一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

doi:10.11983/CBB19239

植物学报 ›› 2020, Vol. 55 ›› Issue (3): 279-286.DOI: 10.11983/CBB19239

一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

管柳蓉^1,²,刘祖培^2,³,徐冉²,段朋根²,张国政^2,³,于海跃^2,³,李静^2,³,罗越华^1,^*(),李云海^2,^*()

¹海南大学热带作物学院, 海南省热带生物资源可持续利用重点实验室, 海口 570228
²中国科学院遗传与发育生物学研究所,植物细胞与染色体工程国家重点实验室, 北京 100101
³中国科学院大学, 北京 100039

收稿日期:2019-12-25 接受日期:2020-03-23 出版日期:2020-05-01 发布日期:2020-07-06
通讯作者: 罗越华,李云海
基金资助:
国家自然科学基金(31960421)

Identification of a New OsBRI1 Weak Allele and Analysis of its Function in Grain Size Control

Liurong Guan^1,²,Zupei Liu^2,³,Ran Xu²,Penggen Duan²,Guozheng Zhang^2,³,Haiyue Yu^2,³,Jing Li^2,³,Yuehua Luo^1,^*(),Yunhai Li^2,^*()

¹Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Tropical Crops, Hainan University, Haikou 570228, China
²State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
³University of Chinese Academy of Sciences, Beijing 100039, China

Received:2019-12-25 Accepted:2020-03-23 Online:2020-05-01 Published:2020-07-06
Contact: Yuehua Luo,Yunhai Li

摘要/Abstract

摘要：

水稻(Oryza sativa)籽粒大小是影响其产量的关键农艺性状, 克隆并研究水稻籽粒大小相关基因对于提高水稻产量具有重要意义。为深入探究水稻籽粒大小的调控机制, 通过EMS诱变品种宽叶粳(KYJ), 分离了一系列水稻籽粒大小改变的突变体, 其中smg12表现为籽粒变小, 株高变矮, 一级枝梗数和二级枝梗数减少。遗传分析表明, 该小粒突变体受隐性单基因控制。细胞学分析显示, 该突变体颖壳纵向细胞长度显著变短, 表明SMG12主要影响细胞扩展。利用Mutmap方法对候选基因进行克隆, 筛选出SMG12的候选基因OsBRI1, 该基因编码油菜素内酯受体激酶。OsBRI1外显子上的第2 074个碱基发生了由C到T的置换, 产生非同义突变, 使得该位置编码的脯氨酸变为丝氨酸, 从而影响OsBRI1的功能。综上, 该研究鉴定了OsBRI1基因的1个新等位变异, 揭示了油菜素内酯途径调控水稻籽粒大小的细胞和分子基础。

关键词: 水稻, 籽粒大小, 细胞扩展, Mutmap, OsBRI1

Abstract:

Rice (Oryza sativa) grain size and grain weight are key agronomic traits that affect rice yield. Cloning and study of grain size genes are helpful to increase rice production. In order to further understand the mechanism of rice grain size control, a set of mutants with altered grain size from an EMS-treated elite japonica cultivar KYJ (Kuanyejing) were isolated. smg12 exhibits small grains, short plants, and reduced number of primary branches and secondary branches. Genetic analyses show that the smg12 mutant phenotypes are controlled by a single recessive gene. Our celluar analyses show that the small grain size phenotype of smg12 is caused by the decrease in cell size of glumes, indicating that SMG12 affects cell expansion. By using the Mutmap method, we reveal that the candidate gene for SMG12 is OsBRI1, which encodes a brassinolide receptor kinase. The smg12 mutant causes a substitution of the 2 074^th base (C to T) in OsBRI1, which results in an amino acid change (proline to serine). Therefore, this study identified a new mutant allele of OsBRI1 and provides a cellular and molecular basis for BR-mediated grain size control in rice.

Key words: rice, grain size, cell expansion, Mutmap, OsBRI1

管柳蓉, 刘祖培, 徐冉, 段朋根, 张国政, 于海跃, 李静, 罗越华, 李云海. 一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究. 植物学报, 2020, 55(3): 279-286.

Liurong Guan, Zupei Liu, Ran Xu, Penggen Duan, Guozheng Zhang, Haiyue Yu, Jing Li, Yuehua Luo, Yunhai Li. Identification of a New OsBRI1 Weak Allele and Analysis of its Function in Grain Size Control. Chinese Bulletin of Botany, 2020, 55(3): 279-286.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19239

https://www.chinbullbotany.com/CN/Y2020/V55/I3/279

图/表 6

图1 水稻突变体smg12的表型分析 (A) KYJ (野生型)和smg12突变体表型(Bar=10 cm); (B) KYJ和smg12的籽粒(Bars=1 cm); (C) 粒宽; (D) 粒长; (E) 一级枝梗数; (F) 二级枝梗数; (G) 株高; (H) 千粒重。显著性分析采用t检验, * 表示差异显著(P<0.05), ** 表示差异极显著(P<0.01)。

Figure 1 Analysis of rice smg12 mutant phenotypes (A) KYJ (wild type) and smg12 mutant phenotype (Bar=10 cm); (B) Grains of KYJ and smg12 (Bars=1 cm); (C) Grain width; (D) Grain length; (E) Number of primary branches; (F) Number of secondary branches; (G) Plant height; (H) Thousand seed weight. Significance is determined using t-test, * indicates significant differences at P<0.05, ** indicates significant differences at P<0.01.

