水稻DnaJ型锌指蛋白OsDJA10正调控叶绿体发育

doi:10.13560/j.cnki.biotech.bull.1985.2026-0023

摘要/Abstract

摘要：

目的 DnaJ型锌指蛋白分子伴侣在水稻中的功能尚不完全清楚。探究OsDJA10基因（LOC_Os08g36140）敲除株系（KO）白化和RNA干扰株系（RNAi）黄化表型的分子机制，评估其过表达株系（OE）的高光耐受性，为水稻抗逆品种培育提供理论参考。方法利用CRISPR/Cas9基因编辑技术、RNAi技术和农杆菌介导转化法分别获得敲除、敲低和过表达株系，通过测定光合色素、叶绿素荧光、叶绿体超微结构、抗氧化酶活性、叶片和根系相对含水量、相对电导率、生长性状等生理指标，并结合亚细胞定位、RT-qPCR和转录组学分析，解析OsDJA10基因的生物学功能。结果 OsDJA10敲除株系幼苗期表现为白化致死表型，几乎不合成植物色素，缺乏成熟叶绿体和类囊体垛叠，叶绿体结构与发育相关基因表达显著下调，差异表达基因显著富集在光合作用通路。RNAi植株叶片黄化，光合色素含量显著低于野生型中花11、F_v/F_m和NPQ显著下调，叶绿体个数显著减少、体积显著变小且重塑类囊体垛叠，叶绿体结构与发育相关基因表达均下调。过表达株系株高、穗长及剑叶长显著降低，未对产量产生积极影响，对高光胁迫的耐受性显著高于野生型中花11。结论 OsDJA10基因通过影响光合基因转录和光合器官稳定性，正调控水稻叶绿体发育，同时增强高光胁迫耐受性。

关键词: 水稻, DnaJ型蛋白, 叶绿体, 转录, 高光胁迫, OsDJA10, 分子伴侣, 光合作用

Abstract:

Objective The function of DnaJ-type zinc finger protein molecular chaperones in rice remains incompletely understood. Using the OsDJA10 gene (LOC_Os08g36140) as a model, this study investigates the molecular mechanisms underlying the albino phenotype in its knockout (KO) lines and the yellowing phenotype in its RNA interference (RNAi) lines. This study also evaluates the high-light tolerance of its overexpression (OE) lines, providing a theoretical reference for breeding stress-resistant rice varieties. Method CRISPR/Cas9 gene editing, RNAi, and Agrobacterium-mediated transformation were employed to generate knockout, knockdown, and overexpression lines, respectively. The biological functions of the OsDJA10 gene were elucidated by measuring photosynthetic pigments, chlorophyll fluorescence, chloroplast ultrastructure, antioxidant enzyme activities, relative water content and relative electrical conductivity of leaves and roots, and growth traits, combined with subcellular localization, RT-qPCR, and transcriptomic analysis. Result OsDJA10 knockout seedlings exhibited a white-leaf lethal phenotype, with near-complete absence of plant pigments, mature chloroplasts, and thylakoid stacks. Expression of genes related to chloroplast structure and development was significantly downregulated, and differentially expressed genes were significantly enriched in photosynthetic pathways. RNAi-treated plants exhibited leaf chlorosis, significantly reduced photosynthetic pigment content compared to wild-type Zhonghua 11, and markedly downregulated F_v/F_m and NPQ. Chloroplast number decreased significantly, volume shrank markedly, thylakoid stacks were remodeled, and expression of chloroplast structure and development-related genes was downregulated. OsDJA10 -overexpressing lines showed significantly reduced plant height, panicle length, and flag leaf length, with no positive effect on yield; however, they exhibited significantly higher tolerance to high-light stress compared to wild-type Zhonghua 11. Conclusion The OsDJA10 gene positively regulates chloroplast development in rice by influencing photosynthetic gene transcription and photosynthetic organ stability, while enhancing tolerance to high-light stress.

Key words: rice, DnaJ-type protein, chloroplast, transcription, high light stress, OsDJA10, molecular chaperone, photosynthesis

任琼, 钟娇, 段玉, 秦童, 康振辉. 水稻DnaJ型锌指蛋白OsDJA10正调控叶绿体发育[J]. 生物技术通报, 2026, 42(9): 93-106.

REN Qiong, ZHONG Jiao, DUAN Yu, QIN Tong, KANG Zhen-hui. DnaJ-type Zinc Finger Protein OsDJA10 Positively Regulates Chloroplast Development in Rice[J]. Biotechnology Bulletin, 2026, 42(9): 93-106.

