水稻叶脉基因OsLVD1的鉴定及功能分析

doi:10.16819/j.1001-7216.2026.251016

摘要/Abstract

摘要：

【目的】光合作用决定水稻干物质积累，叶绿素含量和叶脉密度与光合作用密切相关。本研究在水稻中鉴定调控叶绿素含量和叶脉密度的基因OsLVD1，并对其功能进行解析。【方法】通过玉米叶脉发育基因同源比对获得OsLVD1，并利用生物信息学分析其蛋白特性、系统发育关系、信号肽、跨膜结构域、保守结构域及亚细胞定位。通过CRISPR获得lvd1编辑株，以野生型ZH11为对照，测定叶绿素含量、叶脉密度、可溶性糖含量及株高、分蘖数、每穗粒数等主要农艺性状，并通过qRT-PCR分析相关基因的表达水平。【结果】生物信息学分析表明，OsLVD1编码WRKY家族转录因子，定位于细胞核。lvd1因移码突变导致蛋白提前终止。与ZH11相比，lvd1叶绿素a、叶绿素b和总叶绿素分别下降25.90%、30.75%和27.05%，剑叶小脉密度和泡状细胞数目增加。lvd1株高降低且生育期略延迟，但叶片可溶性糖、结实率、千粒重等均无显著差异。qRT-PCR分析表明，lvd1中叶绿素合成相关基因OsHEMA1和OsCHLI的表达显著下调，而叶绿素降解基因OsNYC1及叶脉发育相关基因OsPIN1b表达上调。组织表达分析显示，OsLVD1在叶片中高表达，且在拔节期达到峰值。【结论】OsLVD1功能缺失导致叶绿素含量降低、叶脉密度增加，可能通过增加叶脉密度和改变泡状细胞发育补偿光合产物运输与水分保持，从而维持产量稳定。本研究揭示OsLVD1在叶片色素代谢与维管系统发育中的新功能，为光合结构优化提供理论基础。

关键词: 水稻, OsLVD1, 叶绿素含量, 叶脉密度, WRKY转录因子, 光合作用

Abstract:

【Objective】Photosynthesis is a major determinant of dry matter accumulation in rice, with chlorophyll content and vein density being critical for photosynthetic efficiency. Here, a rice gene regulating chlorophyll content and vein density, OsLVD1, was identified and functionally analyzed.【Method】OsLVD1 was identified based on homology to maize vein development genes, and its protein features, phylogeny, conserved motifs, and subcellular localization were analyzed bioinformatically. An lvd1 mutant was generated using CRISPR/Cas9, with ZH11 as the wild type. Chlorophyll content, vein density, soluble sugar content, and major agronomic traits were examined. The expression of genes related to chlorophyll metabolism and vein development was analyzed by qRT-PCR.【Result】OsLVD1 encodes a nuclear-localized WRKY transcription factor, and the lvd1 mutation caused a frameshift leading to premature termination. Compared with ZH11, the lvd1 mutant showed reductions of 25.90%, 30.75%, and 27.05% in chlorophyll a, chlorophyll b, and total chlorophyll content, respectively, accompanied by increases in minor vein density and bulliform cell number. Plant height was reduced and growth duration was slightly delayed, whereas soluble sugar content, seed-setting rate, and thousand-grain weight were unaffected. qRT-PCR showed that the expression levels of chlorophyll biosynthesis genes OsHEMA1 and OsCHLI were downregulated, while those of the chlorophyll degradation gene OsNYC1 and the vein development gene OsPIN1b were upregulated in the lvd1 mutant. OsLVD1 was predominantly expressed in leaves, with the highest expression at the jointing stage.【Conclusion】Loss of OsLVD1 resulted in a “low chlorophyll-high vein density” phenotype, which may compensate for reduced photosynthesis by enhancing vein-mediated transport and water retention, thereby maintaining yield stability. This study demonstrates a novel role of OsLVD1 in coordinating leaf pigment metabolism and vascular development in rice.

