陆地棉FAX家族的全基因组鉴定及GhFAX1的功能分析

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

摘要/Abstract

摘要：

【目的】 脂肪酸转运蛋白（FAX）可介导植物细胞内脂肪酸从质体向外运输，在植物生长发育及响应非生物胁迫等方面发挥重要作用。研究陆地棉FAX家族基因及功能分析，为明确陆地棉油脂积累的分子机制和油脂代谢工程提供新思路。【方法】 从全基因组水平对GhFAX基因家族进行鉴定，并对GhFAX1蛋白进行亚细胞定位，通过酵母与烟草遗传转化对GhFAX1进行功能验证。【结果】 在陆地棉基因组中共鉴定到12个GhFAXs基因，家族各成员间具有相似的基因结构及蛋白理化性质。序列比对分析发现，GhFAX蛋白仅有少数氨基酸在进化中高度保守，暗示其对功能的重要性；进化树分析表明，GhFAXs基因与亚洲棉、雷蒙德氏棉的亲缘关系较近。转录组数据表明，GhFAXs可能参与陆地棉响应逆境胁迫的调控过程。通过实时荧光定量PCR发现，GhFAX4在花中表达较高，说明其参与花粉发育过程；GhFAX9在茎中表达较高，GhFAX1在陆地棉各个组织表达量均较高。选择GhFAX1进行亚细胞定位分析，证实GhFAX1定位在质体中。构建GhFAX1载体，使GhFAX1在酿酒酵母中过表达，结果显示，GhFAX1过表达酵母总脂提高了3.53%。底物偏好性试验显示，GhFAX1对C16:0具有选择性。通过烟草遗传转化，培育GhFAX1过表达烟草。结果显示，过表达烟草叶绿素提升了13.24%，荧光参数NPQ提高14.17%，F_m提高34.94%，F₀降低35.28%；叶片总脂肪酸提高了5.2%，种子总脂肪酸提高了6.52%；种子的千粒重增加了近19.1%；同时蛋白含量显著下降。【结论】 GhFAXs提高了酵母与烟草的含油量，参与质体中棕榈酸的转出，使蛋白合成途径上的碳源流向油脂合成途径。

关键词: 陆地棉, 脂肪酸转运蛋白, 基因组鉴定, 表达分析, 功能验证

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

杨伟成, 孙岩, 杨倩, 王壮琳, 马菊花, 薛金爱, 李润植. 陆地棉FAX家族的全基因组鉴定及GhFAX1的功能分析[J]. 生物技术通报, 2024, 40(3): 155-169.

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.

图/表 13

表1 棉花FAX家族蛋白性质分析

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

图1 陆地棉FAX家族蛋白氨基酸序列比对红色特征框代表FAX家族蛋白具有较强保守性的氨基酸

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

图2 棉花FAX家族蛋白进化树分析 GhFAX：陆地棉；GbFAX：海岛棉；GaFAX：亚洲棉；GrFAX：雷蒙德氏棉

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

图3 陆地棉GhFAXs基因在染色体上的分布

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

图4 陆地棉FAX家族基因结构（A）与氨基酸构成分析（B）

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

图5 陆地棉GhFAXs基因的启动子顺式作用元件分析

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

图6 陆地棉FAX家族蛋白跨膜结构域与三维结果预测

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

图7 陆地棉FAX基因在不同胁迫中的表达模式

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

图8 陆地棉FAX家族基因表达模式分析不同小写字母表示在P<0.05水平差异显著

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

图9 GhFAX1蛋白的亚细胞定位分析

Fig. 9 Subcellular localization analysis of GhFAX1 protein

图10 转基因酵母总脂肪酸含量

Fig. 10 Total fatty acid content of transgenic yeast

图11 转基因酵母在外源添加脂肪酸下生长指标

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

图12 转基因烟草叶片叶绿素含量（A）、相关荧光参数（B）和转基因烟草蛋白含量（C）、淀粉（D）、叶片总脂肪酸（E）、可溶性糖（F）及转基因烟草种子千粒重（G）与总脂肪酸含量（H）

