建兰ABC基因家族鉴定及其在花发育过程中的表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0321

摘要/Abstract

摘要：

ABC基因家族编码一类定位于生物膜的转运蛋白，参与多种物质的转运，在植物生长发育和适应胁迫等生命活动中发挥重要作用。通过生物信息学方法对建兰ABC基因家族成员进行全基因鉴定，并基于转录组数据和实时荧光定量PCR技术（RT-qPCR）分析其在建兰花发育过程中的表达模式。结果表明，建兰基因组中共存在121个可分为8个亚族的ABC基因。所有的建兰ABC基因均具有至少1个保守的核苷酸结合结构域。建兰ABC基因家族成员不均匀分布于19条染色体上，2号和8号染色体上成员数目最多。串联重复事件是导致建兰ABC基因家族扩张的主要原因。建兰ABC基因启动子序列上存在多种环境和激素响应元件。CeABCB6、CeABCB30、CeABCG3、CeABCG54和CeABCI7基因的表达水平与建兰主要花香物质的释放量呈正相关。本研究为后续了解建兰ABC基因的功能奠定基础，并为建兰花香研究提供基因资源。

关键词: ABC转运蛋白, 建兰, 全基因组鉴定, 基因表达模式, 花发育, 花香

Abstract:

ATP-binding cassette（ABC）transporters are membrane transporter proteins that participate in the transportation of a huge range of substrates. Plant ABC genes function in many important biological processes of growth and development and abiotic stress resistance. We used bioinformatics methods to perform genome-wide identification of ABC gene family in Cymbidium ensifolium and analyze their expression patterns during flowering development based transcriptome data and RT-qPCR. In total，there were 121 ABC genes classified into 8 subfamilies in C. ensifolium genome. All ABC proteins had at least one conserved nucleotide binding domain（NBD）. The 121 ABC genes were unevenly distributed on 19 chromosomes，and the most ABC genes located on chromosome 2 and 8. Tandem duplication was found to be the main reason for the expansion of the ABC genes. Cis-acting element prediction revealed that the promoter sequences of ABC genes contained phytohormone and abiotic stress responsive elements. The expressions of CeABCB6，CeABCB30，CeABCG3，CeABCG54 and CeABCI7 were positively correlated with the volatilization of the key aromatic components in different flowering stages of the C. ensifolium. This study lays a foundation for further understanding the function of ABC genes and provides genetic resources for the study of C. ensifolium floral fragrance.

Key words: ABC transporters, Cymbidium ensifolium, genome-wide identification, gene expression pattern, flower development, floral fragrance

曹映辉, 胡美娟, 童妍, 张燕萍, 赵凯, 彭东辉, 周育真. 建兰ABC基因家族鉴定及其在花发育过程中的表达模式分析[J]. 生物技术通报, 2022, 38(11): 162-174.

CAO Ying-hui, HU Mei-juan, TONG Yan, ZHANG Yan-ping, ZHAO Kai, PENG Dong-hui, ZHOU Yu-zhen. Identification of the ABC Gene Family and Expression Pattern Analysis During Flower Development in Cymbidium ensifolium[J]. Biotechnology Bulletin, 2022, 38(11): 162-174.

图/表 12

表1 实时荧光定量PCR引物

Table 1 Primers used for quantitative real-time PCR

基因名称Gene name	上游引物Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
CeABCB6	GGAGCTACCCTTGTTCGAGA	TGGACTCTGAGCCTTGTGAG
CeABCB30	AGGCCCTTCGTAACCTTTCA	CAAACTTACGCACGACCACA
CeABCG3	AGGGAAAGAGCAGCAGGAAT	TGGCAGCTGTCCATTCAAAC
CeABCG54	ATCAAGCACACAAGGCCAAC	AAGGTGAGGGAAAGTGGCAT
CeABCI7	TGGGGAAGGATATCAGCGAC	CCATGTGCTTGCCTCCAAAT
TUB	GCAGTTTACGGCGATGTTCA	ACTCTTCCTCGTCAGCTGTG

图1 建兰ABC转运蛋白保守结构域分析

Fig. 1 Conserved domain of the ABC transporters in C. ensifolium

图2 建兰ABC转运蛋白保守基序

Fig. 2 Conserved motif of the ABC transporters in C. ensifolium

图3 建兰ABC基因结构

Fig. 3 Gene structure of the ABCs in C. ensifolium

图4 建兰、水稻和拟南芥的ABC基因家族系统进化树红色字体表示建兰ABC转运蛋白，蓝色字体表示水稻ABC转运蛋白，黑色字体表示拟南芥ABC转运蛋白

Fig. 4 Phylogenetic analysis of ABC gene family in C. ensifolium，O. sativa and A. thaliana The red font refers to C. ensifolium ABC transporter proteins，the blue font refers to O. sativa ABC transporter proteins，the black font refers to A. thaliana ABC transporter proteins

