Identification of SEC1 Complex Components and Functional Validation of the GhSCY1 Gene in Cotton

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

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

Fig. 5 Tissue expression analysis of SEC1 system in cotton

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

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

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

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