HrβVPE1通过调控胚乳外层细胞降解影响沙棘种子萌发

doi:10.13560/j.cnki.biotech.bull.1985.2025-1299

摘要/Abstract

摘要：

目的探讨沙棘（Hippophae rhamnoides）液泡加工酶基因HrβVPE1在种子萌发过程中对胚乳外层细胞程序性死亡（programmed cell death, PCD）及淀粉降解的影响，为阐明其在种子萌发中的分子机制提供依据。方法从沙棘种子中克隆HrβVPE1基因，利用生物信息学方法分析其氨基酸与核苷酸序列，进行系统发育分析与多序列比对；采用实时荧光定量PCR（RT-qPCR）分析其在沙棘种子不同萌发时期的表达模式；采用农杆菌介导法瞬时转化本氏烟草叶片，以确定HrβVPE1亚细胞定位；利用病毒诱导的基因沉默（VIGS）技术下调HrβVPE1表达，通过碘‒碘化钾（I₂-KI）染色法、DAPI（4',6-diamidino-2-phenylindole）染色法及FDA（fluorescein diacetate）与FM4-64双探针染色法，观测其对淀粉降解、胚乳外层细胞液泡动态、细胞核形态、质膜完整性以及细胞活性的影响；构建HrβVPE1过表达载体转化拟南芥，统计种子萌发率，并利用RT-qPCR检测过表达植株中多个PCD相关基因的表达水平。结果 HrβVPE1全长1 482 bp，编码493个氨基酸，与木本植物中杨属、柳属植物亲缘关系较近；HrβVPE1定位于液泡膜，在种子萌发中后期表达量较高；沉默HrβVPE1能显著延缓淀粉的降解，抑制胚乳外层细胞内蛋白质储存液泡（PSV）的融合与细胞核降解，阻碍PCD进程并延长细胞活性；在拟南芥中过表达HrβVPE1可促进种子的萌发并上调PCD正向调控基因AtCEP1、AtXCP2、AtMC9的表达，下调负向调控基因AtMOD1、AtDAD2的表达。结论 HrβVPE1可能通过调控胚乳外层细胞PCD，影响淀粉降解过程，从而调控种子萌发。

关键词: 沙棘, HrβVPE1, 细胞程序性死亡, 胚乳外层细胞, 淀粉降解, 种子萌发, 病毒诱导的基因沉默, 液泡融合

Abstract:

Objective This study aimed to investigate the effects of the Hippophae rhamnoides vacuolar processing enzyme gene HrβVPE1 on programmed cell death (PCD) and starch degradation in outer endosperm cells during seed germination, thereby providing a theoretical basis for clarifying its molecular regulatory mechanism in seed germination. Method The HrβVPE1 gene was cloned from Hippophae rhamnoides seeds. Bioinformatics approaches were adopted to analyze the nucleotide and amino acid sequences of HrβVPE1, followed by multiple sequence alignment and phylogenetic tree construction. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression patterns of HrβVPE1 at different seed germination stages. Agrobacterium-mediated transient transformation was conducted on Nicotiana benthamiana leaves to determine the subcellular localization of HrβVPE1. Virus-induced gene silencing (VIGS) was used to downregulate HrβVPE1 expression in H. rhamnoides seeds. Iodine-potassium iodide (I₂-KI) staining, DAPI (4′,6-diamidino-2-phenylindole) staining, and double-probe staining with FDA (fluorescein diacetate) and FM4-64 were performed to observe the impacts of HrβVPE1 silencing on starch degradation, vacuole dynamics, nuclear morphology, plasma membrane integrity, and cell viability of outer endosperm cells. Additionally, an HrβVPE1 overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate of transgenic A. thaliana was calculated, and RT-qPCR was used to detect the expression levels of multiple PCD-related genes in overexpressing plants. Result The full-length cDNA of HrβVPE1 was 1 482 bp, encoding a polypeptide of 493 amino acids. Phylogenetic analysis revealed that HrβVPE1 had a close genetic relationship with vacuolar processing enzymes from woody plants such as Populus and Salix species. HrβVPE1 was localized to the vacuolar membrane. RT-qPCR analysis showed that HrβVPE1 exhibited high expression levels in the middle and late stages of seed germination. Silencing of HrβVPE1 significantly delayed starch degradation, inhibited the fusion of protein storage vacuoles (PSVs) and the degradation of cell nuclei in outer endosperm cells, blocked the PCD process, and prolonged cell viability. Overexpression of HrβVPE1 in A. thaliana promoted seed germination, upregulated the expression of positive PCD regulatory genes AtCEP1, AtXCP2, AtMC9, and downregulated the expression of negative PCD regulatory genes AtMOD1, AtDAD2. Conclusion HrβVPE1 plays a critical role in H. rhamnoides seed germination by regulating PCD processes in outer endosperm cells and thereby mediating starch degradation.

Key words: Hippophae rhamnoides, HrβVPE1, programmed cell death (PCD), outer endosperm cell, starch degradation, seed germination, virus-induced gene silencing (VIGS), vacuole fusion

程子义, 王沛蓉, 仲子宁, 魏明, 伍国强. HrβVPE1通过调控胚乳外层细胞降解影响沙棘种子萌发[J]. 生物技术通报, 2026, 42(9): 221-230.

