油茶SWEET基因家族的全基因组鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0021

摘要/Abstract

摘要：

【目的】挖掘参与油茶糖代谢及逆境响应的糖外排转运子（sugars will eventually be exported transporters, SWEETs）。【方法】利用生物信息学方法分析油茶SWEETs家族的基因结构、蛋白基序、染色体定位、共线性关系、启动子区顺式作用元件及上游调控因子等，并利用RT-qPCR分析CoSWEETs在不同时期、不同组织及不同逆境胁迫下的基因表达情况。【结果】从油茶中鉴定得到14个CoSWEETs基因，不均匀分布于10条染色体上，不同成员间内含子-外显子数目存在差异。根据系统进化关系，14个CoSWEETs可分为 4个分支，均具有1-2个MtN3 保守结构域，同一分支具有相似的基因结构和基序。根据启动子顺式作用元件和上游转录因子预测的分析结果，CoSWEETs启动子中含有多个与生长发育、植物激素和应激相关的调节元件，其表达可能受到ERF、DOF、BBR-BPC、MYB等转录因子的调控。RT-qPCR分析表明大部分CoSWEETs成员在果实和根中高表达，在种子中的表达水平与发育时期相关，并根据低温、高盐和干旱等非生物胁迫下CoSWEETs的表达模式挖掘出CoSWEET1、CoSWEET2、CoSWEET17等响应油茶低温、干旱或高盐胁迫的基因。【结论】CoSWEET基因的表达受到多种激素及转录因子调控，并在油茶种子发育与逆境胁迫响应中发挥重要作用。

关键词: 油茶, SWEET, 蛋白结构, 表达模式分析, 非生物胁迫

Abstract:

【Objective】To explore the involvement of SWEET（sugars will eventually be exported transporters）in sugar metabolism and response to abiotic stress in Camellia oleifera.【Method】Bioinformatics methods were used to analyze the gene structure, protein motif, chromosome localization, collinearity, cis-acting elements of promoter region and upstream regulators in CoSWEETs family, and RT-qPCR was used to analyze the expression pattern of CoSWEETs in different periods, tissues and stress responses.【Result】Total 14 CoSWEET genes were identified in Camellia oleifera for the first time, they were localized on 10 chromosomes, and there were differences in intron-exon numbers between different members. With 1-2 MtN3 conserved domain, the 14 CoSWEETs were divided into four subgroups by phylogenetic tree, and there were similar gene structures and motif in the same subgroup. There were differences in number of introns and exons among different members. Based on the analysis of promoter cis-acting elements and upstream transcription factor predictions, multiple cis-elements related to development, plant hormones and environmental stresses were found in the promoter region, and their expressions were regulated by transcription factors, like ERF, DOF, BBR-BPC and MYB. RT-qPCR results showed that CoSWEETs highly expressed in the fruit and root, and the expression in the seeds was related to the developmental stages. Moreover, several genes responding to low-temperature, drought or salt stress, such as CoSWEET1, CoSWEET2 and CoSWEET17 were mined based the expression profile of CoSWEETs members under abiotic stresses of low temperature, drought and high salt.【Conclusion】The expression of CoSWEETs is regulated by various hormones and transcription factors, and plays a crucial role in the development of seeds and the response to stress in Camellia oleifera.

Key words: Camellia oleifera, SWEET, protein structure, expression pattern analysis, abiotic stress

杜兵帅, 邹昕蕙, 王子豪, 张馨元, 曹一博, 张凌云. 油茶SWEET基因家族的全基因组鉴定及表达分析[J]. 生物技术通报, 2024, 40(5): 179-190.

DU Bing-shuai, ZOU Xin-hui, WANG Zi-hao, ZHANG Xin-yuan, CAO Yi-bo, ZHANG Ling-yun. Genome-wide Identification and Expression Analysis of the SWEET Gene Family in Camellia oleifera[J]. Biotechnology Bulletin, 2024, 40(5): 179-190.

