油梨乙酰辅酶A 羧化酶基因家族鉴定及表达分析

Identification and expression analysis of the acetyl-CoA carboxylase gene family in Persea americana

摘要

油梨（Persea americana）是一种营养价值丰富的水果，脂肪酸是其关键内在营养物质之一，乙酰辅酶 A 羧化酶（ACC）是脂肪酸合成的关键酶，对油脂积累和储存至关重要。本研究利用生物信息学方法对油梨 PaACC 基因的蛋白结构、进化关系及启动子顺式作用元件等方面进行分析，并通过 qRT-PCR 对油梨不同器官及果实不同发育时期表达量进行分析。油梨中共鉴定出 7 个 PaACC 基因，分别编码 ACC 的 3 个 BC 亚基、3 个 BCCP 亚基和 1 个 α−CT 亚基，氨基酸数为 251～776，平均分子质量为 49.95 kDa，均为亲水蛋白，不具有跨膜结构域。不同物种 ACC 蛋白系统发育分析显示，ACC 蛋白按照亚基组成分为 4 个亚族。基因结构显示，PaACC 都至少含有 5 个外显子，各成员都含有一定数量的内含子。PaACC 基因家族中存在 25 个不同的保守基序，其中，PaACC1、PaACC2 和 PaACC3 具有 motif 14，PaACC4、PaACC5 和 PaACC6 具有 motif 1、2、3、4、5。在 PaACC 基因的启动子区域，鉴定出了一系列与光响应、激素响应、干旱胁迫及低温胁迫响应相关的顺式作用元件，推测该基因在调控植物生长发育进程以及增强植物对非生物胁迫的耐受性等方面发挥重要作用。PaACC 基因在不同组织中表达模式存在差异，其中 PaACC4、PaACC5、PaACC7 在果实 S5 时期表达量显著高于其他器官和时期。本研究为进一步研究 PaACC 基因在油梨脂肪酸合成中的功能提供理论基础。

Abstract

Avocado (Persea americana) is a fruit with high nutritional value, and fatty acids are among its key intrinsic nutrients. Acetyl-CoA carboxylase (ACC) is a crucial enzyme in fatty acid synthesis, essential for oil accumulation and storage. The protein structure, evolutionary relationships, and cis-acting elements of the PaACC gene promoter in avocado were analyzed by employing bioinformatics tools, and the expression levels of the PaACC genes across different avocado organs and developmental stages of the fruit were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A total of seven PaACC genes were identified in avocado, encoding three BC subunits, three BCCP subunits, and one α−CT subunit of ACC. The amino acid lengths of these genes range from 251 to 776, with an average molecular weight of 49.95 kDa. All the encoded proteins are hydrophilic and lack of transmembrane domains. Phylogenetic analysis of ACC proteins from various plant species reveals that ACC proteins are categorized into four subfamilies based on subunit composition. The gene structure shows that all the PaACC genes contain at least 5 exons, each member of which contains a certain number of introns. There are 25 different conserved motifs in the PaACC gene family. Specifically, PaACC1, PaACC2, and PaACC3 all have motif 14, whilePaACC4, PaACC5, and PaACC6 all have motifs 1, 2, 3, 4, and 5. In the promoter region of the PaACC genes, several cis-acting elements associated with light response, hormone response, drought stress, and low-temperature stress were identified. These findings suggest that the PaACC genes play a significant role in regulating plant growth and development and may enhance the plant’s tolerance to abiotic stresses. Expression pattern analysis reveals that PaACC4, PaACC5, and PaACC7 exhibit significantly higher expression levels during the S5 stage of fruit development compared to other organs and stages. This study provides a theoretical foundation for further research on the role of the PaACC genes in avocado fatty acid biosynthesis.

关键词

油梨 / 乙酰辅酶 A 羧化酶 / 基因家族 / 表达模式

Key words

avocado / acetyl-CoA carboxylase / gene family / expression pattern

参考文献

[1] 葛宇, 司雄元, 林兴娥, 等. 油梨研究进展[J]. 中国南方果树, 2017, 46(1): 148-155. https://doi.org/10.13938/j. issn.1007-1431.20160156

[2] 黄少鹏, 任正恺, 唐丽珠, 等. 油梨 Dof 基因家族鉴定及其在生长发育过程中的表达分析[J]. 分子植物育种, 2024, 22(3): 740-750. https://doi.org/10.13271/j.mpb. 022.000740

[3] Ozdemir F, Topuz A. Changes in dry matter, oil content and fatty acids composition of avocado during harvesting time and post-harvesting ripening period[J]. Food Chemistry, 2004, 86(1): 79-83. https://doi.org/10.1016/j.food- chem.2003.08.012

