水稻穗长基因PAL3的克隆及自然变异分析

doi:10.11983/CBB21119

摘要/Abstract

摘要： 穗型是决定水稻(Oryza sativa)产量的关键因素之一。我们从粳稻品种圣稻808 (SD808)的EMS诱变突变体库中发现4份短穗突变体, 这些突变体的穗长、一级枝梗数、二级枝梗数和穗粒数发生不同程度的降低。基因定位和图位克隆表明, 这些突变体的表型受同一基因控制, 将该基因命名为PAL3 (PANICLE LENGTH3)。PAL3编码一个含12个跨膜结构域的多肽转运蛋白。pal3-1和pal3-2的点突变造成保守区域的氨基酸发生非同义突变; pal3-3的点突变造成第1外显子和内含子拼接错误; pal3-4的点突变造成蛋白翻译提前终止, 导致第12个跨膜域缺失。对PAL3进行单倍型分析, 共鉴定出9个单倍型(Hap1-Hap9), 其中Hap1-Hap3为主要单倍型。Hap1以粳稻为主, Hap2同时包含籼稻和粳稻, Hap3则以籼稻为主。Hap1起源于普通野生稻(O. rufipogon), Hap2和Hap3可能起源于一年生普通野生稻(O. nivara)。统计分析结果表明, Hap3的穗长显著高于Hap1和Hap2, 其具有提高穗长的潜力。该研究揭示了多肽转运蛋白对水稻穗型的重要调控作用, 为水稻穗型改良奠定了理论基础。

关键词: 水稻, 穗型, 图位克隆, 多肽转运蛋白, 自然变异

Abstract: Panicle architecture is one of the key factors determining the rice (Oryza sativa) grain yield. In this study, four short panicle mutants were identified from an EMS (ethyl methane sulfonate) mutant library of a japonica vareity Shengdao 808 (SD808). The panicle length, primary branch number, secondary branch number and grain number per panicle of these mutants were decreased in various degrees. Map-based cloning showed that these mutants were controlled by the gene PAL3 (PANICLE LENGTH 3) which encodes a peptide transporter with 12 transmembrane domains. The point mutations of pal3-1 and pal3-2 resulted in non-synonymous mutations of amino acids in the conserved region; the point mutation of pal3-3 resulted in the mis-splicing of the first exon and intron; and the point mutation of pal3-4 resulted in the premature termination of translation and thus the deletion of the 12^th transmembrane domain. Through haplotype analysis, nine haplotypes of PAL3 were identified, including three major haplotypes (Hap1-Hap3). Hap1 is dominated by japonica accessions, Hap2 contains both indica and japonica accessions, while Hap3 is dominated by indica accessions. Hap1 originated from O. rufipogon, while Hap2 and Hap3 may originate from O. nivara. Statistical analysis showed that the panicle length of Hap3 was significantly higher than that of Hap1 and Hap2, indicating that Hap3 may have the potential to improve the panicle length. In conclusion, this study revealed the important role of the peptide transporter in regulating rice panicle architecture and thus provides a new theoretical basis for rice panicle architecture improvement.

Key words: rice, panicle architecture, map-based cloning, peptide transporter, natural variation

尚江源, 淳雁, 李学勇. 水稻穗长基因PAL3的克隆及自然变异分析. 植物学报, 2021, 56(5): 520-532.

Jiangyuan Shang, Yan Chun, Xueyong Li. Map-based Cloning and Natural Variation Analysis of the PAL3 Gene Controlling Panicle Length in Rice. Chinese Bulletin of Botany, 2021, 56(5): 520-532.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21119

https://www.chinbullbotany.com/CN/Y2021/V56/I5/520

图/表 11

表1 定位引物

Table 1 Primers used for mapping

Primer name	Forward primer (5′→3′)	Reverse primer (5′→3′)
R11-3	AGAGAGACATCCGGAGACAA	TAAGACGAAAGGTCAAACGT
R11-5	GTGCTAACGTTTCGTCTAAC	AATAGCCTTCGGTGGTCTCA
R11-10	GTTCGTAATGTGGGCGTCTT	TGGGCACTCTTCTCACACTG
R11-12	CAATCTTGCTCTACTAGCTAGTG	GTGGCAACTAACAGATTAGATG
M1	GCAGTATATATTCGGCGGCG	GCCGTCGCCATATAGCTG
M2	GAGCCTCTCCTACTGTGCTA	AGAGCCCTCAGTTCCTCAAT
M3	GCTGACTACAGTAAGATCATGC	AGACAAACGGTCAAACATGT
M4	AAGGATCCAAGCTAGCCTCC	CCTGACAGCAAGCGAGAGAT
M5	CTTCAGCAAGTGAACTACGA	CCTAAACTAGCACGGATCATAGC
M6	TGTGAGGTTTAGGTTCTCGGA	TGAATAGAGATGCGGTCCAAC
M7	GGATTCGGCCACTGGTTGTT	GAATGTACTCGGATAAACCC

