Global identification of key genes for pollen germination in rice through high-throughput screening and gene editing.

IF 9.3 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Eui-Jung Kim, Woo-Jong Hong, Yu-Jin Kim, Eun Young Kim, Sang Dae Yun, Sunok Moon, Su-Kyoung Lee, Soon Ki Park, Ki-Hong Jung
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引用次数: 0

Abstract

Successful reproduction depends on the stable germination and growth of the pollen tubes (PT). However, the molecular mechanisms involved in rice PT growth and development remain largely unknown. In a previous study, microarray transcriptome analysis identified 627 genes preferentially expressed in the tricellular and germinating pollen of rice (i.e., Oryza sativa ssp. japonica). To elucidate key genes involved in the gene transfer process facilitated by male gametophytes, we systematically screened T-DNA lines containing disrupted sequences that corresponded to these 627 genes and analyzed the genotypes of heterozygote progeny from 107 T-DNA-indexed lines covering 105 genes. We found that 42 lines exhibited a distorted segregation ratio among the wild-type (WT), heterozygote (HT), and homozygote (HM) genotypes, which deviated from the expected Mendelian ratio of 1:2:1 (WT:HT:HM). Further characterization using CRISPR/Cas9 mutants revealed that knockout mutants of certain genes that exhibited segregation distortion in the T-DNA insertion region were completely sterile. Moreover, even when T-DNA insertion lines followed Mendelian segregation patterns, sterility could be induced by simultaneously mutating functionally redundant genes, thereby overcoming genetic compensation. Interestingly, although some T-DNA insertion lines exhibited segregation ratios approximating 1:1:0, the corresponding CRISPR/Cas9 mutants produced homozygous seeds and showed partial sterility. Partial sterility suggests that despite mutant pollen grains being less competitive than WT pollen, they retain their fertilization potential under relaxed competition from WT pollen. Beyond mutant-based analysis, transcriptomic profiling of sterile mutant lines provided additional insight into the regulatory relationship between key germination regulators and the 105 target genes studied here. Overall, this study demonstrates the effectiveness of a multi-pronged strategy to accelerate the identification of defective phenotypes using mutant studies and provides valuable genetic resources for inducing novel male sterility in rice.

水稻花粉萌发关键基因的高通量筛选与基因编辑研究
成功的繁殖取决于花粉管(PT)的稳定发芽和生长。然而,水稻PT生长发育的分子机制仍不清楚。在之前的一项研究中,微阵列转录组分析鉴定出627个基因在水稻(即Oryza sativa ssp)的三细胞和萌发花粉中优先表达。粳稻)。为了阐明参与雄性配子体基因转移过程的关键基因,我们系统地筛选了包含这627个基因对应的中断序列的T-DNA系,并分析了107个T-DNA索引系(涵盖105个基因)的杂合子基因型。结果发现,42个品种的野生型(WT)、杂合子(HT)和纯合子(HM)基因型的分离比例存在扭曲,偏离了孟德尔基因型1:2:1 (WT:HT:HM)的预期分离比例。利用CRISPR/Cas9突变体的进一步表征表明,在T-DNA插入区表现出分离扭曲的某些基因的敲除突变体是完全无菌的。此外,即使T-DNA插入系遵循孟德尔分离模式,也可以通过同时突变功能冗余基因来诱导不育,从而克服遗传补偿。有趣的是,尽管一些T-DNA插入系的分离比例接近1:1:0,但相应的CRISPR/Cas9突变体产生纯合子种子,并表现出部分不育。部分不育性表明,尽管突变体花粉粒的竞争力不如WT花粉,但它们在与WT花粉的竞争中保持了较弱的受精潜力。除了基于突变体的分析之外,不育突变系的转录组学分析为研究105个目标基因与关键发芽调节因子之间的调控关系提供了额外的见解。总之,本研究证明了多管齐下的策略在利用突变体研究加速缺陷表型鉴定方面的有效性,并为诱导水稻新型雄性不育提供了宝贵的遗传资源。
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来源期刊
Journal of Integrative Plant Biology
Journal of Integrative Plant Biology 生物-生化与分子生物学
CiteScore
18.00
自引率
5.30%
发文量
220
审稿时长
3 months
期刊介绍: Journal of Integrative Plant Biology is a leading academic journal reporting on the latest discoveries in plant biology.Enjoy the latest news and developments in the field, understand new and improved methods and research tools, and explore basic biological questions through reproducible experimental design, using genetic, biochemical, cell and molecular biological methods, and statistical analyses.
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