图/表 10

图1 OsDJA10基因的鉴定A：LOC_Os08g36140的CDS编码序列和氨基酸序列，图中红色框代表DnaJ型“CxxCxGxG”锌指结构域的保守基序；B：水稻DnaJ基因的染色体定位，图中红色字母代表OsDJA10的染色体位置；C：水稻和拟南芥的DnaJ家族系统进化树

Fig. 1 Identification of OsDJA10A: The coding sequence and amino acid sequence of LOC_Os08g36140. The red box indicates the conserved motif of the DnaJ-type “CxxCxGxG” zinc finger domain. B: Chromosomal localization of the rice DnaJ gene. The red letters denote the chromosomal position of OsDJA10. C: Phylogenetic tree of the DnaJ family in rice and Arabidopsis

图2 OsDJA10的表达模式和亚细胞定位A：OsDJA10在水稻不同组织中的表达；B：OsDJA10的节律表达；C：OsDJA10在不同胁迫处理下的表达量，冷：4 ℃，高温：42 ℃；D：OsDJA10在高光胁迫下不同时间点的表达水平；E：OsDJA10在水稻原生质体中的亚细胞定位；绿色荧光表示GFP信号，红色荧光表示叶绿体自发荧光；Bright：明场。数据以平均值±SD表示，n=3。比例尺= 10 μm。数据采用t检验。**** P< 0.000 1，下同

Fig. 2 Expression pattern and subcellular localization of OsDJA10A: Expression of OsDJA10 in different tissues of rice. B: Circadian expression of OsDJA10. C: Expression analysis of OsDJA10 under different stress conditions. Cold stress: 4 ℃; high temperature: 42 ℃. D: Expression of OsDJA10 at different time points under high light stress. E: Subcellular localization of OsDJA10 in rice protoplasts. Green fluorescence indicates GFP signal, red fluorescence indicates chloroplast autofluorescence. Bright: Bright field. Data are presented as mean ± SD. n=3. Scale bar = 10 μm. Data were analyzed using t-tests. **** P < 0.000 1. The same below

图3 OsDJA10敲除突变体的分析鉴定A：OsDJA10的sgRNA靶点位置，蓝色方框代表外显子，绿色方框代表起始密码子，红色方框代表终止密码子；B：株系中各CRISPR/Cas9基因组编辑频率汇总；C：CRISPR/Cas9编辑T0代的序列对比图；D：CRISPR/Cas9编辑T1代的序列对比图。图中划线序列代表sgRNA和PAM序列，蓝色字体代表碱基替换，虚线代表缺失碱基，红色字体代表插入碱基序列

Fig. 3 Analysis and identification of OsDJA10 knockout mutantsA: sgRNA target site location for OsDJA10. The blue boxes represent exons, the green boxes represent start codons, and the red boxes represent stop codons. B: Summary of CRISPR/Cas9 genome editing frequencies across strains. C: Sequence alignment of CRISPR/Cas9-edited T0 generation. D: Sequence alignment of CRISPR/Cas9-edited T1 generation. Underlined sequences denote sgRNA and PAM sequences; blue letters indicate base substitutions, dashed lines represent deleted bases, and red letters indicate inserted nucleotide sequences

图4 OsDJA10纯合突变体显示白化表型A：水稻种子萌发后3 d的表型；标尺为1 cm，KO1、KO2和KO3分别为阳性编辑株系E387-3、E387-18和E387-20；B：水稻种子萌发后9 d的表型，标尺为2 cm；C：生长周期为21 d，标尺为2 cm；D：Chl a、Chl b和Car含量测定；E：叶绿体超微结构，CP：叶绿体。数据为6个生物学重复的均值±SD。数据采用t检验

Fig. 4 Homozygous mutant of OsDJA10 displays an albino phenotypeA: Phenotype of rice seeds 3 days after germination, scale bar = 1 cm; KO1, KO2, and KO3 represent positive edit lines E387-3, E387-18, and E387-20, respectively. B: Phenotype of rice seeds 9 days after germination, scale bar = 2 cm. C: Growth cycle at 21 days post-germination, scale bar = 2 cm. D: Chl a, Chl b, and Car content measurements. E: Chloroplast ultrastructure. CP: Chloroplast. Data represent the mean ± SD of six biological replicates. All data were analyzed using Student’s t-test

图5 叶绿体相关基因表达及蛋白水平分析A：WT和OSDJA10叶绿体基因和核编码的叶绿体基因的RT-qPCR分析；I类代表PEP依赖基因；II类代表PEP和NEP依赖基因；III类代表NEP依赖基因；B：Western blot分析WT和osdja10幼苗的叶绿体编码蛋白，以β-actin作为内参蛋白；数据为3个生物重复的均值±SD；所有数据采用t检验；***P<0.001，下同

Fig. 5 Analysis of chloroplast-associated gene expression and protein levelsA: RT-qPCR analysis of chloroplast-encoded genes and nucleus-encoded chloroplast genes in WT and OsDJA10. Class I: PEP-dependent genes; Class II: PEP- and NEP-dependent genes; Class III: NEP-dependent genes. B: Western blot analysis of chloroplast-encoded proteins in WT and osdja10 seedlings, with β-actin as the internal control protein. Data represent the mean ± SD of three biological replicates. All data were analyzed using Student’s t-test. ***P < 0.001. The same below