图1 水稻野生型与pal3突变体的表型比较 (A) 野生型与pal3突变体的成熟期株型(Bar=20 cm); (B) 闭合状态下野生型与pal3突变体的穗(Bar=2 cm); (C) 展开状态下野生型与pal3突变体的穗(右) (Bar=2 cm); (D)-(G) 野生型与pal3突变体的穗长(D)、一级枝梗数(E)、二级枝梗数(F)和穗粒数(G)的统计分析。WT: 野生型。图中数据为平均值±标准差(n=20)。**表示在P<0.01水平差异显著(经t检验)。

Figure 1 Comparison of the rice phenotype between wild type and the pal3 mutants (A) Gross morphology of wild type and the pal3 mutants at mature stage (Bar=20 cm); (B) Closed panicle of wild type and the pal3 mutants (Bar=2 cm); (C) Spread panicles of wild type and the pal3 mutants (right) (Bar=2 cm); (D)-(G) Statistic analysis of panicle length (D), primary branch number (E), secondary branch number (F) and grain number per panicle (G) of wild type and the pal3 mutants. WT: Wild type. Data in figure are means±SD (n=20). ** indicate the significant differences at P<0.01 level by Students’t test.

表2 pal3-1、pal3-2、pal3-3和pal3-4水稻突变体的遗传分析

Table 2 Genetic analysis of the pal3-1, pal3-2, pal3-3 and pal3-4 rice mutants

Hybrid combi- nations	Phenotype of F₁	F₂ population			χ²_3:1
Hybrid combi- nations	Phenotype of F₁	Wild-type plant number	Mutant-phenotypic plant number	Total number	χ²_3:1
pal3-1 × WT	WT	190	60	250	0.13
pal3-2 × WT	WT	196	68	264	0.08
pal3-3 × WT	WT	180	63	243	0.11
pal3-4 × WT	WT	201	69	270	0.04

图2 PAL3基因的图位克隆 (A) PAL3基因定位于第11号染色体InDel标记R11-3和R11-5之间; (B), (C) PAL3的精细定位。进一步将PAL3基因定位于M3和M5标记之间, 该区间包含7个开放阅读框(ORFs)。标记下方的数字表示重组个体的数目; (D) PAL3基因的结构示意图及pal3突变体的各突变位点。WT: 野生型

Figure 2 Map-based cloning of the PAL3 gene (A) The PAL3 gene was mapped to a region between InDel markers R11-3 and R11-5 on chromosome 11; (B), (C) Fine-mapping of PAL3. The PAL3 gene was further delimited to the region between markers M3 and M5, which contains seven open reading frames (ORFs). The number of recombinants is indicated beneath the marker positions; (D) Schematic structure of the PAL3 gene and the mutation sites of pal3 mutants. WT: Wild type

表3 精细定位区间候选基因注释

Table 3 Annotations of candidate genes in the fine-mapping region

Gene ID	Annotation
LOC_Os11g12680	Expressed protein
LOC_Os11g12690	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12700	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12710	Retrotransposon protein, putative, unclassified, expressed
LOC_Os11g12720	Retrotransposon, putative, centromere-specific
LOC_Os11g12730	Transposon protein, putative, CACTA, En/Spm sub-class, expressed
LOC_Os11g12740	Peptide transporter PTR2, putative, expressed

图3 PAL3跨膜域及系统发育分析 (A) PAL3的12个跨膜域及pal3-1、pal3-2和pal3-4突变体的突变位点; (B) PAL3及其同源蛋白的系统发育分析。系统发育树采用MEGA X软件, bootstrap重复1 000次, 最大似然距离法构建。

Figure 3 Transmembrane regions and phylogenetic analysis of PAL3 (A) 12 transmembrane domains of PAL3 and the mutation sites of the pal3-1, pal3-2 and pal3-4 mutants; (B) Phylogenetic analysis of PAL3 and its homologs. The phylogenetic analysis was carried out by MEGA X with 1 000 bootstrap replicates and was constructed using the distance method with maximum likelihood.