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

参考文献 31

[1]	王美霞, 周大云, 马磊, 等. 棉籽油脂肪酸组成分析与评价[J]. 食品科学, 2016, 37(22): 136-141. doi: 10.7506/spkx1002-6630-201622020
	Wang MX, Zhou DY, Ma L, et al. Analysis and evaluation of fatty acid composition in cottonseed oil[J]. Food Sci, 2016, 37(22): 136-141. doi: 10.1111/jfds.1972.37.issue-1 URL
[2]	刘大川. 棉籽油加工及其营养[J]. 中国油脂, 2012, 37(11): 8-10.
	Liu DC. Processing and nutrition of cottonseed oil[J]. China Oils Fats, 2012, 37(11): 8-10.
[3]	Walther TC, Farese RV. Lipid droplets and cellular lipid metabolism[J]. Annu Rev Biochem, 2012, 81: 687-714. doi: 10.1146/annurev-biochem-061009-102430 pmid: 22524315
[4]	Li-Beisson Y, Neunzig J, Lee Y, et al. Plant membrane-protein mediated intracellular traffic of fatty acids and acyl lipids[J]. Curr Opin Plant Biol, 2017, 40: 138-146. doi: S1369-5266(17)30136-X pmid: 28985576
[5]	Li N, Gügel IL, Giavalisco P, et al. FAX1, a novel membrane protein mediating plastid fatty acid export[J]. PLoS Biol, 2015, 13(2): e1002053. doi: 10.1371/journal.pbio.1002053 URL
[6]	金龙飞, 李睿, 曹红星. 油棕脂肪酸外运蛋白基因FAX1的克隆及表达分析[J]. 热带作物学报, 2022, 43(1):27-33. doi: 10.3969/j.issn.1000-2561.2022.01.004
	Jin LF, Li R, Cao HX, et al. Cloning and expression analysis of oil palm fatty acid transport protein gene FAX1[J]. Journal of Tropical Crops, 2022, 43(1):27-33.
[7]	Li N, Zhang Y, Meng H, et al. Characterization of Fatty Acid Exporters involved in fatty acid transport for oil accumulation in the green alga Chlamydomonas reinhardtii[J]. Biotechnol Biofuels, 2019, 12: 14. doi: 10.1186/s13068-018-1332-4
[8]	Takemura T, Imamura S, Tanaka K. Identification of a chloroplast fatty acid exporter protein, CmFAX1, and triacylglycerol accumulation by its overexpression in the unicellular red alga Cyanidioschyzon merolae[J]. Algai Res, 2019, 38: 101396.
[9]	高岩, 郭东林, 郭长虹. 三烯脂肪酸在高等植物逆境胁迫应答中的作用[J]. 分子植物育种, 2010, 8(2): 365-369.
	Gao Y, Guo DL, Guo CH. Role of trienoic fatty acids in higher plants stress responses[J]. Mol Plant Breed, 2010, 8(2): 365-369.
[10]	Routaboul JM, Fischer SF, Browse J. Trienoic fatty acids are required to maintain chloroplast function at low temperatures[J]. Plant Physiol, 2000, 124(4): 1697-1705. doi: 10.1104/pp.124.4.1697 pmid: 11115886
[11]	Iba K. Acclimative response to temperature stress in higher plants: Approaches of gene engineering for temperature tolerance[J]. Annu Rev Plant Biol, 2002, 53: 225-245. pmid: 12221974
[12]	Shi JL, Cao YP, Fan XR, et al. A rice microsomal delta-12 fatty acid desaturase can enhance resistance to cold stress in yeast and Oryza sativa[J]. Mol Breeding, 2012, 29(3): 743-757. doi: 10.1007/s11032-011-9587-5 URL
[13]	Yamauchi Y, Furutera A, Seki K, et al. Malondialdehyde generated from peroxidized linolenic acid causes protein modification in heat-stressed plants[J]. Plant Physiol Biochem, 2008, 46(8/9): 786-793. doi: 10.1016/j.plaphy.2008.04.018 URL
[14]	韩二琴, 李健春, 李英双, 等. 转运蛋白调控植物脂质运输研究进展[J]. 中国油料作物学报, 2017, 39(2): 260-268. doi: 10.7505/j.issn.1007-9084.2017.02.018
	Han EQ, Li JC, Li YS, et al. Research advance in the regulation of plant lipid trafficking by transporters[J]. Chin J Oil Crop Sci, 2017, 39(2): 260-268.
[15]	Wang X, Ma Q, Dou L, et al. Genome-wide characterization and comparative analysis of the MLO gene family in cotton[J]. Plant Physiol Biochem, 2016, 103: 106-119. doi: 10.1016/j.plaphy.2016.02.031 URL
