水稻矮化基因WLD1的图位克隆

doi:10.11983/CBB16010

植物学报 ›› 2017, Vol. 52 ›› Issue (1): 54-60.DOI: 10.11983/CBB16010

所属专题：水稻生物学专辑 (2017年52卷1期)

水稻矮化基因WLD1的图位克隆

王文乐, 冯丹, 吴金霞, 张治国, 路铁刚^*()

中国农业科学院生物技术研究所, 农作物基因资源及遗传改良国家重大科学工程, 北京 100081

收稿日期:2016-01-15 接受日期:2016-04-29 出版日期:2017-01-01 发布日期:2017-01-23
通讯作者: 路铁刚
作者简介:
# 共同第一作者
基金资助:
农业部重大转基因专项(No.2014Ex08001-003)

Gene Mapping of a Dwarf Gene WLD1 in Rice

Wang Wenle, Feng Dan, Wu Jinxia, Zhang Zhiguo, Lu Tiegang^*()

Biotechnology Research Institute/National Key Facility for Genetic Resources and Gene Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2016-01-15 Accepted:2016-04-29 Online:2017-01-01 Published:2017-01-23
Contact: Lu Tiegang
About author:
# Co-first authors

摘要/Abstract

摘要： 水稻(Oryza sativa)矮化是与光合效率及产量等密切相关的重要农艺性状。发掘更多的水稻矮秆资源, 不仅能够进一步加深对水稻株高分子遗传机制的认识, 而且还能为水稻新品种培育提供新的种质资源。在水稻T-DNA插入突变体库中筛选到1个矮化、宽叶小粒突变体(wld1)。经图位克隆将WLD1基因定位在第5号染色体长臂, 位于分子标记InDel37与InDel48之间, 基因编号为LOC_Os05g32270, 属于AP2转录因子家族。该基因第6外显子处胸腺嘧啶缺失, 造成转录提前终止。石蜡切片观察结果显示, 茎部第2节间横向细胞数目增加, 而纵向细胞数目未变。RT-PCR检测结果表明, LOC_Os05g32270在突变体wld1中不表达, 造成功能缺失。该基因与已报道的水稻OsSMOS1 (SMALL ORGAN SIZE1)为等位基因。水稻突变体wld1的矮秆遗传效应可直接应用于育种中。该研究结果进一步明确了突变体wld1的表型特征与遗传基础, 为解析其参与的信号途径提供参考。

Abstract: Dwarf rice (Oryza sativa) is the most important agronomic trait closely related to photosynthetic efficiency and yield. Exploring more dwarf resources can deepen our understanding of the genetic mechanisms of plant height and provide new germplasm for cultivating new varieties. A wide-leaf, wide-stem, small-kernel and dwarf mutant, wld1, was identified from the rice T-DNA insertion mutant library. We used map-based cloning to locate the gene on rice chromosome 5 between the markers InDel37 and InDel48, with 91 kb physical distance. The mutant wld1 resulted from deletion of the base T in LOC_Os05g32270, which causes a reading error and early termination of the peptide chain encoding. We used microscopy to observe the top-second internal structure of vegetative organs of the mutant and the wild type in paraffin sections. In the wld1, cells were significantly increased in size in cross-sections but reduced in size in longitudinal sections. The expression of LOC_Os05g32270 was completely inactivated. The gene is an allele of the reported rice gene OsSMOS1 (SMALL ORGAN SIZE1). The agronomic properties of wld1 are good (lodging) and it can be directly applied to practice. In this study, we defined the inheritance and phenotype characteristics of this mutant, which lays the foundation for further analysis of the signaling pathway.

王文乐, 冯丹, 吴金霞, 张治国, 路铁刚. 水稻矮化基因WLD1的图位克隆. 植物学报, 2017, 52(1): 54-60.