图/表 9

表1 本研究所用RT-qPCR 引物

Table 1 RT-qPCR primers used in this study

基因名称Gene	上游引物Forward primer sequence（5'-3'）	下游引物Reverse primer sequence（5'-3'）
CoSWEET1a	GGTGAAGCCAAGAAACCTCCT	CTACTTGCTCGATCGCTTCTCT
CoSWEET1b	TTGGAGGAAGAGAAGCTATCTG	ATGATTTCTTGACATGCGATTGAG
CoSWEET2a	GCAGAGAACGCGAAAAAGGT	ATATCCAACGAAGATCTGTCGATT
CoSWEET2b	ATGGTTTCTTTGGCTTTGTCCC	AGATATGAGTGGCGTGCCGT
CoSWEET5	CACCAGTCCCGACGTTCTTT	GGACGAAGGGAAGGCCATAG
CoSWEET7	CCAAGCTCCGGTCACTCATT	ATTAGCACAGGAAGCCAGGG
CoSWEET9a	AGGTTCTACCAGAAGTCAAGCTG	TGGATTCGCTCGGTTTGATA
CoSWEET9b	AAGAGCGTGGAGTACATGCC	GCAAATCCCGAGAGACGACA
CoSWEET10	TGGAGGAAGAGAAGCTATCTGAAT	TGAGGCATGACTGGAATGATTTCT
CoSWEET15	TCACCACCCACTGGTGTTTAC	ACATCCAAAGCATGGCACTG
CoSWEET16a	TTCACCACCCACTGGTGTTT	GGCTGTGTGGTTCTTAGCCT
CoSWEET16b	AGACTGCAACACTAGCTGGG	TCCTCAACAGAGGACACTGC
CoSWEET17a	ATGTTGGGTTTCTTGGGGCA	GACAGCTAGAGGTGCTGCAT
CoSWEET17b	GCAAGGCCTCGATGACCAC	TTTGTCGATGCTGTATTGCCG
CoGAPDH	GGTGCCAAGAAGGTGGTAATA	GTTGTGCAGCTTGCATTAGAG
CoActin	AAACTACGGTTGCGGATAGAG	CTCCGGTGCATCCTTCATAAT

表2 油茶CoSWEETs家族的理化特征分析

Table 2 Physical and chemical characteristics analysis of CoSWEETs family in C. oleifera

Name	Gene ID	Chrom	CDS size	Protein length/aa	TMHs	Molecular weight（Average）/Da	pI	Instability index	GRAVY
CoSWEET1a	KAI8013233.1	4	756	251	7	27681.96	9.64	36.09	0.534
CoSWEET1b	KAI8003830.1	9	726	241	7	26377.37	9.71	36.9	0.671
CoSWEET2a	KAI8001534.1	8	708	235	7	25982.12	8.69	40.43	0.944
CoSWEET2b	KAI8008303.1	7	708	235	7	25933.89	9.39	39.6	0.888
CoSWEET5	KAI8018837.1	2	723	240	7	27247.42	6.72	32.52	0.702
CoSWEET7	KAI7995081.1	12	771	256	7	27737.2	9.26	40.41	0.668
CoSWEET9a	KAI7982657.1	11	828	275	7	30777.62	8.71	31.73	0.638
CoSWEET9b	KAI7982326.1	11	831	276	6	31463.67	9.37	27.9	0.769
CoSWEET10	KAI8016974.1	2	984	327	7	36875.13	9.01	42.64	0.606
CoSWEET15	KAI8023305.1	6	894	297	7	33239.44	6.19	36.79	0.638
CoSWEET16a	KAI8028642.1	3	750	249	7	27594.03	4.98	52.73	0.492
CoSWEET16b	KAI7980775.1	1	789	262	3	28591.53	4.75	51.82	0.012
CoSWEET17a	KAI8028641.1	3	930	309	7	33939.77	8.41	38.05	0.342
CoSWEET17b	KAI7980777.1	1	909	302	7	33108.94	9.27	35.38	0.429

图1 不同物种间（拟南芥、水稻、茶树和油茶）SWEETs系统发育树

Fig. 1 Phylogenetic analysis of the SWEET proteins from four plant species（Arabidopsis thaliana, Oryza sati-va, Camellia sinensis and C. oleifera）