[16]	Niu EL, Cai CP, Zheng YJ, et al. Genome-wide analysis of CrRLK1L gene family in Gossypium and identification of candidate CrRLK1L genes related to fiber development[J]. Mol Genet Genomics, 2016, 291(3): 1137-1154. doi: 10.1007/s00438-016-1169-0 URL
[17]	Li FG, Fan GY, Lu CR, et al. Genome sequence of cultivated Upland cotton(Gossypium hirsutum TM-1)provides insights into genome evolution[J]. Nat Biotechnol, 2015, 33(5): 524-530. doi: 10.1038/nbt.3208
[18]	Liu X, Zhao B, Zheng HJ, et al. Gossypium barbadense genome sequence provides insight into the evolution of extra-long staple fiber and specialized metabolites[J]. Sci Rep, 2015, 5(1): 1-14.
[19]	Zhao LF, Katavic V, Li FL, et al. Insertional mutant analysis reveals that long-chain acyl-CoA synthetase 1(LACS1), but not LACS8, functionally overlaps with LACS9 in Arabidopsis seed oil biosynthesis[J]. Plant J, 2010, 64(6): 1048-1058. doi: 10.1111/tpj.2010.64.issue-6 URL
[20]	Nilsson R, Schultz IJ, Pierce EL, et al. Discovery of genes essential for heme biosynthesis through large-scale gene expression analysis[J]. Cell Metab, 2009, 10(2): 119-130. doi: 10.1016/j.cmet.2009.06.012 pmid: 19656490
[21]	陈雨沁, 王道阳, 刘艳艳, 等. 水稻与拟南芥跨膜蛋白14家族的分子进化特征与功能分析[J]. 基因组学与应用生物学, 2020, 39(3): 1229-1238.
	Chen YQ, Wang DY, Liu YY, et al. Bioinformatics analysis on evolutionary properties and functions of transmembrane 14 family in Arabidopsis and rice[J]. Genom Appl Biol, 2020, 39(3): 1229-1238.
[22]	李濯雪, 陈信波. 植物诱导型启动子及相关顺式作用元件研究进展[J]. 生物技术通报, 2015, 31(10): 8-15. doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.006
	Li ZX, Chen XB. Research progress of plant inducible promoters and related cis-acting elements[J]. Biotechnology Bulletin, 2015, 31(10): 8-15.
[23]	Morsomme P, Chami M, Marco S, et al. Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast[J]. J Biol Chem, 2002, 277(33): 29608-29616. doi: 10.1074/jbc.M203871200 pmid: 12048206
[24]	Zwiers LH, Stergiopoulos I, Gielkens MMC, et al. ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds[J]. Mol Genet Genomics, 2003, 269(4): 499-507. pmid: 12768412
[25]	Ton VK, Mandal D, Vahadji C, et al. Functional expression in yeast of the human secretory pathway Ca⁽²⁺⁾, Mn⁽²⁺⁾-ATPase defective in Hailey-Hailey disease[J]. J Biol Chem, 2002, 277(8): 6422-6427. doi: 10.1074/jbc.M110612200 URL
[26]	Liang F, Cunningham KW, Harper JF, et al. ECA1 complements yeast mutants defective in Ca²⁺ pumps and encodes an endoplasmic reticulum-type Ca²⁺-ATPase in Arabidopsis thaliana[J]. Proc Natl Acad Sci USA, 1997, 94(16): 8579-8584. doi: 10.1073/pnas.94.16.8579 pmid: 9238019
[27]	王丽艳. 油莎豆油脂合成相关基因的挖掘、表达分析及功能鉴定[D]. 长春: 吉林农业大学, 2022.
	Wang LY. Extraction, expression analysis and functional identification of genes related to oil synthesis in Yousha bean[D]. Changchun: Jilin Agricultural University, 2022.
[28]	王昌陵, 王文斌, 曹永强, 等. 农杆菌介导的植物遗传转化机制研究进展[J]. 辽宁农业科学, 2013, 53(6): 56-61.
	Wang CL, Wang WB, Cao YQ, et al. Research of transformation mechanism mediated by Agrobacterium in plant[J]. Liaoning Agric Sci, 2013(4): 56-61.
[29]	Siloto RMP, Kim F, Arturo LV, et al. The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis[J]. Plant Cell, 2006, 18(8): 1961-1974. doi: 10.1105/tpc.106.041269 URL
[30]	Andre C, Froehlich JE, Moll MR, et al. A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis[J]. Plant Cell, 2007, 19(6): 2006-2022. doi: 10.1105/tpc.106.048629 pmid: 17557808
[31]	Sébastien B, Sylvie W, Bertrand D, et al. Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana[J]. Plant J, 2007, 52(3): 405-419. doi: 10.1111/j.1365-313X.2007.03232.x pmid: 17892448