[4] Ge Y, Dong X, Liu Y, et al. Molecular and biochemical analyses of avocado (Persea americana) reveal differences in the oil accumulation pattern between the mesocarp and seed during the fruit developmental period [J]. Scientia Horticulturae, 2021, 276: 109717. https://doi.org/ 10.1016/j.scienta.2020.109717

[5] Kilaru A, Cao X, Dabbs P B, et al. Oil biosynthesis in a basal angiosperm: transcriptome analysis of Persea americana mesocarp [J]. BMC Plant Biology, 2015, 15: 203. https://doi.org/10.1186/s12870-015-0586-2

[6] 高博, 李宗宝. 植物乙酰辅酶 A 羧化酶性质和抑制剂研究[J]. 医学信息, 2008, 21(7): 1215-1217. https://doi. org/10.3969/j.issn.1006-1959.2008.07.115

[7] 赵虎基, 王国英. 植物乙酰辅酶 A 羧化酶的分子生物学与基因工程[J]. 中国生物工程杂志, 2003, 23(2): 12-16. https://doi.org/10.13523/j.cb.20030203

[8] Roesler K R, Shorrosh B S, Ohlrogge J B. Structure and expression of an Arabidopsis acetyl-coenzyme A carboxylase gene [J]. Plant Physiology, 1994, 105(2): 611-617. https://doi.org/10.1104/pp.105.2.611

[9] Reverdatto S, Beilinson V, Nielsen N C. A multisubunit acetyl coenzyme A carboxylase from soybean[J]. Plant Physiology, 1999, 119(3): 961-978. https://doi.org/10. 1104/pp.119.3.961

[10] 武玉永, 谭秀华, 马立新. 甘蓝型油菜乙酰辅酶 A 羧化酶 3 个亚基基因的克隆及其表达[J]. 安徽农业科学, 2008, 36(10): 4002-4006. https://doi.org/10.3969/j.issn. 0517-6611.2008.10.030

[11] Nakkaew A, Chotigeat W, Eksomtramage T, et al. Cloning and expression of a plastid-encoded subunit, beta-carboxyltransferase gene (accD) and a nuclear-encoded subunit, biotin carboxylase of acetyl-CoA carboxylase from oil palm (Elaeis guineensis Jacq.) [J]. Plant Science, 2008, 175(4): 497-504. https://doi.org/10.1016/j. plantsci.2008.05.023

[12] 王哲. 油桐异质型 ACCase 基因的克隆及功能表达研究 [D]. 长沙: 中南林业科技大学, 2015.

[13] 刘浩, 鲁清, 李海芬, 等. 花生酰基-CoA 羧化酶基因 ACC1 的克隆与表达分析 [J]. 山东农业科学, 2019, 51(9): 21-27. https://doi.org/10.14083/j.issn.1001-4942. 2019.09.003

[14] 王保明. 油茶 ACCase 基因的克隆及功能研究[D]. 长沙: 中南林业科技大学, 2012.

[15] 杨婷, 张建平, 刘自刚, 等. 胡麻异质型 ACCase 亚基基因的克隆与表达分析[J]. 草业学报, 2020, 29(4): 111-120. https://doi.org/10.11686/cyxb2019520

[16] 李欣叶. 亚麻 LuAccD 基因在种子油脂积累中的功能研究 [D]. 杨凌: 西北农林科技大学, 2023. https://doi. org/10.27409/d.cnki.gxbnu.2023.002617

[17] 崔燕. 棉花乙酰辅酶 A 羧化酶 β 亚基 accD 基因的克隆及在拟南芥中超量表达的研究[D]. 石河子: 石河子大学, 2008.

[18] Pham T, Walden E, Huard S, et al. Fine-tuning acetyl-CoA carboxylase 1 activity through localization: functional genomics reveals a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization[J]. Genetics, 2022, 221(4): iyac086. https://doi.org/10.1093/genetics/iyac0 86

[19] Gudenschwager O, García-rojas M, Defilippi B G, et al. Identification and characterization of two putative genes encoding acetyl-coenzyme A carboxylase subunits that are possibly associated with internal browning during cold storage of ‘Hass’ avocados (Persea americana Mill. ) [J]. Postharvest Biology and Technology, 2013, 84: 74-80. https://doi.org/10.1016/j.postharvbio.2013.04.011

[20] Ford N A, Spagnuolo P, Kraft J, et al. Nutritional composition of hass avocado pulp[J]. Foods, 2023, 12(13): 2516. https://doi.org/10.3390/foods12132516

[21] Rendón-anaya M, ibarra-laclette E, Méndez-bravo A, et al. The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(34): 17081-17089. https://doi.org/10.1073/pnas.1822129116