表4 PAL3及其同源家族基因的基因号和名称

Table 4 Locus ID and gene name of PAL3 and its homologs

Species	Locus ID	Gene name
Rice	LOC_Os11g12740	PAL3/SP1
	LOC_Os07g01070	OsPTR1
	LOC_Os12g44100	OsPTR2
	LOC_Os10g33210	OsPTR3
	LOC_Os07g41250	OsPTR4
	LOC_Os04g50940	OsPTR5
	LOC_Os04g50950	OsPTR6
	LOC_Os01g04950	OsPTR7
	LOC_Os03g51050	OsPTR8
	LOC_Os06g49250	OsPTR9
	LOC_Os03g13274	OsNTR1.1
	LOC_Os10g40600	OsNRT1.1B
	LOC_Os01g54515	OsNPF4.5
Arabidopsis	At1g12110	AtNRT1.1
	At5g62680	AtNRT1.10
	At1g52190	AtNRT1.11
	At1g33440	AtNRT1.13
	At1g59740	AtNRT1.14
	At1g72120	AtNRT1.15
	At1g72125	AtNRT1.16
	At1g69850	AtNRT1.2
	At3g21670	AtNRT1.3
	At2g26690	AtNRT1.4
	At1g32450	AtNRT1.5
	At1g27080	AtNRT1.6
	At1g69870	AtNRT1.7
	At4g21680	AtNRT1.8
	At1g18880	AtNRT1.9
	At3g54140	AtPTR1
	At2g02040	AtPTR2/AtNTR1
	At5g46050	AtPTR3
	At2g02020	AtPTR4
	At5g01180	AtPTR5
	At1g62200	AtPTR6
	At1g27040	AIT2
	At3g25260	AIT3
	At3g25280	AIT4
	At3g47960	GTR1
	At3g45650	NAXT1
	At1g68570	Nitr

图4 PAL3及其同源蛋白序列比对黑色背景表示所有氨基酸序列都高度同源, 灰色部分表示部分氨基酸序列同源, 白色部分表示氨基酸序列完全不同源。*依次为pal3-1、pal3-2和pal3-4突变体的突变位点; △为Hap1和Hap3的差异位点。

Figure 4 Sequences alignment of PAL3 and its homologs The black background represent that all amino acid sequences are homologous. The gray part represent that some of the amino acid sequences are homologous. The white part represent that the amino acid sequences are completely different. * indicate the mutation sites of the pal3-1, pal3-2 and pal3-4 mutants in turn; △: The variation sites between Hap1 and Hap3.

图5 PAL3基因的单倍型分析 (A) PAL3基因结构及各单倍型变异所在位置; (B) PAL3在栽培稻中的单倍型。单倍型数据通过水稻分子育种整合组学知识库(MBKbase) (Peng et al., 2020)分析而来, 共包含2 187个栽培稻品种, 分析时去除了内含子变异、杂合变异和同义变异(-: 碱基对缺失); (C) Hap1和Hap3差异位点的保守性分析(Δ: Hap1和Hap3的差异位点)。

Figure 5 Haplotype analysis of PAL3 (A) Structure of PAL3 gene and the location of haplotypes; (B) Haplotypes of PAL3 in the cultivated rice. Haplotype data were analyzed by Rice Molecular Breeding Knowledgebase (MBKbase) (Peng et al., 2020), including 2 187 cultivated rice varieties. Variations on intron, heterozygous variations and synonymous variations were removed during analysis (-: The deletion of base pair); (C) Sequence alignment around the variation sites between Hap1 and hap3 (Δ: The variation sites between Hap1 and Hap3).

图6 PAL3在野生稻中的单倍型野生稻单倍型数据来源于禾本科基因组数据库(Gramene)和水稻相关物种基因组数据库(Rice Relatives-GD) (Mao et al., 2019)。*: D475和G476缺失; ●: 无数据

Figure 6 Haplotype of PAL3 in the wild rice Haplotype data of wild rice were from Gramene and Rice Relatives-GD (Mao et al., 2019). *: D475 and G476 deletion; ●: No datum

图7 PAL3基因Hap1、Hap2和Hap3的亚种组成及穗型分析 (A) Hap1、Hap2和Hap3的亚种构成; (B) Hap1、Hap2和Hap3的穗长比较; (C) Hap1、Hap2和Hap3的穗长统计。不同小写字母表示差异显著(P<0.05)。

Figure 7 Subspecies composition and panicle phenotype analysis of Hap1, Hap2 and Hap3 of the PAL3 gene (A) Subspecies composition of Hap1, Hap2 and Hap3; (B) Comparison of panicle length among Hap1, Hap2 and Hap3; (C) Statistic of panicle length of Hap1, Hap2 and Hap3. Different lowercase letters indicate significant differences (P<0.05).

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