图5 建兰ABC基因染色体定位蓝色基因的为串联重复基因，红线连接的为片段复制基因

Fig. 5 Chromosomal locations of ABC genes in C. ensifolium Tandem arrays of genes are marked in blue，and red lines by fragment duplications

表2 建兰ABC同源基因对的非同义与同义替换率

Table 2 Nonsynonymous and synonymous substitution ratio calculation of ABC hologous genes in C. ensifolium

基因名称Gene name	基因名称Gene name	复制类型Duplication type	非同义替换率Ka	同义替换率Ks	非同义替换率/同义替换率Ka/Ks
CeABCG31	CeABCG29	片段复制	0.148 156 999	0.608 282 47	0.243 566 117
CeABCG19	CeABCG25	片段复制	0.368 169 066	NaN	NaN
CeABCB18	CeABCB16	片段复制	0.069 986 027	0.674 387 106	0.103 777 231
CeABCG10	CeABCG46	串联复制	0.017 153 167	0.023 289 543	0.736 517 994
CeABCG5	CeABCG8	串联复制	0.108 286 131	0.156 200 883	0.693 249 159
CeABCG8	CeABCG50	串联复制	0.016 788 187	0.022 178 207	0.756 967 701
CeABCG47	CeABCG1	串联复制	0.207 255 225	2.066 524 894	0.100 291 666
CeABCC4	CeABCC3	串联复制	0.212 818 781	1.459 333 628	0.145 832 849
CeABCG18	CeABCG21	串联复制	0.006 383 502	0.021 424 752	0.297 949 888
CeABCI2	CeABCI3	串联复制	0.005 878 541	NaN	NaN
CeABCC1	CeABCC5	串联复制	0.291 628 981	NaN	NaN
CeABCB12	CeABCB21	串联复制	0.024 644 867	0.050 781 874	0.485 308 334
CeABCB17	CeABCB16	串联复制	0.644 437 783	NaN	NaN
CeABCB10	CeABCB31	串联复制	0.039 511 484	0.03 561 5	1.109 405 7
CeABCG30	CeABCG39	串联复制	0.106 510 279	0.544 293 256	0.195 685 466
CeABCG24	CeABCG27	串联复制	0.040 625 407	0.058 053 477	0.699 792 83
CeABCG32	CeABCG44	串联复制	0.064 248 894	0.099 652 01	0.644 732 544

图6 建兰与拟南芥、水稻、蝴蝶兰中ABC基因共线性分析红色线表示共线性ABC基因对

Fig. 6 Collinearity analysis of ABC genes between C. ensifolium and A. thaliana，O. sativa，P. aphrodite Red line indicates the collinearity of pair of ABC genes

图7 建兰ABC基因启动子顺式作用元件分析结果 Abscisic acid responsive element：脱落酸响应元件. Anaerobic induction：厌氧感应元件. Anoxic inducibility：缺氧诱导元件. Auxin responsive element：生长素响应元件. Circadian control：昼夜节律调控元件. Defense and stress responsive element：防御和应激响应元件. Endosperm expression：胚乳表达调控元件. Gibberellin responsive element：赤霉素响应元件. Light responsive element：光响应元件. Low temperature responsive element：低温响应元件. MeJA responsive element：茉莉酸甲酯响应元件. Meristem expression：分生组织表达调控元件. MYB binding site：MYB结合位点. MYBHv1 binding site：MYBHv1结合位点. Salicylic acid responsive elemen：水杨酸响应元件. Wound responsive element：创伤响应元件

Fig. 7 Cis-acting elements in the promoters of ABC genes in C. ensifolium

图8 建兰ABC基因家族在不同时期的花中表达模式 A：不同亚家族基因表达模式；B：基因表达模式聚类。ZL：蕾期；CK：初开期；SK：盛开期；SB：衰败期

Fig. 8 Expression profiles of ABC genes of C. ensifolium in flower development A：Expression patterns related to ABC subfamilies. B：Hierarchical clusters based on gene expression. ZL：Budding flower. CK：Half flowering stage. SK：Full bloomed stage. SB：Senescence stage