Wang Wenle, Feng Dan, Wu Jinxia, Zhang Zhiguo, Lu Tiegang. Gene Mapping of a Dwarf Gene WLD1 in Rice. Chinese Bulletin of Botany, 2017, 52(1): 54-60.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB16010

https://www.chinbullbotany.com/CN/Y2017/V52/I1/54

图/表 7

图1 水稻野生型(WT)与突变体wld1成熟期表型^(A) 野生型与突变体wld1成熟期植株形态比较(Bar=20 cm); (B) 野生型与突变体wld1叶宽比较(Bar=0.8 cm); (C) 野生型与突变体wld1穗型比较(Bar=4 cm); (D) 野生型与突变体wld1种子大小比较(Bar=0.5 mm)

Figure 1 Phenotype comparison between wild type (WT) and the mutant wld1 of rice at mature stage^(A) The morphology comparison between wild type and wld1 mutant at mature stage (Bar=20 cm); (B) The leaf blade comparison between wild type and wld1 mutant (Bar=0.8 cm); (C) Panicle comparison between wild type and wld1 mutant (Bar=4 cm); (D) Kernel comparison between wild type and wld1 mutant (Bar=0.5 mm)

表1 水稻突变体wld1与野生型的农艺性状比较

Table 1 The agronomic traits comparison between the mutant wld1 and wild type of rice

Agronomic trait	Wild type	Mutant wld1
Plant height (cm)	103.47±0.89	78±1.38**
Leaf blade (cm)	1.32±0.33	2.12±0.46**
Panicle length (cm)	18.79±0.68	14.87±0.33**
No. of effective tillers per plant	25.65±1.79	24.08±1.76
Seed setting percentage (%)	90.01±1.78	88.03±2.34
1000-grain weight (g)	24.86±0.16	22.89±0.27**

图2 水稻野生型和突变体wld1成熟期茎节石蜡切片对比^(A) 野生型第2节间石蜡横截面(Bar=80 µm); (B) 突变体wld1第2节间石蜡横截面(Bar=80 µm); (C) 野生型第2节间石蜡纵截面(Bar=20 µm); (D) 突变体wld1第2节间石蜡纵截面(Bar=20 µm)

Figure 2 The paraffin section comparison of stem between the mutant wld1 and wild type in rice at mature stage^(A) Cross-section of second internode in wild type (Bar=80 µm); (B) Cross-section of second internode in wld1 mutant (Bar=80 µm); (C) Longitudinal section of second internode in wild type (Bar=20 µm); (D) Longitudinal section of second internode in wld1 mutant (Bar=20 µm)

表2 F2代分离群体统计结果

Table 2 The statistics of the phenotype of F2 segregating population

Cross combination	Nipponbare × Mutant	Mutant × Nipponbare
Number of plants	1691	1304
Number of normal plants	1264	986
Number of dwarf plants	427	318
Segregation of ratio	3:1	1:3
χ²_0.05(1)=3.841	0.057	0.261

表3 本实验开发的多态性InDel标记

Table 3 Polymorphic InDel markers developed in this study

Marker	Forward primer (5'-3')	Reverse primer (5'-3')
rm5-4	CAGAAGCCTTCTTACGAACTT	TAATCTTTGAGCGAGCAATT
rm5-30	CCATGCATGATTGAAAGCAT	TTCTCCCATCTCTTGTATGTGG
InDel48	TGCAGCAAATGTATGCAGTG	TGGTGATGTCCTTTGGTTGA
InDel82	GCCGCAAAACCGAGTATAGA	CGGTGTCACGTCAATCAAAA
InDel46	CGTGACAGTTTTGACCATGC	TAACAAGGCCCAAGATCACC
InDel37	GAAGAAGGCGAGGGAAACC	ACCTGGGCTAGTTGATCTGC

图3 水稻OsWLD1基因的初定位和精细定位

Figure 3 The rough and fine mapping of OsWLD1 gene in rice

图4 水稻候选基因的克隆及表达^(A) LOC_Os05g32270基因在突变体wld1的序列分析; (B) LOC_Os05g32270基因在野生型和突变体的半定量分析

Figure 4 Cloning and expression analysis of candidate gene in rice^(A) LOC_Os05g32270 sequence analysis of wld1; (B) The LOC_Os05g32270 RT-PCR analysis in wld1 mutant and wild type

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水稻矮化基因WLD1的图位克隆

Gene Mapping of a Dwarf Gene WLD1 in Rice

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