棉花SEC1复合体组分的鉴定与GhSCY1基因功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2024-0923

摘要/Abstract

摘要：

目的 SEC1复合体是位于类囊体上的蛋白质转运系统，由SCY1、SECE1和SECA1共同组成。SEC1系统参与类囊体蛋白的转运和整合，对维持叶绿体结构的稳定起着非常重要的作用。利用生物信息学从陆地棉全基因组中鉴定SEC1复合体基因，旨在为光合机制研究和棉花高产抗逆育种提供新的基因资源。方法基于拟南芥SEC1系统组分基因蛋白序列，从陆地棉基因组中鉴定棉花SEC1系统组分，分析该系统组分各基因的理化性质、跨膜结构、蛋白互作、启动子元件及表达模式。利用VIGS和透射电镜技术，研究叶绿体主要通道蛋白组分GhSCY1对叶绿素合成、叶绿体结构和形态的影响。结果在陆地棉中鉴定出了SEC1转运系统的组分，GhSCY1A/D、GhSECE1A/D和GhSECA1A/D。GhSCY1A/D含有1个SecY保守结构域，7个跨膜结构域，GhSECE1D只含有1个跨膜结构域。预测GhSCY1A/D与GhSECE1D在SEC1转运系统中共同形成叶绿体跨膜通道蛋白。GhSECA1A/D拥有1个SecA结构域，不含跨膜结构域，主要为蛋白转运过程提供能量。SEC1系统各基因在叶片中的表达量较高，且能够参与温度胁迫的响应。沉默GhSCY1基因（GhSCY1A和GhSCY1D）后，棉花叶片出现斑驳的淡黄色表型，叶绿素含量明显下降，叶片叶绿体结构和形态异常，没有淀粉粒累积。结论棉花SEC1系统分别由GhSCY1A/D、GhSECE1A/D和GhSECA1A/D构成，其中GhSCY1A/D基因在维持叶绿体结构和形态及叶绿素生物合成方面发挥重要作用。

关键词: 棉花, SEC1系统, GhSCY1, 叶色, 叶绿体结构, 温度胁迫, 叶绿体蛋白转运

Abstract:

Objective The SEC1 complex is a protein transport system located on the thylakoid membrane, composed of SCY1, SECE1, and SECA1. The SEC1 system is involved in the transport and integration of thylakoid proteins, playing a very important role in maintaining the stability of chloroplast structure. Identifying SEC1 complex genes from the whole genome of upland cotton using bioinformatics aims to provide new gene resources for photosynthesis mechanism research and high-yield, stress-resistant cotton breeding. Method Based on the protein sequences of the Arabidopsis SEC1 system component genes, cotton SEC1 system components were identified from the upland cotton genome, and then the physicochemical properties, transmembrane structures, protein interactions, promoter elements, and expression patterns of these system components were analyzed. The effects of the main channel protein component GhSCY1 on chlorophyll synthesis, chloroplast structure, and morphology were studied using VIGS and transmission electron microscopy techniques. Result Components of the SEC1 transport system in upland cotton were identified, GhSCY1A/D, GhSECE1A/D, and GhSECA1A/D. GhSCY1A/D contains one SecY conserved domain and seven transmembrane domains, while GhSECE1D only contains one transmembrane domain. It is predicted that GhSCY1A/D and GhSECE1D together form the chloroplast transmembrane channel protein in the SEC1 transport system. GhSECA1A/D possesses a SecA domain and lacks transmembrane domains, mainly providing energy for the protein transport process. The expressions of the SEC1 system genes were high in the leaves and could participate in the response to temperature stress. After silencing the GhSCY1 genes (GhSCY1A and GhSCY1D), the cotton leaves demonstrated a mottled pale yellow phenotype, a significant decrease in chlorophyll content, abnormal chloroplast structure and morphology, and no starch granule accumulation. Conclusion The cotton SEC1 system is constituted by GhSCY1A/D, GhSECE1A/D, and GhSECA1A/D, among which the GhSCY1A/D genes play an important role in maintaining chloroplast structure and morphology and in the biosynthesis of chlorophyll.