图6 OsDJA10敲低赋予水稻黄绿色表型A：RT-qPCR分析SiRNAi1-i8的干扰水平，数据为3个生物重复的均值±SD，数据采用单因素方差分析；B：WT和RNAi株系的表型，Bar=2 cm；C：WT和RNAi株系的叶片表型对比，Bar=1 cm；D：Chla、Chlb和Car含量；E：鲜重；F：根长；G：地上部高度；H：PSII最大光量子效率；I：PSII的实际光量子效率；J：光化学淬灭；K：非光化学淬灭；数据为6个生物学重复的均值±SD，数据采用单因素方差分析；L：叶绿体超微结构；M：叶绿体基因和核编码的叶绿体基因的RT-qPCR分析；I类代表PEP依赖基因；II类代表PEP和NEP依赖基因；III类代表NEP依赖基因；数据为6个生物重复的均值±SD。所有数据均采用t检验，*P <0.05，**P<0.01，下同

Fig. 6 Knockdown of OsDJA10 confers a yellowish-green phenotype in riceA: RT-qPCR analysis of siRNAi1-i8 interference levels. Data represent the mean ± SD of three biological replicates analyzed by one-way ANOVA. B: Phenotype of WTand RNAi lines. Bar = 2 cm. C: Comparison of leaf phenotypes between WT and RNAi lines. Bar = 1 cm. D: Chla, Chlb, and Car content. E: Fresh weight. F: Root length. G: Above-ground height. H: Maximum quantum efficiency of PSII. I: Actual quantum efficiency of PSII. J: Photochemical quenching. K: Non-photochemical quenching. Data represent the mean ± SD of six biological replicates, analyzed by one-way ANOVA. L: Chloroplast ultrastructure. M: RT-qPCR analysis of chloroplast genes and nucleus-encoded chloroplast genes; class I represents PEP-dependent genes; class II represents PEP- and NEP-dependent genes; class III represents NEP-dependent genes. Data represent the mean ± SD from six biological replicates. All data were analyzed using Student’s t-test, *P <0.05,**P < 0.01. The same below

图7 OsDJA10过表达株系在高光胁迫下的表型及生理生化指标A‒D：正常（A、B）和2 d高光处理（C、D）条件下的表型；Bar=2 cm；E：OsDJA10过表达株系的基因组水平分子鉴定；F：OsDJA10过表达株系的转录水平分子鉴定；G‒R：8 h高光处理后WT和OE株系的生理生化指标；数据均为平均数±标准误，统计分析采用双因素方差分析

Fig. 7 Phenotypic and physiological and biochemical indicators of OsDJA10 overexpression lines under high light stressA-D: Phenotype under normal conditions (A, B) and 2 d high light treatment (C, D). Scale bar = 2 cm. E: Genomic-level molecular characterization of the OsDJA10 overexpressing line. F: Transcriptional-level molecular characterization of the OsDJA10 overexpressing line. G-R: Physiological and biochemical indicators of WT and OE lines after 8 h high light treatment. Data are presented as mean ± standard error (SE). Statistical analysis was performed using two-way ANOVA

图8 WT和KO的转录组分析A：WT与KO基因表达水平的相关性，Wt1、Wt2、Wt3分别代表3株独立的野生型ZH11株系，Mt1、Mt2、Mt3分别代表白化突变体KO1、KO2、KO3；B：WT与KO之间差异表达基因（DEGs）的统计分析；C：KEGG通路富集分析；D：GO通路富集分析

Fig. 8 Transcriptome analysis of WT and KO plantsA: Correlation analysis of gene expression levels between WT and KO, Wt1, Wt2, and Wt3 represent three independent wild-type ZH11 lines, while Mt1, Mt2, and Mt3 represent the albino mutants KO1, KO2, and KO3 respectively. B: Statistical analysis of differentially expressed genes (DEGs) between WT and KO. C: KEGG pathway enrichment analysis. D: GO pathway enrichment analysis

图9 OsDJA10过表达株系相关农艺性状A‒B：WT和OE株系成熟期的表型，Bar=30 cm；C‒D：WT和OE株系成熟期穗长的表型，Bar=2 cm；E：WT和OE的粒长表型，Bar=1 cm；F：WT和OE的粒宽表型，Bar=1 cm；G：株高；H：穗长；I：剑叶长；J：剑叶宽；K：分蘖数；L：粒长；M：粒宽；N：千粒重；数据表示为平均值±标准差；10次生物学重复；采用t检验进行统计分析

Fig. 9 Agronomic traits of OsDJA10 OE linesA‒B: Maturity-stage phenotypes of WT and OE lines, bar=30 cm. C‒D: Mature spike length phenotypes of WT and OE lines, bar=2 cm. E: Grain length phenotypes of WT and OE, bar=1 cm. F: Grain width phenotypes of WT and OE, bar=1 cm. G: Plant height. H: Spike length. I: Flag leaf length. J: Flag leaf width. K: Tillering number. L: Grain length. M: Grain width. N: Thousand-grain weight. Data are presented as mean ± standard deviation; ten biological replicates. Experimental data were analyzed using Student’s t-test

图10 OsDJA10参与叶绿体发育及白化形成的作用模型

Fig. 10 Model of OsDJA10 involvement in chloroplast development and albino formation

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