图9 花发育过程中CeABC基因的表达分析 XL：小蕾；ZL：中蕾；DL：大蕾；CK：初开；SK：盛开期；SB：衰败期

Fig. 9 Express analysis of CeABC genes during flowering development XL：Small bud stage. ZL：Medium bud stage. DL：Big bud stage. CK：Half flowering stage. SK：Full bloomed stage. SB：Senescence stage

图10 建兰花香物质释放量与CeABC基因表达量的相关性分析 Methyl jasmonate：茉莉酸甲酯；beta-selinene：β-瑟林烯；aromadendrene：香橙烯

Fig. 10 Correlation analysis between the release of floral aroma components and CeABC genes expression

参考文献 32

[1]	Theodoulou FL, Kerr ID. ABC transporter research:going strong 40 years on[J]. Biochem Soc Trans, 2015, 43(5):1033-1040. doi: 10.1042/BST20150139 URL
[2]	Garcia O, Bouige P, Forestier C, et al. Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette(ABC)systems[J]. J Mol Biol, 2004, 343(1):249-265. doi: 10.1016/j.jmb.2004.07.093 URL
[3]	Andolfo G, Ruocco M, di Donato A, et al. Genetic variability and evolutionary diversification of membrane ABC transporters in plants[J]. BMC Plant Biol, 2015, 15:51. doi: 10.1186/s12870-014-0323-2 pmid: 25850033
[4]	Do THT, et al. Functions of ABC transporters in plant growth and development[J]. Curr Opin Plant Biol, 2018, 41:32-38. doi: S1369-5266(17)30151-6 pmid: 28854397
[5]	Hwang JU, Song WY, Hong D, et al. Plant ABC transporters enable many unique aspects of a terrestrial plant’s lifestyle[J]. Mol Plant, 2016, 9(3):338-355. doi: 10.1016/j.molp.2016.02.003 URL
[6]	Zhang ZL, Tong T, Fang YX, et al. Genome-wide identification of barley ABC genes and their expression in response to abiotic stress treatment[J]. Plants(Basel), 2020, 9(10):1281.
[7]	Chen PJ, Li Y, Zhao LH, et al. Genome-wide identification and expression profiling of ATP-binding cassette(ABC)transporter gene family in pineapple(Ananas comosus(L.)merr. )reveal the role of AcABCG38 in pollen development[J]. Front Plant Sci, 2017, 8:2150. doi: 10.3389/fpls.2017.02150 URL
[8]	Pang KY, Li YJ, Liu MH, et al. Inventory and general analysis of the ATP-binding cassette(ABC)gene superfamily in maize(Zea mays L.)[J]. Gene, 2013, 526(2):411-428. doi: 10.1016/j.gene.2013.05.051 URL
[9]	Gani U, Vishwakarma RA, Misra P. Membrane transporters:the key drivers of transport of secondary metabolites in plants[J]. Plant Cell Rep, 2021, 40(1):1-18. doi: 10.1007/s00299-020-02599-9 URL
[10]	Borghi L, Kang J, de Brito Francisco R. Filling the gap:functional clustering of ABC proteins for the investigation of hormonal transport in planta[J]. Front Plant Sci, 2019, 10:422. doi: 10.3389/fpls.2019.00422 URL
[11]	Ai Y, et al. The Cymbidium genome reveals the evolution of unique morphological traits[J]. Hortic Res, 2021, 8(1):255. doi: 10.1038/s41438-021-00683-z URL
[12]	Verrier PJ, et al. Plant ABC proteins—a unified nomenclature and updated inventory[J]. Trends Plant Sci, 2008, 13(4):151-159. doi: 10.1016/j.tplants.2008.02.001 pmid: 18299247
[13]	Lefèvre F, Boutry M. Towards identification of the substrates of ATP-binding cassette transporters[J]. Plant Physiol, 2018, 178(1):18-39. doi: 10.1104/pp.18.00325 pmid: 29987003
[14]	Sánchez-Fernández R, Davies TG, Coleman JO, et al. The Arabidopsis thaliana ABC protein superfamily, a complete inventory[J]. J Biol Chem, 2001, 276(32):30231-30244. doi: 10.1074/jbc.M103104200 pmid: 11346655
[15]	Lopez-Ortiz C, Dutta SK, et al. Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp[J]. PLoS One, 2019, 14(4):e0215901.
[16]	Ofori PA, Mizuno A, Suzuki M, et al. Genome-wide analysis of ATP binding cassette(ABC)transporters in tomato[J]. PLoS One, 2018, 13(7):e0200854.