Key words: Oryza sativa, OsLVD1, chlorophyll content, leaf vein density, WRKY transcription factor, photosynthesis

邱一洁, 齐飞扬, 杜何为, 黄敏, 李曼菲. 水稻叶脉基因OsLVD1的鉴定及功能分析[J]. 中国水稻科学, 2026, 40(4): 447-459.

QIU Yijie, QI Feiyang, DU Hewei, HUANG Min, LI Manfei. Identification and Functional Analysis of the Rice Vein Development Gene OsLVD1[J]. Chinese Journal OF Rice Science, 2026, 40(4): 447-459.

图/表 9

表1 本研究使用的在线平台

Table 1. Online platforms used in the research

序号 No.	软件名称 Software name	网址 Website
1	国家水稻数据中心National Rice Data Center	https://www.ricedata.cn/
2	NCBI	https://www.ncbi.nlm.nih.gov/
3	SMART	http://smart.embl.de/
4	ProtParam	https://web.expasy.org/protparam/
5	SOPMA	https://npsa-prabi.ibcp.fr/
6	SWISS-MODEL	https://swissmodel.expasy.org/
7	EvolView	http://evolgenius.info/
8	SignalP4.1	http://www.cbs.dtu.dk/services/SignalP-4.1/
9	TMHMM	http://www.cbs.dtu.dk/services/TMHMM/
10	Predict Protein	https://www.predictprotein.org
11	Plant-mPLoc	http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/
12	CELLO	http://cello.life.nctu.edu.tw/

图1 OsLVD1蛋白的生物信息学分析图A为LVD1蛋白结构域分析，该蛋白含多个低复杂度区域（紫色方块）、1个保守结构区段（绿色方框）及WRKY保守结构域（绿色五边形）；图B为LVD1蛋白的疏水性分析，该蛋白主要为亲水蛋白；图C是LVD1蛋白的二级结构预测，α螺旋（蓝色）与无规则卷曲（紫色）比例较高；图D为OsLVD1蛋白三级结构预测模型；图E是不同物种WRKY1蛋白的聚类分析；图F含有WRKY1结构域的不同物种蛋白序列比对分析；图G为染色体定位分析。

Fig. 1. Bioinformatic analysis of the OsLVD1 protein A, Domain analysis of LVD1 protein, including multiple low-complexity regions (purple squares), one conserved segment (green box), and a WRKY conserved domain (green pentagon); B, Hydrophobicity analysis of LVD1 protein, which is mainly hydrophilic; C, Secondary structure prediction of LVD1 protein, with high proportions of α-helix (blue) and random coil (purple); D, Predicted tertiary structure model of OsLVD1 protein; E, Phylogenetic analysis of WRKY1 proteins from different species; F, Multiple sequence alignment of WRKY1 domain-containing proteins from different species; G, Chromosomal localization analysis.

表2 水稻OsLVD1基因启动子顺式作用元件

Table 2. Cis-acting elements of promoters of rice OsLVD1 genes

元件类别 Element category	元件名称 Element name	核心序列 Core sequence	数量 Number	功能注释 Function annotation
光响应元件 Light-responsive elements	GT1-motif	GGTTAAT/GCTTAA	2	光响应Light responsiveness
	G-Box	CACGTC/CACGAC	13	光响应Light responsiveness
	BOX4	ATTAAT	1	光响应Light responsiveness
	TCT-motif	TCTTAC	2	光响应Light responsiveness
	Spl	GGCCGG	1	光响应Light responsiveness
	AE-BOX	AGAAACTT	2	光响应Light responsiveness
	ATC-motif	AGTAATCT	1	光响应Light responsiveness
激素响应元件 Hormone-responsive elements	ABRE	ACGTG/CACGTG	13	脱落酸响应Abscisic acid responsiveness
	TGACG-motif	TGACG	2	茉莉酸响应MeJA responsiveness
	CGTCA-motif	CGTCA	2	茉莉酸响应MeJA responsiveness
	TGA-element	AACGAC	1	生长素响应Auxin responsiveness
	P-box	CCTTTTG	1	赤霉素响应Gibberellin responsiveness
	TATC-box	TATCCCA	1	赤霉素响应Gibberellin responsiveness
胁迫响应元件 Stress-responsive elements	LTR	CCGAAA	1	低温响应Low-temperature responsiveness
胁迫响应元件 Stress-responsive elements	MBS	CAACTG	1	干旱诱导Drought inducibility
生长发育相关元件 Growth and development-related elements	CAT-box	GCCACT	2	分生组织发育相关 Meristem expression and development

