J-野生型武运粳31和突变体nal12的大脉数和小脉数。误差线表示标准差（n=20）,**0.01极显著水平。

Fig. 2. Leaf microstructure of Wuyunjing 31(wild type, WT) and mutant nal12. A, Morphology of top four leaves in the wild-type Wuyunjing 31 and mutant nal12, scale bar = 1 cm; B, The main vein (MV), large vein (LV) and small vein (SV) of leaves, scale bar = 1 cm; C and D, Paraffin section of main vein, scale bar = 200 μm; E and F, Paraffin section of large veins and small veins, scale bar=200 μm; G and H, Leaf width and length of top four leaves; I and J, Number of large veins and small veins of top four leaves; FL, Flag leaf; L2, The second leaf from the top; L3, The third leaf from the top; L4, The fourth leaf from the top. Bars represents standard deviations (n=20), **significant difference between WT and nal12 by t-test (P<0.01).

图3 窄叶突变基因NAL12的精细定位 A-NAL12初定位在第10染色体上;B-NAL12精细定位在64.7 kb的区间内;C-定位区间的10个预测基因。

Fig. 3. Fine mapping of NAL12. A, NAL12 was primarily mapped on chromosome 10; B, NAL12 was narrowed to a 64.7 kb genomic region; C, Total ten genes were predicted in the region.

表1 用于基因定位和定量分析的分子标记

Table 1 Markers developed for mapping of NAL12 and quantitative real-time PCR (qRT-PCR) analysis.

分子标记 Marker	正向引物序列（5'-3'） Forward primer sequence (5'-3')	反向引物序列（5'-3'） Reverse primer sequence (5'-3')
RF13	GTGTACATTTCGTGCGTACCA	TTGACTGATGAAGCAGAATGC
RF16	GCGTACGTTCAACAAGACCC	CAGTTCAACACAAGTAATGCAGAG
RF21	ATTATTCAACAACGAAGGAG	AAATTAAGAAAGATGGAGGG
P42299-1	AACGGAACAAGTCCACTAAACC	ATCCTCCTCCTTCTTGACCCT
P42430-1	TTCAGGAATGCCAAGAAGACG	CGCTGCTATCAGTATCAAAAACAAG
P42490-1	GATGCGGGCACTCCTTGAACA	ATGCCATTGCCAGCCGTTTCG
LC2-RF37	CATATTGCCTTTTGCCTTTGT	AGCGAGCATGGTGTTATCTT
LC4R-RF39	TTAAGCAGACAAGAGTCAACA	CCCCATCCAGATGTGAATCCGCCAT
H42800-1	AGGGTGCCGGATCAGAACAGC	CCATGGGGAATGGGGATTTAGTTT
P43040-2	CTCGCCGAGGTGAACCTATTT	AGGAGGGTGGTGGAAAGCAAA

表2 目标区域预测基因

Table 2 Predicted genes in fine-mapping region.

基因Locus name	功能注释Function annotation
LOC_Os10g42690	jmjC结构域蛋白 jmjC domain containing protein, expressed
LOC_Os10g42700	假定的CS结构域蛋白 CS domain containing protein, putative, expressed
LOC_Os10g42710	RCD1酶 RCD1, putative, expressed
LOC_Os10g42720	假定的酰基转移酶 Acyltransferase, putative, expressed
LOC_Os10g42724	编码VHS和GAT结构域蛋白 VHS and GAT domain containing protein, expressed
LOC_Os10g42730	未知蛋白 Expressed protein
LOC_Os10g42750	CSLD1纤维素合成酶 CSLD1-cellulose synthase-like family D, expressed
LOC_Os10g42754	未知蛋白 Expressed protein
LOC_Os10g42760	PPR重复结构域蛋白 PPR repeat domain containing protein, putative, expressed
LOC_Os10g42770	编码假定的a_IG002N01.7蛋白 a_IG002N01.7, putative, expressed

图4 野生型及突变体nal12生长素相关基因的表达分析 Bar值代表标准误（n=3）,**表示0.01极显著水平,*表示0.05显著差异。

Fig. 4. Expression analysis of auxin-related genes in the wild-type Wuyunjing 31 and mutant nal12. Bars represent standard error (n=3). ** and * indicate significant difference between WT and nal12 by t-test (P<0.01, P<0.05) respectively.

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水稻窄叶突变体nal12的鉴定与基因精细定位

Identification and Fine Mapping of a Narrow Leaf Mutant nal12 in Rice

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