表1 用于dCAPS分析的引物和酶

Table 1 Primer and enzyme for dCAPS analysis

Primer name	Primer sequence (5'→3')	DNA fragment length (bp)	Enzyme
smg12-1	CTTTCTCGGCACTTTCCTTG	154	HphI
smg12-1	CTATGGTCACATGGTGGCGGTG	154	HphI

图2 SMG12调控水稻颖壳细胞大小 (A) 颖壳纵向细胞数目; (B) 颖壳横向细胞数目; (C) 颖壳单个细胞纵向长度; (D) 颖壳单个细胞横向宽度; (E) 细胞学分析图(Bars=0.1 mm)。** 表示突变体与野生型之间差异极显著(P< 0.01)。

Figure 2 SMG12 regulates cell size in grain hulls of rice (A) Outer epidermal cell number in the grain-length direction of the glume; (B) Outer epidermal cell number in the grain-width direction of the glume; (C) The longitudinal length of a single cell in the glume; (D) Transverse width of a single cell in the glume; (E) Cytological analysis diagram (Bars= 0.1 mm). ** indicates extremely significant difference between the mutant and the wild type (P<0.01).

表2 水稻突变体smg12的遗传分析

Table 2 Genetic analysis of the rice mutant smg12

Hybrid combinations	Phenotype of F₁	F₂ generation			χ²_3:1
Hybrid combinations	Phenotype of F₁	Wild-phenotypic plant number	Mutant-phenotypic plant number	Total number	χ²_3:1
smg12/KYJ	Wild type	168	52	220	0.151

表3 smg12突变体的候选SNPs分析

Table 3 Analysis of the candidate SNPs for the smg12 mutant

Chromosomal position	Physical location (bp)	Genotype (KYJ/smg12)	Gene location	Gene locus name	Frequency of sequencing (KYJ/smg12)	Mutation type
Chr. 1	15686971	G/A	Upstream of the gene	LOC_Os01g28040	0/6	/
Chr. 1	27750517	G/A	In the gene compartment	LOC_Os01g48420; LOC_Os01g48430	0/12	/
Chr. 1	28534819	G/A	Upstream and downstream of the gene	LOC_Os01g49630; LOC_Os01g49614	0/14	/
Chr. 1	29397508	G/A	In the gene compartment	LOC_Os01g51140; LOC_Os01g51154	0/12	/
Chr. 1	29788896	G/A	Upstream of the gene	LOC_Os01g51810	0/17	/
Chr. 1	29929259	G/A	Exon	LOC_Os01g52050	0/19	Changes in amino acids
Chr. 1	33892012	T/A	Upstream of the gene	LOC_Os01g58620	0/1	/
Chr. 1	39503252	G/A	Downstream of the gene	LOC_Os01g67980	0/11	/
Chr. 1	30392222	G/A	Upstream of the gene	LOC_Os01g52840; LOC_Os01g52851	0/5	/
Chr. 1	33745383	G/A	Intron	LOC_Os01g58400	0/8	/
Chr. 3	19627588	C/A	Upstream of the gene	LOC_Os03g35390	0/6	/
Chr. 6	17710551	G/A	Intron	LOC_Os06g30610	0/8	/

图3 候选基因的鉴定 (A) smg12突变的dCAPS标记验证; (B) 候选基因LOC_Os01g52050的群体连锁分析; (C) LOC_Os01g52050的基因结构图, 开放的方框表示基因5'和3'端的未翻译区, 封闭的方框表示编码区, 且图中标注起始密码子(ATG)和终止密码子(TAG)以及LOC_Os01g52050中的突变位点(C/T); (D) LOC_Os01g52050的蛋白结构域。P/S表示LOC_Os01g52050编码蛋白结构域中的突变位点。

Figure 3 Identification of candidate genes (A) The dCAPS marker was developed to detect the smg12 mutation; (B) Population linkage analysis of the candidate gene LOC_Os01g52050; (C) The LOC_Os01g52050 gene structure, open boxes show the 5' and 3' untranslated regions, the closed box shows the coding sequence, and the start codon (ATG), the stop codon (TAG) and the LOC_Os01g52050 mutation site (C/T) are indicated; (D) Schematic of the LOC_Os01g52050 protein. P/S indicated the LOC_Os01g52050 mutation site.

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一个新的OsBRI1弱等位突变体的鉴定及其调控种子大小的功能研究

Identification of a New OsBRI1 Weak Allele and Analysis of its Function in Grain Size Control

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