图2 OsLVD1突变对相关基因表达及其组织表达特征的影响 A：在拔节期采集ZH11和lvd1突变体叶片，采用qRT-PCR检测叶绿素合成相关基因OsHEMA1和OsCHLI，叶绿素降解相关基因OsNYC1和OsPAO，以及叶脉发育相关基因OsPIN1b的相对表达水平。各基因表达量以ZH11为对照进行标准化处理。B：qRT-PCR分析ZH11在不同生育时期根、茎和叶中OsLVD1的相对表达水平。数据以平均值 ± 标准差表示（mean ± SD, n = 3）。单因素方差分析和LSD多重比较检验差异显著性。

Fig. 2. Effects of OsLVD1 mutation on the expression of related genes and its tissue-specific expression pattern A, Relative expression levels of chlorophyll synthesis genes (OsHEMA1, OsCHLI), chlorophyll degradation genes (OsNYC1, OsPAO), and vein development gene (OsPIN1b) in leaves of ZH11 and lvd1 mutant at jointing stage by qRT-PCR, with ZH11 as the control for normalization. B, Relative expression of OsLVD1 in roots, stems, and leaves of ZH11 at different growth stages analyzed by qRT-PCR. Data are presented as mean ± SD (n = 3). Significant differences were determined by one-way ANOVA and LSD multiple comparison test (P < 0.05).

图3 ZH11与lvd1表型鉴定 A：拔节期ZH11（左）和lvd1（右）植株整体形态比较；B：ZH11（左）与lvd1（右）植株剑叶形态比较；C：拔节期ZH11与lvd1植株株高统计，数据为平均值 ± 标准差（mean ± SD，n = 30）。ns表示差异不显著（P < 0.05），采用t检验进行统计分析；D：拔节期ZH11与lvd1植株剑叶叶宽的统计（mean ± SD，n = 30），***表示差异显著（P < 0.05），采用t检验进行统计分析。E：泳道1~2为ZH11基因组DNA，泳道3~5为lvd1基因组DNA；F：lvd1蛋白三级结构；G：OsLVD1基因的CRISPR编辑位点及突变序列比对，黄色高亮处表示lvd1中相较于野生型插入的1 bp碱基，PAM序列以红色标示。

Fig. 3. Phenotypic characterization of ZH11 and lvd1 A, Overall morphology of ZH11 (left) and lvd1 (right) plants at jointing stage; B, Flag leaf morphology of ZH11 (left) and lvd1 (right) plants; C, Plant height of ZH11 and lvd1 at jointing stage (mean ± SD, n = 30). ns, not significant (P < 0.05), analyzed by Student’s t-test; D, Flag leaf width of ZH11 and lvd1 at jointing stage (mean ± SD, n = 30). ***, Significant (P < 0.05) by Student’s t-test; E, Genomic DNA PCR. Lanes 1-2, ZH11; Lanes 3-5: lvd1; F, Tertiary structure of lvd1 protein; G, CRISPR editing sites and mutant sequence alignment of OsLVD1. Yellow highlight indicates a 1 bp insertion in lvd1 compared with wild type; PAM sequence is marked in red.