CHENG Zi-yi, WANG Pei-rong, ZHONG Zi-ning, WEI Ming, WU Guo-qiang. HrβVPE1 Regulates Seed Germination in Hippophae rhamnoides by Controlling Degradation of Outer Endosperm Cells[J]. Biotechnology Bulletin, 2026, 42(9): 221-230.

图/表 9

图1 HrβVPE1系统发生树

Fig. 1 Phylogenetic tree of HrβVPE1

图2 HrβVPE1表达分析A：沙棘种子萌发0‒10 d的HrβVPE1相对表达水平；B：HrβVPE1的亚细胞定位观察结果；GFP-K（GFP蛋白通道）、VPE（HrβVPE1与GFP融合蛋白通道）、γ-TIP-mcherry（液泡膜标记通道）、CHI（叶绿体标记荧光通道）、DIC（微分干涉相差明场图像）以及Merge（各荧光通道与明场的叠加图），图中比例尺为20 μm。不同小写字母表示差异显著（P<0.05），下同

Fig. 2 Expression analysis of HrβVPE1A: Relative expression levels of HrβVPE1 in H. rhamnoides seeds during 0-10 d of germination. B: Subcellular localization observation results of HrβVPE1, GFP-K (GFP protein channel), VPE (HrβVPE1-GFP fusion protein channel), γ-TIP-mcherry (tonoplast marker channel), CHI (chloroplast marker fluorescence channel), DIC (differential interference contrast bright-field image), and Merge (merged image of all fluorescence channels and bright field). The scale is 20 μm. Different lowercase letters indicate significant differences (P<0.05), the same below

图3 HrβVPE1沉默对沙棘种子萌发的基因表达及表型影响A：HrβVPE1的mRNA相对表达量；EV：空载体对照处理组；VIGS：HrβVPE1基因沉默处理组；B：EV组与VIGS处理组的种子萌发率。**P<0.01

Fig. 3 Effects of HrβVPE1 silencing on gene expression and phenotype during seed germination in H. rhamnoidesA: Relative expression of HrβVPE1 mRNA. EV: Empty vector control group; VIGS: HrβVPE1 gene silencing treatment group. B: Seed germination rates of the EV group and VIGS group. **P<0.01

图4 不同处理组种子萌发1-6 d淀粉降解与淀粉含量的变化A：不同处理组种子萌发1-6 d 的淀粉降解变化；各行依次展示3组种子萌发1‒6 d样品，每一样品左侧为未染色种子，右侧为I2-KI染色种子；比例尺为2 mm；CK：正常萌发种子组；EV：空载体对照处理组；VIGS：HrβVPE1基因沉默处理组，下同；B：不同处理组种子萌发1-6 d的淀粉含量变化

Fig. 4 Changes in starch degradation and starch content of seeds in different treatment groups during 1-6 d of germinationA: Starch degradation dynamics of seeds in different treatment groups during 1-6 d of germination. Each row shows seed samples from three groups at 1-6 d of germination, with unstained seeds on the left and I2-KI stained seeds on the right for each sample. The scale is 2 mm. CK: normally germinating seed group; EV: empty vector control group; VIGS: HrβVPE1 gene-silenced group; the same applies hereinafter. B: Changes in starch content of seeds in different treatment groups during 1-6 d of germination

图5 不同处理组种子萌发1-6 d胚乳外层细胞液泡与细胞核形态特征a行为明场观察结果，b行为DAPI染色后的细胞核荧光观察结果；比例尺为50 μm；箭头所指区域为红色方框的放大观察图（放大倍数×2.5）

Fig. 5 Morphological characteristics of vacuoles and nuclei in outer endosperm cells in seeds from different treatment groups during 1-6 d of germinationRow a shows the bright-field observation, and row b shows nuclear fluorescence observation results after DAPI staining. The scale bar is 50 μm. The area indicated by the arrow shows an enlargement of the red box at ×2.5 magnification

图6 不同处理组种子萌发1-6 d胚乳外层细胞死亡状态a行：明场观察结果；b行：FDA染色荧光图像；c行：FM4-64染色荧光图像；d行：FDA与FM4-64染色的叠加荧光图像；a行比例尺为为50 μm，b、c、d行比例尺为100 μm

Fig. 6 Cell death status of outer endosperm cells in seeds from different treatment groups during 1-6 d of germinationRow a: Bright-field observation. Row b: FDA-stained fluorescence images. Row c: FM4-64-stained fluorescence images. Row d: Merged fluorescence images of FDA and FM4-64 staining. The scale bar for row a is 50 μm, and that for rows b, c, and d is 100 μm

图7 HrβVPE1过表达拟南芥的基因表达（A）、酶活性（B）与种子萌发特性（C）分析WT：野生型拟南芥；2-1、5-2：HrβVPE1过表达株系

Fig. 7 Analysis of gene expression (A), enzyme activity (B) and seed germination characteristics (C) of A. thaliana overexpression HrβVPE1WT: Wild-type A. thaliana. 2-1, 5-2: HrβVPE1 overexpression lines

图8 过表达HrβVPE1拟南芥中与PCD相关基因的表达量

Fig. 8 Relative expressions of PCD-related genes in overexpression HrβVPE1 A. thaliana

图9 HrβVPE1调控沙棘种子胚乳外层细胞PCD与萌发的机制模式图

Fig. 9 Schematic diagram of the mechanism of HrβVPE1 regulating PCD and seed germination in outer endosperm cells of H. rhamnoides seeds

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