Key words: Gossypium hirsutum, SEC1 system, GhSCY1, leaf color, chloroplast structure, temperature stress, chloroplast protein translocation

冯小康, 梁倩, 王学峰, 孙杰, 薛飞. 棉花SEC1复合体组分的鉴定与GhSCY1基因功能验证[J]. 生物技术通报, 2025, 41(3): 112-122.

FENG Xiao-kang, LIANG Qian, WANG Xue-feng, SUN Jie, XUE Fei. Identification of SEC1 Complex Components and Functional Validation of the GhSCY1 Gene in Cotton[J]. Biotechnology Bulletin, 2025, 41(3): 112-122.

图/表 10

表1 所用相关的引物序列

Table 1 Sequences of relevant primers used

名称 Name	序列 Sequence （5′‒3′）	用途 Purpose
V-GhSCY1-F	aaggttaccgaattTTGGATCAGAACAGTCTACTAAGCAC	VIGS
V-GhSCY1-R	ctcggtaccggatcGTGCTGAAATCGTTGACATATGGAC
q-GhSCY1-F	GTATCAACCCAGACGAGTCTTCAG	RT-qPCR
q-GhSCY1-R	AACTCCAAGACGAGATAAAGCC

图1 棉花SEC1系统组分进化分析与鉴定A：GhSCY1（GH_A05G0068和GH_D05G0074）的进化分析；B：GhSECA1（GH_A12G1366和GH_D12G1384）的进化分析；C：GhSECE1（GH_A09G2370和GH_D09G2311）的进化分析；D：SEC1组分保守结构域分析。图1-A-C中蛋白质序列来源于拟南芥、玉米、水稻、烟草、雷蒙德氏棉、瑟伯氏棉、黄褐棉、海岛棉、陆地棉、亚洲棉、毛棉和达尔文棉；红色符号表示内部节点符号，表示的是假定祖先；绿色符号表示是位于树的最末端的节点（叶节点），代表基因

Fig. 1 Identification and evolutionary analysis of SEC1 system in cotton (Gossypium hirsutum)A: Evolutionary analysis of GhSCY1 (GH_A05G0068 and GH_D05G0074). B: Evolutionary analysis of GhSECA1 (GH_A12G1366 and GH_D12G1384). C: Evolutionary analysis of GhSECE1 (GH_A09G2370 and GH_D09G2311). D: Analysis of conserved domains in the SEC1 component. The protein sequences in Fig.1 A-C are derived from Arabidopsis thaliana, Zea mays, Oryza sativa, Nicotiana attenuata, Gossypium raimondii, Gossypium thurberi, Gossypium mustelinum, Gossypium barbadense, Gossypium hirsutum, Gossypium arboreum, Gossypium tomentosum, and Gossypium darwinii. The red symbols indicate internal node symbols, which denote the presumed ancestors; the green symbols indicate the nodes at the very end of the tree (leaf nodes), representing genes

表2 棉花SEC1系统理化性质及亚细胞定位分析

Table 2 Analysis of physicochemical properties and subcellular localization of the SEC1 system in cotton

基因名称 Gene name	基因ID Gene ID	氨基酸数 Number of amino acids	分子量 Molecular weight/kD	理论等电点 Theoretical pI	不稳定系数 Instability index	亲水性 GRAVY	亚细胞定位 Subcellular location
GhSCY1A	GH_A05G0068	542	58.612 73	9.48	37.39	0.258	叶绿体Chloroplast
GhSCY1D	GH_D05G0074	542	58.709 83	9.56	36.39	0.244	叶绿体Chloroplast
GhSECA1A	GH_A12G1366	1 037	116.944 51	6.23	42.82	-0.368	叶绿体Chloroplast
GhSECA1D	GH_D12G1384	1 037	116.993 61	6.33	43.19	-0.366	叶绿体Chloroplast
GhSECE1A	GH_A09G2370	174	18.592 99	5.53	46.77	-0.208	叶绿体Chloroplast
GhSECE1D	GH_D09G2311	174	18.489 82	5.34	50.07	-0.208	叶绿体Chloroplast