表3 ZH11及lvd1生育期统计表

Table 3. Statistical table of growth periods of ZH11 and lvd1

材料 Material	出芽期 Germination stage	幼苗期 Seedling stage	分蘖期 Tillering stage	拔节期 Jointing stage	孕穗期 Booting stage	抽穗期 Heading stage	扬花期 Anthesis stage	灌浆期 Grain filling stage	成熟期 Maturity stage
ZH11	3.67±0.99	10.47±1.33	40.00±1.53	61.77±1.99	70.70±1.15	84.93±1.84	99.37±1.47	119.63±1.92	155.47±1.93
lvd1	4.03±1.03	11.00±1.41	40.50±1.31	62.03±1.85	73.03±1.07	88.63±1.56	102.20±1.69	122.03±1.99	161.03±1.75
P值 P value	0.856	0.14	0.21	0.60	6.65×10⁻⁹	5.47×10⁻⁸	4.74×10⁻⁷	3.63×10⁻⁵	1.13×10⁻¹¹

图4 ZH11与lvd1剑叶的生理及解剖特征比较 A：叶绿素含量测定；B~E：剑叶石蜡切片观察，B, D为ZH11和lvd1中脉，C, E为ZH11和lvd1泡状细胞（黑色三角形示），黑色方框为小脉形态；比例尺为400 μm；F：ZH11和lvd1泡状细胞数量；G：ZH11和lvd1可溶性糖含量。数据以平均值 ± 标准差（mean ± SD）表示，（n ≥ 3）。统计分析采用GraphPad Prism进行单因素方差分析，ns表示无显著差异，**、***和****分别表示P < 0.01、P < 0.001和P < 0.0001。

Fig. 4. Comparison of physiological and anatomical features of flag leaves between ZH11 and lvd1 A, Chlorophyll content measurement (chla: chlorophyll a, chlb: chlorophyll b, chls: total chlorophyll); B−E, Paraffin sections of flag leaves. B, D, midribs of ZH11 and lvd1; C, E, Bulliform cells of ZH11 and lvd1 (marked by black triangles), with small veins indicated by black boxes. Scale bar; 400 μm. F, Bulliform cell number in ZH11 and lvd1; G, Soluble sugar content measurement in ZH11 and lvd1. Data are presented as mean ± SD (n≥3). Statistical analysis was performed by one-way ANOVA using GraphPad Prism, ns indicates no significant difference; **, ***, and **** indicate P < 0.01, P < 0.001, and P < 0.0001, respectively.

表4 ZH11和lvd1叶脉密度

Table 4. Leaf vein density of flag leaves of ZH11 and lvd1 mm−2

材料 Material	大脉密度 Major vein density	小脉密度 Minor vein density
ZH11	3.6 ± 0.5	21.0 ± 0.6
lvd1	4.2 ± 0.4	22.9 ± 0.7
P值P value	0.16	8.00×10⁻³

图5 ZH11与lvd1水稻主要农艺性状比较 A：ZH11（左）与lvd1（右）的穗部形态；B：ZH11（上）与lvd1（下）籽粒长度比较；C：ZH11（上）与lvd1（下）籽粒宽度比较；D~K：ZH11和lvd1株高，分蘖数，穗长，粒长，粒宽，结实率，每穗粒数，千粒重。各农艺性状数据以平均值 ± 标准差（mean ± SD）表示（n = 30）。ZH11与lvd1植株间差异采用GraphPad Prism软件进行单因素方差分析，*表示在P < 0.05水平上差异显著。

Fig. 5. Statistical analysis of agronomic traits of ZH11 and lvd1 in field conditions A, Panicle morphology of ZH11 (left) and lvd1 (right); B, Grain length comparison of ZH11 (top) and lvd1 (bottom); C, Grain width comparison of ZH11 (top) and lvd1 (bottom); D-K, Plant height, tiller number, panicle length, grain length, grain width, seed-setting rate, grains per panicle, and 1000-grain weight of ZH11 and lvd1. Data are presented as mean ± SD (n = 30). Differences between ZH11 and lvd1 were analyzed by one-way ANOVA using GraphPad Prism; * indicates significant difference at P < 0.05.

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