Genome-wide Identification of the FAX family in Gossypium hirsutum and Functional Analysis of GhFAX1

doi:10.13560/j.cnki.biotech.bull.1985.2023-1075

Abstract

Abstract:

【Objective】 Fatty acid export（FAX）can mediate the transport of fatty acids from plant cells to the outer plastid, and play an important role in plant growth and development and response to abiotic stress. This work is to study the FAX family gene and function analysis of upland cotton（Gossypium hirsutum）, and to provide new ideas for clarifying the molecular mechanism of oil accumulation and oil metabolism engineering of upland cotton. 【Method】 In order to reveal the potential function of the upland cotton FAX gene family, the GhFAX gene family was identified at the whole genome level, and the GhFAX1 protein was to have subcellular localization, and the function of GhFAX1 was verified by the genetic transformation of yeast and tobacco.【Result】 A total of 12 GhFAXs genes were identified in the genome of upland cotton, and all members of the family had similar gene structure and protein physicochemical properties. Sequence alignment analysis showed that only a few amino acids of GhFAX protein were highly conserved in evolution, suggesting their importance to function. Phylogenetic tree analysis showed that GhFAX gene was closely related to Asian cotton（Gossypium arboreum）and Raymond's cotton（G. raimondii）. Transcriptome data suggested that GhFAXs may be involved in the regulation of upland cotton response to stress. Real-time fluorescence quantitative PCR showed that GhFAX4 was highly expressed in flowers, suggesting that GhFAX4 was involved in pollen development. The expression of GhFAX9 was high in the stems and GhFAX1 was high in all tissues of upland cotton. GhFAX1 was selected for subcellular localization analysis, and it was confirmed that GhFAX1 was localized in plastid. The GhFAX1 vector was constructed to overexpress GhFAX1 in Saccharobacteria cerevisiae. The results showed that the total fat of GhFAX1 overexpressed yeast was increased by 3.53%. Substrate bias experiments showed that GhFAX1 was selective to C16:0. GhFAX1 overexpressing tobacco was cultivated through genetic transformation of tobacco. The results showed that the chlorophyll of overexpressed tobacco increased by 13.24%, the fluorescence parameters NPQ increased by 14.17%, F_m increased by 34.94%, F₀decreased by 35.28%. The total fatty acids of the leaves and seeds increased by 5.2% and 6.52% respectively. The 1 000-grain weight of seeds increased by 19.1%. At the same time, the protein content decreased significantly.【Conclusion】 GhFAXs can increase the oil content of yeast and tobacco, participated in the transfer of palmitic acid from plastid, and causing the carbon source from protein synthesis pathway to oil synthesis pathway.

Key words: Gossypium hirsutum, fatty acid transport, genome identification, expression analysis, functional verification

YANG Wei-cheng, SUN Yan, YANG Qian, WANG Zhuang-lin, MA Ju-hua, XUE Jin-ai, LI Run-zhi. Genome-wide Identification of the FAX family in Gossypium hirsutum and Functional Analysis of GhFAX1[J]. Biotechnology Bulletin, 2024, 40(3): 155-169.

Figures/Tables 13

Table 1 Analysis of the properties of the FAX family proteins in G. hirsutum

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	信号肽Signal peptide
GhFAX1	Gohir.1Z091100	226	24.766	9.945	43.96	0.037	叶绿体Chloroplast	无None
GhFAX2	Gohir.A01G079000	92	9.800	10.205	26.53	0.608	叶绿体Chloroplast	无None
GhFAX3	Gohir.A02G003100	325	35.350	7.082	48.29	-0.086	叶绿体Chloroplast	无None
GhFAX4	Gohir.A03G078700	167	17.689	10.572	31.56	0.475	叶绿体Chloroplast	无None
GhFAX5	Gohir.A04G018500	119	12.694	9.914	19.20	0.497	叶绿体Chloroplast	无None
GhFAX6	Gohir.A10G151100	203	21.714	8.691	26.02	0.246	叶绿体Chloroplast	无None
GhFAX7	Gohir.D01G066100	119	12.652	9.979	24.77	0.640	叶绿体Chloroplast	无None
GhFAX8	Gohir.D02G003500	323	35.316	7.368	51.03	-0.124	叶绿体Chloroplast	无None
GhFAX9	Gohir.D03G089300	174	18.350	10.151	29.59	0.571	叶绿体Chloroplast	无None
GhFAX10	Gohir.D03G073000	228	25.046	6.885	46.30	-0.113	叶绿体Chloroplast	无None
GhFAX11	Gohir.D05G367500	119	12.846	9.914	20.92	0.541	叶绿体Chloroplast	无None
GhFAX12	Gohir.D10G115200	316	34.020	8.984	38.16	0.005	叶绿体Chloroplast	无None

Fig. 1 Multiple sequence alignment of FAX family protein in G. hirsutum The red box indicates the highly conserved amino acids of the FAX family proteins

Fig. 2 Evolutionary tree analysis of FAX family proteins in cotton GhFAX: Gossypium hirsutum; GbFAX: G. barbadense; GaFAX：G. arboreum; GrFAX: G. raimondi

Fig. 3 Distribution of GhFAXs genes on the chromosomes of G. hirsutum

Fig. 4 Analysis of FAX family gene structure（A）and amino acid composition（B）in G. hirsutum

Fig. 5 Cis-acting elements analysis of GhFAXs genes in G. hirsutum

Fig. 6 Transmembrane domain and 3D result prediction of the FAX family proteins in G. hirsutum

Fig. 7 Expression patterns of FAX genes in G. hirsutum under different stresses

Fig. 8 Analysis of gene expression patterns of FAX family Different lower letters indicate significant differences at P<0.05 level

Fig. 9 Subcellular localization analysis of GhFAX1 protein

Fig. 10 Total fatty acid content of transgenic yeast

Fig. 11 Transgenic yeast growth indicators under exogenous added fatty acids added fatty acids

Fig. 12 Chlorophyll content（A）, relevant fluorescence parameters（B）, total fatty acid（C）, soluble sugar（D）, starch（E）, protein content（F）, 1 000-grain weight（G）and total fatty acid content（H）in transgenic tobacco leaves

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