[17]	Zhang XD, Zhao KX, Yang ZM. Identification of genomic ATP binding cassette(ABC)transporter genes and Cd-responsive ABCs in Brassica napus[J]. Gene, 2018, 664:139-151. doi: S0378-1119(18)30434-7 pmid: 29709635
[18]	Huang JJ, Li XY, Chen X, et al. Genome-wide identification of soybean ABC transporters relate to aluminum toxicity[J]. Int J Mol Sci, 2021, 22(12):6556. doi: 10.3390/ijms22126556 URL
[19]	Yan L, Zhang JH, Chen HY, et al. Genome-wide analysis of ATP-binding cassette transporter provides insight to genes related to bioactive metabolite transportation in Salvia miltiorrhiza[J]. BMC Genomics, 2021, 22(1):315. doi: 10.1186/s12864-021-07623-0 pmid: 33933003
[20]	Khan N, You FM, Datla R, et al. Genome-wide identification of ATP binding cassette(ABC)transporter and heavy metal associated(HMA)gene families in flax(Linum usitatissimum L.)[J]. BMC Genomics, 2020, 21(1):722. doi: 10.1186/s12864-020-07121-9 URL
[21]	Yan C, et al. Genome-wide identification, evolution, and expression analysis of the ATP-binding cassette transporter gene family in Brassica rapa[J]. Front Plant Sci, 2017, 8:349.
[22]	Cui BL, Li Y, Zhang YJ. A comparative genome-wide analysis of the ABC transporter gene family among three Gossypium species[J]. Crop Sci, 2021, 61(4):2489-2509. doi: 10.1002/csc2.20525 URL
[23]	Lane TS, Rempe CS, Davitt J, et al. Diversity of ABC transporter genes across the plant kingdom and their potential utility in biotechnology[J]. BMC Biotechnol, 2016, 16(1):47. doi: 10.1186/s12896-016-0277-6 pmid: 27245738
[24]	Geisler M, Aryal B, di Donato M, et al. A critical view on ABC transporters and their interacting partners in auxin transport[J]. Plant Cell Physiol, 2017, 58(10):1601-1614. doi: 10.1093/pcp/pcx104 pmid: 29016918
[25]	Panikashvili D, Shi JX, Schreiber L, et al. The Arabidopsis ABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis[J]. New Phytol, 2011, 190(1):113-124. doi: 10.1111/j.1469-8137.2010.03608.x pmid: 21232060
[26]	Kuromori T, Miyaji T, et al. ABC transporter AtABCG25 is involved in abscisic acid transport and responses[J]. Proc Natl Acad Sci USA, 2010, 107(5):2361-2366. doi: 10.1073/pnas.0912516107 URL
[27]	Kang J, Hwang JU, Lee M, et al. PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid[J]. Proc Natl Acad Sci USA, 2010, 107(5):2355-2360. doi: 10.1073/pnas.0909222107 URL
[28]	Demissie ZA, Tarnowycz M, Adal AM, et al. A lavender ABC transporter confers resistance to monoterpene toxicity in yeast[J]. Planta, 2019, 249(1):139-144. doi: 10.1007/s00425-018-3064-x pmid: 30535718
[29]	Adebesin F, Widhalm JR, Boachon B, et al. Emission of volatile organic compounds from Petunia flowers is facilitated by an ABC transporter[J]. Science, 2017, 356(6345):1386-1388. doi: 10.1126/science.aan0826 pmid: 28663500
[30]	彭红明. 中国兰花挥发及特征花香成分研究[D]. 北京: 中国林业科学研究院, 2009.
	Peng HM. Study on the volatile, characteristic floral fragrance components of Chinese Cymbidium[D]. Beijing: Chinese Academy of Forestry, 2009.
[31]	Huang MK, Ma CP, Yu RC, et al. Concurrent changes in methyl jasmonate emission and the expression of its biosynthesis-related genes in Cymbidium ensifolium flowers[J]. Physiol Plant, 2015, 153(4):503-512. doi: 10.1111/ppl.12275 URL
[32]	李梦雅. 茉莉酸转运蛋白的发现及其在系统伤害抗性中传递系统信号的功能分析[D]. 北京: 中国农业大学, 2014.
	Li MY. The identification of jasmonate transporters and their essential role of signal relay in systemic wound resistance. Beijing: China Agricultural University, 2014.