图2 油茶CoSWEETs家族基序，保守结构域及基因结构分析 A：CoSWEET基因家族保守基序分析；B：CoSWEET基因家族保守结构域分析；C：CoSWEET基因家族基因结构分析；D：CoSWEET基因家族保守基序的序列 Logo

Fig. 2 Conserved motifs and exon-intron structure analysis of the CoSWEETs family in C. oleifera A: Conservative domain of CoSWEET gene family; B: conserved domain analysis of CoSWEET gene family; C: gene structure map of CoSWEET gene family; D: Logo of conserved motif of CoSWEET gene family

图3 油茶和拟南芥物种间SWEETs共线性分析

Fig. 3 Synteny of SWEETs in Arabidopsis thaliana and C. oleifera

图4 油茶CoSWEET基因家族启动子区的顺式作用元件分析和上游转录因子预测 A：CoSWEET基因家族启动子区的顺式作用元件分析；B-C：CoSWEET基因家族的上游转录因子预测

Fig. 4 Cis-element analysis and upstream transcription factor prediction of CoSWEET gene family in C. oleifera A: Cis-acting elements analysis in the promotors of CoSWEET gene family. B-C: Prediction about the upstream transcription factors of CoSWEET gene family

图5 油茶种子中蔗糖含量变化及CoSWEETs表达特性分析 A：不同发育时期的油茶种子中蔗糖含量变化；B：不同发育时期的油茶种子中CoSWEETs的表达模式分析

Fig. 5 Content changes of sucrose in seeds and expression analysis of CoSWEETs genes in C. oleifera A: Content changes of sucrose in seeds at different stages. B: The expression profiles of CoSWEETs in seeds at differential stages

图6 油茶CoSWEETs基因家族的表达特性分析 A：CoSWEETs在油茶不同组织中的表达模式分析；B：CoSWEETs在不同非生物胁迫下的表达模式分析

Fig. 6 Expression analysis of CoSWEETs gene family in C. oleifera A: Expression profiles of CoSWEETs in different C. oleifera tissues. B: Expression profiles of CoSWEETs under various abiotic stress

图7 CoSWEETs在油茶不同组织和不同非生物胁迫下的表达模式图

Fig. 7 A model for CoSWEETs expression in different tissues of C. oleifera and various abiotic stress