[22] 卜宪辰， 郭龙飞， 祝嫦晔， 等. 构树 bZIP 基因家族鉴定及硒代谢相关基因筛选[J]. 南方农业学报, 2025, 56(1): 135-148. https://doi.org/http://nfnyxb.xml-journ- al.net/article/doi/10.3969/j.issn.2095-1191. 2025.01.012

[23] 林胜男, 刘杰玮, 张晓妮, 等. 香石竹 WRKY 家族全基因组鉴定及其表达分析[J]. 园艺学报, 2021, 48(9): 1768-1784. https://doi.org/10.16420/j.issn.0513-353x. 2019-0986

[24] 陆雯佳， 汪军成， 姚立蓉， 等. 大麦 PRX 基因家族全基因组鉴定及其干旱胁迫下的表达分析[J]. 作物学报, 2025, 51(5): 1198-1214. https://doi.org/10.3724/SP.J. 1006.2025.41053

[25] 马春泉, 孙培琳, 李海英. BvM14-GAI 基因的克隆及亚细胞定位[J]. 中国农学通报, 2020, 36(16): 28-33.

[26] 刘梦迪, 倪守飞, 王月雪, 等. 大麦 TIFY 基因家族成员鉴定及表达分析 [J]. 南方农业学报, 2022, 53(2): 417-429. https://doi.org/10.3969/j.issn.2095-1191.2022.02.015

[27] 黄俊, 江羽宸, 张云川, 等. 橡胶草 SRPP/ REF 家族基因的鉴定及表达分析 [J]. 植物生理学报, 2020, 56(7): 1541-1552. https://doi.org/10.13592/j.cnki.ppj.2020.0041

[28] 许晓兰， 黎洁， 许慧娴， 等. 三色堇 VwF3H 基因启动子克隆及其功能分析 [J]. 南方农业学报, 2025, 56(2): 368-377. https://doi.org/10.3969/j.issn.2095-1191.2025. 02.003

[29] 郑飞雄, 陈俊宇, 江林琪, 等. 掌叶覆盆子 4CL 基因家族鉴定及表达分析[J]. 农业生物技术学报, 2024, 32(2): 311-321. https://doi.org/10.3969/j.issn.1674-7968. 2024.02.006

[30] 于爽, 田海莹, 李生梅, 等. 花生乙酰辅酶 A 羧化酶家 族基因在不同胁迫下的响应模式分析[J]. 植物生理学报 , 2021, 57(10): 2011-2023. https://doi.org/10. 13592/j.cnki.ppj.2021.0154

[31] Wu X, Yang Z, Zhu Y, et al. Bioinformatics identification and expression analysis of acetyl-CoA carboxylase reveal its role in isoflavone accumulation during soybean seed development[J]. International Journal of Molecular Sciences, 2024, 25(18): 10221. https://doi.org/10. 3390/ijms251810221

[32] 肖勇, 雷新涛, 王永, 等. 椰子乙酰 CoA 羧化酶 (ACC) 基因的鉴定及表达分析[J]. 安徽农业科学, 2017, 45(35): 128-129. https://doi.org/10.3969/j.issn. 0517-6611.2017.35.039

[33] Hu Y, Gao C, Yang X, et al. Genome-scale identification and analysis of acetyl-coenzyme a carboxylase genes in Arabidopsis and soybean [C]// 2011 International Conference on Electrical and Control Engineering. September 16-18, 2011, Yichang, China. IEEE, 2011: 3474-3477. https://doi.org/10.1109/ICECENG.2011. 6058473

[34] Elborough K M, Winz R, Deka R K, et al. Biotin carboxyl carrier protein and carboxyltransferase subunits of the multi-subunit form of acetyl-CoA carboxylase from Brassica napus: cloning and analysis of expression during oilseed rape embryogenesis [J]. Biochemical Journal, 1996, 315(1): 103-112. https://doi. org/10.1042/bj3150103

[35] Wang M, Garneau M G, Poudel A N, et al. Overexpression of pea α-carboxyltransferase in Arabidopsis and Camelina increases fatty acid synthesis leading to improved seed oil content [J]. The Plant Journal, 2022, 110(4): 1035-1046. https://doi.org/10.1111/tpj.15721

[36] Zhou L, Du Y, Zhang M, et al. Regulator of fatty acid synthesis proteins regulate de novo fatty acid synthesis by modulating hetACCase distribution [J]. The Plant Cell, 2024, 37(1): koae295. https://doi.org/10.1093/pl- cell/koae295

[37] Salie M J, Zhang N, Lancikova V, et al. A family of negative regulators targets the committed step of de novo fatty acid biosynthesis [J]. The Plant Cell, 2016, 28(9): 2312-2325. https://doi.org/10.1105/tpc.16.00317

基金资助

科技部、财政部项目(NTPGRCC2024-010)

农业农村部项目(2124013)

海南省研究生创新科研课题(Qhys2023-277)