图2 棉花SEC1系统组分跨膜结构分析图中紫色方框个数表示跨膜结构的数量；A‒F分别表示GhSCY1A含有7个跨膜结构、GhSCY1D含有7个跨膜结构、GhSECE1A不含跨膜结构、GhSECE1D含有1个跨膜结构、GhSECA1A和GhSECA1D不含跨膜结构

Fig. 2 Analysis of the transmembrane structure of the SEC1 systemic component in cottonThe number of purple squares in the figure indicates the number of transmembrane structures; A‒F indicate that GhSCY1A contains 7 transmembrane structures, GhSCY1D contains 7 transmembrane structures, GhSECE1A does not contain any transmembrane structures, GhSECE1D contains 1 transmembrane structure, and both GhSECA1A and GhSECA1D do not contain transmembrane structures

图3 棉花SEC1系统组分三维结构互作分析A：GhSCY1、GhSECE1 和GhSECA1之间的互作；B：GhSCY1与GhSECA1的互作；C：GhSCY1与GhSECE1的互作；D：GhSECE1 和GhSECA1之间的互作；绿色：GhSCY1蛋白结构；蓝色：GhSECE1蛋白结构；黄色：GhSECA1蛋白结构；红色：蛋白间的互作区域

Fig. 3 Analysis on three-dimensional structural interactions analysis of SEC1 system components in cottonA: Interaction between GhSCY1, GhSECE1 and GhSECA1. B: Interaction between GhSCY1 and GhSECA1. C: Interaction between GhSCY1 and GhSECE1. D: Intercropping between GhSECE1 and GhSECA1. Green color: Protein structure of GhSCY1; blue color: protein structure of GhSECE1; yellow color: protein structure of GhSECA1; red color: region of interactions between proteins

图4 棉花SEC1系统基因顺式作用元件分析

Fig. 4 Analysis of cis-acting elements of SEC1 system in cotton

图5 棉花SEC1系统基因组织表达分析

Fig. 5 Tissue expression analysis of SEC1 system in cotton

图6 棉花SEC1系统胁迫表达模式分析

Fig. 6 Analysis of stress expression pattern of SEC1 system in cotton

图7 棉花GhSCY1A/D基因沉默后的表型、表达量及色素含量A：GhCHLI沉默后表型；B：TRV:00植株和TRV:GhSCY1植株沉默后的整株表型；C：GhSCY1在TRV:00和TRV:GhSCY1植株中的相对表达量；D:TRV:00和TRV:GhSCY1沉默后叶片表型；E：TRV:00和TRV:GhSCY1沉默后总叶绿素含量；F：TRV:00和TRV:GhSCY1沉默后的胡萝卜素含量；***：在0.01水平上存在显著差异，****：在0.001水平上存在显著差异

Fig. 7 Phenotype, expression and pigment content after silencing of GhSCY1A/D gene in cottonA: Phenotype of GhCHLI silencing. B: Whole plant phenotype after silencing with TRV:00 and TRV:GhSCY1. C: Relative expressions of GhSCY1 in TRV:00 and TRV:GhSCY1 plants. D: Leaf phenotype after silencing with TRV:00 and TRV:GhSCY1. E: Total chlorophyll content after silencing with TRV:00 and TRV:GhSCY1. F: Carotenoid content after silencing with TRV:00 and TRV:GhSCY1. ***: Significant difference at the 0.01 level, ****: significant difference at the 0.001 level

图8 棉花GhSCY1A/D基因沉默后的叶绿体结构

Fig. 8 Chloroplast structure after silencing of GhSCY1A/D gene in cotton

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