水稻维生素B1合成相关基因OsTHIC的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0797

摘要/Abstract

摘要：

【目的】维生素B₁（vitamin B₁, VB₁）是生物所必需的微量元素，其作为多个酶的辅因子，参与重要的细胞代谢途径，而水稻中维生素B₁合成途径还有待深入研究。本研究旨在解析水稻维生素B₁合成相关基因OsTHIC的生物学功能。【方法】综合运用诱变技术、色素测定、Mutmap+、基因编辑及非靶向代谢组分析等手段克隆目的基因并解析其功能。【结果】从EMS诱变的水稻突变体库中发现一个心叶白化致死突变体wll1（white leaf and lethal 1）。wll1从四叶期开始出现心叶白化表型，白化表型逐渐扩展到其他叶片并导致幼苗死亡。与野生型相比，wll1的叶绿素含量与类胡萝卜素含量显著降低。利用Mutmap+及基因编辑技术，确定目的基因为维生素B₁合成相关基因OsTHIC。OsTHIC在叶片具有较高表达量，OsTHIC蛋白定位于叶绿体。wll1及OsTHIC的敲除突变体中维生素B₁含量均显著低于野生型，外施维生素B₁可以恢复wll1的突变表型。外施维生素B₁及OsTHIC突变均会影响维生素B₁合成相关基因的表达。非靶向代谢组测序分析表明，野生型与wll1差异代谢物在氨基酸的生物合成、辅因子的生物合成、ABC转运器及氨酰基-tRNA的生物合成等方面显著富集。【结论】OsTHIC通过调控维生素 B₁ 含量，参与氨基酸合成等代谢过程，在水稻生长发育过程中发挥重要作用。

关键词: OsTHIC, 维生素B₁, 叶色, Mutmap+, 水稻

Abstract:

【Objective】 Vitamin B₁ (VB₁) is a biologically essential micronutrient that acts as a cofactor for several enzymes and is involved in important cellular metabolic pathways. However, the synthesis pathway of vitamin B₁ in rice remains elusive. The aim of this study is to elucidate the biological functions of OsTHIC, a gene related to vitamin B₁ synthesis in rice.【Method】 We used a combination of mutagenesis techniques, pigment measurement, Mutmap+, gene editing, and non-targeted metabolomics analysis to clone the target gene and analyze its biological functions.【Result】 An albino lethal mutant, wll1 (white leaf and lethal 1), was obtained from EMS-induced mutant pool of japonica rice variety Nipponbare. The fourth leaf of wll1 had albino phenotype and the albino phenotype spread to other leaves, ultimately leading to seedling death. The photosynthetic chlorophyll and carotenoid contents of the wll1 mutant was significantly lower than that of the wild type (WT). The target gene OsTHIC involved in VB₁ synthesis was determined by MutMap+ and gene editing technology. OsTHIC gene was expressed in various tissues of rice, with the highest expression in the leaves and OsTHIC protein was localized in chloroplasts. The VB₁ content in the wll1 was significantly lower than that of the WT. Exogenous VB₁ restored the mutated phenotype of wll1. Exogenous VB₁ and OsTHIC mutation both affected the expressions of genes related to VB₁ synthesis. Non-targeted metabolome sequencing analysis showed that differential metabolites between wild type and wll1 were significantly enriched in amino acid biosynthesis, cofactor biosynthesis, ABC transporters, and aminoacyl-tRNA biosynthesis pathways.【Conclusion】 OsTHIC plays an important role in the growth and development of rice by regulating the content of VB₁ and participating in metabolic processes such as amino acid synthesis.

Key words: OsTHIC, vitamin B₁, leaf color, Mutmap+, rice

张超, 王子瑞, 孙亚丽, 毛馨晨, 唐家琪, 于恒秀. 水稻维生素B₁合成相关基因OsTHIC的功能研究[J]. 生物技术通报, 2024, 40(2): 99-108.

ZHANG Chao, WANG Zi-rui, SUN Ya-li, MAO Xin-chen, TANG Jia-qi, YU Heng-xiu. Functional Study of Vitamin B₁ Synthesis-related Gene OsTHIC in Rice[J]. Biotechnology Bulletin, 2024, 40(2): 99-108.

图/表 8

表1 RT-qPCR所用引物序列

Table 1 Sequences of primers used in RT-qPCR

Primer name	Forward primer sequence（5'-3'）	Reverse primer sequence（5'-3'）
OsTHIC-RT	AAGATGTGCAGGACAGGAGC	ACATGGCCAGATTCCTCGTG
OsTH1-RT	CGCCGTCGAGGACCTCAT	GACTGCGTGTCGTGGTTCATC
OsTHI1-RT	TACGACGAGCAGGAGGACTAC	CGTTGAACAGCTTCACGTTGG
OsPALE1-RT	GCCAATACATCGCCCAGG	CGACGATGGTGGCCTTGT
TPK1-RT	AGGTCCGCATGAGGTACAAG	TCAACTATTTCGGCACCCAG
TPK2-RT	GCCCCGCCTGTGGACTCA	CGGGTGCTTTCATAGTCTGGA
TPK3-RT	TCAGGACACCACCGATTTAC	CATCTCGTGGTCAAACCTTC
Ubiquitin	CAAGATGATCTGCCGCAAATGC	TTAACCAGTCCATGAACCCG

图1 wll1表型及光合色素测定 A: 野生型与wll1在一叶期、二叶期和三叶期的表型。标尺：1 cm。B: 野生型与wll1四叶期表型。箭头指示叶片白化位置。标尺：1 cm；C: 野生型与wll1四叶期第三叶光合色素测定。D: 野生型与wll1四叶期第四叶光合色素测定。n.s. 表示无显著性差异。**表示显著性差异（P <0.01），下同