参考文献 40

[1]	Ruan YL. Sucrose metabolism: gateway to diverse carbon use and sugar signaling[J]. Annu Rev Plant Biol, 2014, 65: 33-67.
[2]	Yoon J, Cho LH, Tun W, et al. Sucrose signaling in higher plants[J]. Plant Sci, 2021, 302: 110703.
[3]	Ayre BG. Membrane-transport systems for sucrose in relation to whole-plant carbon partitioning[J]. Mol Plant, 2011, 4(3): 377-394. doi: 10.1093/mp/ssr014 pmid: 21502663
[4]	Doidy J, Grace E, Kühn C, et al. Sugar transporters in plants and in their interactions with fungi[J]. Trends Plant Sci, 2012, 17(7): 413-422. doi: 10.1016/j.tplants.2012.03.009 pmid: 22513109
[5]	Chen LQ, Qu XQ, Hou BH, et al. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport[J]. Science, 2012, 335(6065): 207-211.
[6]	Tao YY, Cheung LS, Li S, et al. Structure of a eukaryotic SWEET transporter in a homotrimeric complex[J]. Nature, 2015, 527(7577): 259-263.
[7]	Xuan YH, Hu YB, Chen LQ, et al. Functional role of oligomerization for bacterial and plant SWEET sugar transporter family[J]. Proc Natl Acad Sci USA, 2013, 110(39): E3685-E3694.
[8]	Chen LQ, Hou BH, Lalonde S, et al. Sugar transporters for intercellular exchange and nutrition of pathogens[J]. Nature, 2010, 468(7323): 527-532.
[9]	Yuan M, Wang SP. Rice MtN3/saliva/SWEET family genes and their homologs in cellular organisms[J]. Mol Plant, 2013, 6(3): 665-674. doi: 10.1093/mp/sst035 pmid: 23430047
[10]	Gao Y, Wang ZY, Kumar V, et al. Genome-wide identification of the SWEET gene family in wheat[J]. Gene, 2018, 642: 284-292. doi: S0378-1119(17)31009-0 pmid: 29155326
[11]	Patil G, Valliyodan B, Deshmukh R, et al. Soybean(Glycine max)SWEET gene family: insights through comparative genomics, transcriptome profiling and whole genome re-sequence analysis[J]. BMC Genomics, 2015, 16(1): 520.
[12]	Wei XY, Liu FL, Chen C, et al. The Malus domestica sugar transporter gene family: identifications based on genome and expression profiling related to the accumulation of fruit sugars[J]. Front Plant Sci, 2014, 5: 569.
[13]	Jiang L, Song C, Zhu X, et al. SWEET transporters and the potential functions of these sequences in tea(Camellia sinensis)[J]. Front Genet, 2021, 12: 655843.
[14]	Eom JS, Chen LQ, Sosso D, et al. SWEETs, transporters for intracellular and intercellular sugar translocation[J]. Curr Opin Plant Biol, 2015, 25: 53-62.
[15]	Wang SD, Yokosho K, Guo RZ, et al. The soybean sugar transporter GmSWEET15 mediates sucrose export from endosperm to early embryo[J]. Plant Physiol, 2019, 180(4): 2133-2141. doi: 10.1104/pp.19.00641 pmid: 31221732
[16]	Shen S, Ma S, Chen XM, et al. A transcriptional landscape underlying sugar import for grain set in maize[J]. Plant J, 2022, 110(1): 228-242.
[17]	Guan YF, Huang XY, Zhu J, et al. RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis[J]. Plant Physiol, 2008, 147(2): 852-863.
[18]	徐磊, 王伟伟, 苏世超, 等. 小麦糖转运蛋白基因TaSWEET6的克隆与表达分析[J]. 麦类作物学报, 2016, 36(11): 1411-1418.
	Xu L, Wang WW, Su SC, et al. Cloning and expression analysis of a sugar transporter protein gene TaSWEET6 in wheat[J]. J Triticeae Crops, 2016, 36(11): 1411-1418.
[19]	张璐, 李明, 叶广继, 等. 马铃薯糖转运蛋白基因的克隆及表达分析[J]. 西北植物学报, 2019, 39(9): 1528-1533.
	Zhang L, Li M, Ye GJ, et al. Cloning and expression analysis of a sugar transporter protein gene in potato[J]. Acta Bot Boreali Occidentalia Sin, 2019, 39(9): 1528-1533.
[20]	马来. 水稻蔗糖转运蛋白OsSWEET11和OsSWEET14功能的研究[D]. 南京: 南京农业大学, 2018.
	Ma L. Functional analysis of sucrose transporter genes OsSWEET11 and OsSWEET14 in rice[D]. Nanjing: Nanjing Agricultural University, 2018.
[21]	Durand M, Porcheron B, Hennion N, et al. Water deficit enhances C export to the roots in Arabidopsis thaliana plants with contribution of sucrose transporters in both shoot and roots[J]. Plant Physiol, 2016, 170(3): 1460-1479.
[22]	Valifard M, Le Hir R, Müller J, et al. Vacuolar fructose transporter SWEET17 is critical for root development and drought tolerance[J]. Plant Physiol, 2021, 187(4): 2716-2730. doi: 10.1093/plphys/kiab436 pmid: 34597404
