Characterization and Transcriptome Analysis of a Mutant with Short Panicle and Small Grain from Zhejing 99

doi:10.16819/j.1001-7216.2024.231111

Abstract

Abstract:

【Objective】This study aims to explore the formation mechanism of the superior panicle type in Zhejing 99 and lays a scientific foundation for understanding the developmental mechanisms of rice panicle types and their breeding applications.【Method】We used ethyl methanesulfonate (EMS) to induce mutations in Zhejing 99, resulting in a short panicle and small grain mutant. Molecular markers were employed to obtain linkage intervals, and candidate genes were identified through RNA sequencing (RNA-Seq) and DNA sequencing. The subcellular localization of the mutant gene was observed using a laser confocal microscopy. Additionally, transcriptome sequencing was conducted to analyze gene expression and the expression characteristics of related genes.【Result】Phenotypic identification revealed differences in plant type, panicle type, and grain type of the Ossp3 mutant compared to Zhejing 99. The mutant phenotype was caused by a base insertion in the candidate gene Os02g0450000. Subcellular localization showed that the gene product is located in the nucleus. In the young panicles of the Ossp3 mutant, 329 differentially expressed genes (DEGs) were identified, primarily distributed on chromosomes 1, 3, and 4. KEGG enrichment analysis highlighted significant biological processes, including plant hormone signaling pathways and MAPK signaling pathways in plants. Analysis of related gene expression suggests that the evolutionarily conserved plant-specific gene OsSP3 influences auxin and cytokinin content and their corresponding signaling pathways through the MAPK signaling pathway or by interacting with bZIP transcription factors, thereby regulating rice panicle development.【Conclusion】The gene mutation in the superior panicle type variety Zhejing 99, specifically in Os02g0450000, results in the phenotype of short panicles and small grains. Its biological function is mediated through nuclear localization. RNA-Seq analysis indicates that the mutated gene may synergistically regulate the growth and development of rice panicles by affecting auxin and cytokinin signaling pathways and may also directly or indirectly influence grain type through interaction with OsbZIP47.

Key words: short panicle and small grain, phenotypic identification, RNA-Seq, transcriptome analysis

摘要：

【目的】探明浙粳99优良穂型的形成机制，为水稻穗型发育机制研究及其育种应用提供科学依据。【方法】利用EMS诱变浙粳99获得短穗小粒突变体，借助分子标记获得连锁区间，采用RNA-Seq及DNA测序确定候选基因，并利用激光共聚焦显微镜观察突变基因的亚细胞定位。使用转录组测序的方法对该突变体的基因表达情况进行分析。【结果】Ossp3与浙粳99在株型、穗型以及粒型上均存在差异。该突变表型由候选基因Os02g0450000发生碱基插入引起。亚细胞定位结果显示该基因产物定位于细胞核。Ossp3幼穗出现329个差异表达基因（DEG），主要分布在1、3和4号染色体上，KEGG富集分析显示，这些差异表达基因主要集中在植物激素信号转导途径、MARK信号通路-植物等生物学过程。相关基因表达分析显示进化上保守的植物特异性基因OsSP3通过MAPK信号通路或与bZIP转录因子作用影响生长素、细胞分裂素含量及其对应信号途径，实现对稻穗发育的调控。【结论】优良穗型品种浙粳99 Os02g0450000基因突变会产生短穗小粒的表型，其生物学功能通过核定位实现，RNA-Seq分析表明该突变基因可能通过影响生长素与细胞分裂素信号通路协同调控稻穗的生长发育，同时很可能通过与OsbZIP47直接或间接作用调控粒型。

关键词: 短穗小粒, 表型鉴定, RNA-Seq, 转录组分析

LIANG Chuyan, ZENG Wei, WANG Jiebing, YE Jing, WU Mingming, ZHAI Rongrong, ZHANG Xiaoming, ZHANG Hengmu, YE Shenghai. Characterization and Transcriptome Analysis of a Mutant with Short Panicle and Small Grain from Zhejing 99[J]. Chinese Journal OF Rice Science, 2025, 39(1): 67-81.

梁楚炎, 曾维, 王洁冰, 叶靖, 巫明明, 翟荣荣, 张小明, 张恒木, 叶胜海. 一个浙粳99短穗小粒突变体的鉴定及转录组分析[J]. 中国水稻科学, 2025, 39(1): 67-81.

Figures/Tables 15

References 61

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引物名称 Name	引物序列 Sequence (5’-3’)
OsSP3-F1	ATGTCGACGGCCGCCAAGGAGGA
OsSP3-R1	TTACACTGAGGAGCGCTCCAAAC
OsSP3-F2	AACACGGGGGACTTTGCAACATGTCGACGGCCGCCAAGG
OsSP3-R2	GACAGAGCTAGTTACATTACACTGAGGAGCGCTCCAAACT
OsSP3-F3	GGGGCCTGGGGTACCCATGTCGACGGCCGCCAAGG
OsSP3-R3	CGCCCTTGCTCACGTCGACCACTGAGGAGCGCTCCAAACT

引物名称 Name	引物序列 Sequence (5’-3’)
OsSP3-F1	ATGTCGACGGCCGCCAAGGAGGA
OsSP3-R1	TTACACTGAGGAGCGCTCCAAAC
OsSP3-F2	AACACGGGGGACTTTGCAACATGTCGACGGCCGCCAAGG
OsSP3-R2	GACAGAGCTAGTTACATTACACTGAGGAGCGCTCCAAACT
OsSP3-F3	GGGGCCTGGGGTACCCATGTCGACGGCCGCCAAGG
OsSP3-R3	CGCCCTTGCTCACGTCGACCACTGAGGAGCGCTCCAAACT

组合 Combination	F₁正常表型植株 Number of normal plants in F₁	F₂					χ²_（3:1）
组合 Combination	F₁正常表型植株 Number of normal plants in F₁	总株数 Total plant number	正常表型植株 Normal plants		突变表型植株 Mutant plants		χ²_（3:1）
Ossp3/WT	5	355		265		90	0.0117

组合 Combination	F₁正常表型植株 Number of normal plants in F₁	F₂					χ²_（3:1）
组合 Combination	F₁正常表型植株 Number of normal plants in F₁	总株数 Total plant number	正常表型植株 Normal plants		突变表型植株 Mutant plants		χ²_（3:1）
Ossp3/WT	5	355		265		90	0.0117

变异位点 Mutation site	SNP/InDel	参考基因组碱基 Reference genome bases	样品wt碱基 Sample wt base	样品sp3碱基 Sample sp3 base	候选基因 Candidate gene	原因 Reason
17287987	SNP	A	A	G	否No	同义突变GGT/GGC→Gly
17489881	SNP	C	C	T	否No	其他参考基因组碱基为T
18059754	SNP	A	A	G	否No	其他参考基因组碱基为G
18121601	SNP	C	C	A	否No	其他参考基因组碱基为A
19270791	SNP	T	T	G	否No	其他参考基因组碱基为G
19309819	SNP	A	A	G	否No	其他参考基因组碱基为G
20348604	SNP	G	G	A	否No	其他参考基因组碱基为A
14683027	InDel	A	A	AAC	是Yes	移码突变