Fig. 1 Phenotypic characterization and photosynthesis pigment measurement of wll1 A: Phenotypes of of the seedlings at one-, two-, and three-leaf stage of wild type and wll1. Bar=1 cm. B: Phenotypes of the seedlings at four-leaf stage of wild type and wll1. Arrows indicate white leaves. Bar=1 cm. C: Photosynthesis pigment content of the third leaves of wild type and wll1 at four-leaf stage. D: Photosynthesis pigment measurement of the fourth leaves of wild type and wll1 at four-leaf stage. n.s. indicates no significant differencest. Asterisks indicate significant differences（**P <0.01）， the same below

表2 候选突变位点

Table 2 Candidate mutation sites in wll1

Position	Mutation type	Index^WT	Index^Mutant	Annotation
Chr3-24858822	G to A	0.29	1	LOC_Os03g44240 Intron
Chr3-25176848	G to A	0.41	1	Intergenic region
Chr3-26049430	G to A	0.11	1	Intergenic region
Chr3-26101874	G to A	0.15	1	Intergenic region
Chr3-26268328	G to A	0.37	1	Intergenic region
Chr3-26295456	G to A	0.39	1	LOC_Os03g46480 Intron
Chr3-26372631	G to A	0.36	1	LOC_Os03g46590 Exon
Chr3-26862420	T to C	0.22	1	Intergenic region
Chr3-26955051	G to A	0.25	1	LOC_Os03g47610 Exon
Chr3-27059507	G to A	0.36	1	LOC_Os03g47740 Exon
Chr3-27128816	G to A	0.32	1	LOC_Os03g47790 Intron
Chr3-27371505	G to A	0.35	1	Intergenic region
Chr3-27377064	G to A	0.31	1	LOC_Os03g48140 Exon
Chr3-27379000	G to A	0.29	1	LOC_Os03g48140 Intron
Chr3-27418905	G to A	0.35	1	LOC_Os03g48180 Promoter

图2 敲除OsTHIC基因产生类wll1表型 A: OsTHIC基因结构。三角形指示wll1中的突变位置，核苷酸及氨基酸变化标于下方。基因编辑靶序列位置以箭头指示，蓝色线表示“核糖开关”序列；B: CRISPR/Cas9基因编辑靶序列及敲除突变体OsTHIC-Cr中靶位点序列。插入的核苷酸标为红色；C：OsTHIC-Cr表型，标尺：5 cm

Fig. 2 wll1-like phenotype by knocking out OsTHIC A： Gene structure of OsTHIC. The triangle indicates the position of mutation in wll1. Nucleotide and amino acid modification in wll1 are listed below. The position of target sequence for gene editing is marked with arrow. Blue line indicates the position of riboswitch. B: Target sequence for CRISPR/Cas9 gene editing and genotype of knock-out mutants of OsTHIC（OsTHIC-Cr）. Inserted nucleotide is shown in red. C：Phenotype of OsTHIC-Cr. Bar=5 cm

图3 OsTHIC表达模式及OsTHIC亚细胞定位模式 A: OsTHIC在水稻不同组织的相对表达量。Seed 1：授粉后2 d的种子；Seed 2：授粉后7 d的种子；Seed 3：授粉后25 d的种子；不同字母表示具有显著性差异（P≤0.01）；B: OsTHIC的亚细胞定位模式。红色信号为叶绿体自发荧光

Fig. 3 Expression pattern of OsTHIC and subcellular localization of OsTHIC A: Relative expressions of OsTHIC in different tissues. Seed 1: Seeds 2 DAF（days after pollination）. Seed 2: Seeds 7 DAF. Seed 3: Seeds 25 DAF. Different letters indicate significant difference. B: Subcellular localization of OsTHIC in rice protoplasts. Chloroplast auto-fluorescence is in red

图4 OsTHIC突变体维生素B1含量及外施维生素B1表型 A: 野生型、wll1及OsTHIC-Cr第四叶中维生素B1含量。B：wll1外施维生素B1后表型。标尺：5 cm

Fig. 4 Vitamin B1 content and phenotype after exogenous vitamin B1 treatment of OsTHIC mutant A: Vitamin B1 content in the fourth leaf of wild type, wll1, and OsTHIC-Cr. B: Phenotype of wll1 plant sprayed with exogenous vitamin B1. Bar=5 cm

图5 OsTHIC突变及外施维生素B1对维生素B1合成相关基因表达量影响 A:野生型及wll1维生素B1合成相关基因表达量比较；B:外施维生素B1植株与对照植株维生素B1合成相关基因表达量比较

Fig. 5 Influences on expressions of vitamin B1 biosynthesis genes by mutation of OsTHIC and exogenous vitamin B1 treatment A: Comparison of expression level of vitamin B1 biosynthesis genes between wild type and wll1. B: Comparison of expression level of vitamin B1 biosynthesis genes between plants treated with vitamin B1 and control plants

图6 野生型与wll1非靶向代谢组分析 A: 代谢物主成分分析图；B：野生型与wll1差异代谢物火山图；C：差异代谢物 KEGG 通路富集分析

Fig. 6 Analysis of untargeted metabolomics of wild type and wll1 A: PCA of metabolite between wild type and wll1. B: Volcano map of differential metabolite between wild type and wll1. C: Enrichment analysis of differential metabolite KEGG pathway

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水稻维生素B₁合成相关基因OsTHIC的功能研究

Functional Study of Vitamin B₁ Synthesis-related Gene OsTHIC in Rice

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