[23]	叶洲辰, 吴友根, 于靖, 等. 不同产地油茶籽油提取物的抗氧化活性比较分析及其营养评价[J]. 生物技术通报, 2019, 35(10): 80-88. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0433
	Ye ZC, Wu YG, Yu J, et al. Comparative analysis of antioxidant activities of Camellia oleifera oil extracts from different areas and their nutritional assessments[J]. Biotechnol Bull, 2019, 35(10): 80-88.
[24]	Zhang FH, Li Z, Zhou JQ, et al. Comparative study on fruit development and oil synthesis in two cultivars of Camellia oleifera[J]. BMC Plant Biol, 2021, 21(1): 348.
[25]	Song QL, Gong WF, Yu XR, et al. Transcriptome and anatomical comparisons reveal the effects of methyl jasmonate on the seed development of Camellia oleifera[J]. J Agric Food Chem, 2023, 71(17): 6747-6762.
[26]	He ZL, Liu CX, Zhang Z, et al. Integration of mRNA and miRNA analysis reveals the differentially regulatory network in two different Camellia oleifera cultivars under drought stress[J]. Front Plant Sci, 2022, 13: 1001357.
[27]	Wu YT, Wu PZ, Xu SM, et al. Genome-wide identification, expression patterns and sugar transport of the physic nut SWEET gene family and a functional analysis of JcSWEET16 in Arabidopsis[J]. Int J Mol Sci, 2022, 23(10): 5391.
[28]	Hu Z, Tang ZJ, Zhang YM, et al. Rice SUT and SWEET transporters[J]. Int J Mol Sci, 2021, 22(20): 11198.
[29]	Zeng Z, Lyu T, Lyu YM. LoSWEET14, a sugar transporter in lily, is regulated by transcription factor LoABF2 to participate in the ABA signaling pathway and enhance tolerance to multiple abiotic stresses in tobacco[J]. Int J Mol Sci, 2022, 23(23): 15093.
[30]	Yu MX, Liu JL, Du BS, et al. NAC transcription factor PwNAC11 activates ERD1 by interaction with ABF3 and DREB2A to enhance drought tolerance in transgenic Arabidopsis[J]. Int J Mol Sci, 2021, 22(13): 6952.
[31]	Dai ZR, Yan PY, He SZ, et al. Genome-wide identification and expression analysis of SWEET family genes in sweet potato and its two diploid relatives[J]. Int J Mol Sci, 2022, 23(24): 15848.
[32]	孙全喜, 苑翠玲, 牟艺菲, 等. 花生SWEET基因全基因组鉴定及表达分析[J]. 作物学报, 2023, 49(4): 938-954. doi: 10.3724/SP.J.1006.2023.24066
	Sun QX, Yuan CL, Mou YF, et al. Genome-wide identification and expression analysis of SWEET genes from peanut genomes[J]. Acta Agron Sin, 2023, 49(4): 938-954.
[33]	Yang J, Luo DP, Yang B, et al. SWEET11 and 15 as key players in seed filling in rice[J]. New Phytol, 2018, 218(2): 604-615. doi: 10.1111/nph.15004 pmid: 29393510
[34]	Saddhe AA, Manuka R, Penna S. Plant sugars: Homeostasis and transport under abiotic stress in plants[J]. Physiol Plant, 2021, 171(4): 739-755. doi: 10.1111/ppl.13283 pmid: 33215734
[35]	Wu YT, Wang SS, Du WH, et al. Sugar transporter ZmSWEET1b is responsible for assimilate allocation and salt stress response in maize[J]. Funct Integr Genomics, 2023, 23(2): 137.
[36]	路静, 马齐军, 康慧, 等. 苹果糖转运蛋白基因MdSWEET1在番茄中异源表达提高其耐盐性[J]. 园艺学报, 2019, 46(3): 433-443. doi: 10.16420/j.issn.0513-353x.2018-0744
	Lu J, Ma QJ, Kang H, et al. Ectopic expressing MdSWEET1 in tomato enhanced salt tolerance[J]. Acta Hortic Sin, 2019, 46(3): 433-443.
[37]	Ye ZH, Du BS, Zhou J, et al. Camellia oleifera CoSWEET10 is crucial for seed development and drought resistance by mediating su-gar transport in transgenic Arabidopsis[J]. Plants, 2023, 12(15): 2818.
[38]	胡晓波. 柑橘SWEET11d对果实蔗糖积累的转录调控机制研究[D]. 杭州: 浙江大学, 2022.
	Hu XB. Transcriptional regulation mechanism of citrus SWEET11d on fruit sucrose accumulation[D]. Hangzhou: Zhejiang University, 2022.
[39]	Wu YF, Lee SK, Yoo Y, et al. Rice transcription factor OsDOF11 modulates sugar transport by promoting expression of sucrose transporter and SWEET genes[J]. Mol Plant, 2018, 11(6): 833-845.
[40]	Sun WJ, Gao ZY, Wang J, et al. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate transportation into expanding fibers[J]. New Phytol, 2019, 222(2): 864-881.