Identification and bioinformatics analysis of <i>AQP</i> gene family in <i>Cucumis sativus</i>

LAI Mengxia; DU Changxia; FAN Huaifu

doi:10.11833/j.issn.2095-0756.20210361

Volume 39 Issue 2

Mar. 2022

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LAI Mengxia, DU Changxia, FAN Huaifu. Identification and bioinformatics analysis of AQP gene family in Cucumis sativus[J]. Journal of Zhejiang A&F University, 2022, 39(2): 318-328. doi: 10.11833/j.issn.2095-0756.20210361

Citation:

LAI Mengxia, DU Changxia, FAN Huaifu. Identification and bioinformatics analysis of AQP gene family in Cucumis sativus[J]. Journal of Zhejiang A&F University, 2022, 39(2): 318-328. doi: 10.11833/j.issn.2095-0756.20210361

Identification and bioinformatics analysis of AQP gene family in Cucumis sativus

doi: 10.11833/j.issn.2095-0756.20210361

College of Horticulture Science, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2021-05-10
Rev Recd Date: 2021-10-13

Available Online: 2022-03-25

Publish Date: 2022-03-25

Abstract

Objective This study aims to explore the functions of aquaporin (AQP) gene family (CsAQP) in Cucumis sativus. Method The family members were identified by whole genome analysis, and their protein physical and chemical properties, phylogenetic relationships, selection pressure, gene structure, conserved motifs, cis-acting elements and protein interactions were analyzed. Result There were 33 AQP genes in C. sativus genome, which contained 2−5 exons and were unevenly distributed on chromosomes. According to the evolutionary relationship, the AQP gene family was divided into 5 subfamilies. Genome repeat analysis showed that there were 2−3 pairs of gene tandem repeats on chromosome 5 and 6 respectively. The ratios of synonymous substitution (K_s) and nonsynonymous substitution (K_a) of these genes were calculated. The results showed that they were less than 1, indicating that their evolution was affected by purification selection. The analysis of cis-acting regulatory elements showed that most of the elements in the gene promoter region were closely related to hormone regulation, light response, and stress resistance. Conclusion Through the whole genome scanning, 33 AQP family members are obtained from C. sativus genome, which belong to 5 subfamilies and map on 7 chromosomes. The upstream promoter region contains stress-related elements, and some genes participate in tandem replication and undergo purification selection. [Ch, 6 fig. 4 tab. 26 ref.]
- Cucumis sativus,
- aquaporin,
- gene family,
- whole genome identification

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植物常遭受由生物或非生物环境因子所带来的伤害。胁迫因子在抑制植物生长发育的同时，亦触发其系统性防御反应。例如，为适应干旱胁迫，植物在漫长的进化过程中演化出一系列调控水分吸收、关闭气孔降低水分散失量等机制。深入了解植物对外界的防御反应，对提高植物抗性和农业生产具有十分重要的意义。目前，研究者已在不同植物中发现多个响应外界环境胁迫的基因家族，如荔枝Litchi chinensis Dof基因家族^[1]、黄瓜 Cucumis sativus DnaJ基因家族^[2]、水稻Oryza sativa ABC1基因家族^[3]及MIP基因家族等。其中MIP家族的各种蛋白质成员具有独特和特定的运输功能，是近年来的研究热点。水通道蛋白(AQP)家族属于跨膜通道蛋白MIP超家族^[4]。大量研究表明：AQP参与水分子跨膜、调节植物细胞渗透势及响应多种逆境胁迫。AQP已知分类主要为PIPs (plasma membrane intrinsic proteins)、TIPs (tonoplast intrinsic proteins)、NIPs (noduLin 26-like intrinsic proteins)、SIPs (small and basic intrinsic proteins)、XIPs (uncharacterized intrinsic proteins)以及HIPs (hybrid intrinsic proteins)、GIPs (glycerolfacilitato) ^[5]。 POU等^[6]研究发现：拟南芥Arabidopsis thaliana中ATPIP2;7在盐胁迫后，表达丰度降低；烟草Nicotiana tabacum中NtPIP1;1和NtPIP2;1在植株根系的水分运输中起重要作用^[7]; 拟南芥ATPIP2;1在胁迫条件下，其胞吞作用受到不同程度的影响^[8]；烟草NtAQP1参与二氧化碳的渗透^[9]；拟南芥^[10]、玉米Zea mays^[11]等植物的TIP亚家族参与植物跨细胞长距离运输水分的过程。随着越来越多物种的测序的完成，在拟南芥、水稻等模式植物上的AQP基因家族信息已比较明确，在番茄Lycopersicon esculentum^[12]、龙眼Ferocactus viridescens^[13]等植物中也识别出许多AQP基因家族。黄瓜是世界性的重要蔬菜作物，但因其根系分布较浅，因此对水分、盐渍化等胁迫敏感。本研究采用生物信息学方法得到33个CsAQP基因家族的氨基酸序列，在染色体上定位了CsAQP基因家族，对编码的蛋白质理化性质、结构特点等进行分析比较，并建立系统进化树，为CsAQP基因功能研究和响应逆境机制提供理论基础和参考。

1. 材料与方法

1.1. 数据来源

所用各物种AQP基因家族数据及黄瓜基因组数据集均来源于美国国家生物信息中心 (https://www.ncbi.nlm.nih.gov/)，并从Pfam数据库中下载种子文件PF00230，于BIO-Linux系统中使用hmmsearch扫描黄瓜蛋白数据库，得到含MIP蛋白保守结构域的基因^[14]。运用Perl程序对其进一步筛选(e值为1E−20)，利用hmmbuild构建蛋白质特异保守结构域的隐马可夫模型。基于新隐马可夫模型，使用hmmsearch再次扫描黄瓜蛋白数据库，保留结果。同时，以拟南芥AQP基因家族序列及番茄、马铃薯Solanum tuberosum XIPs序列为检索靶标，BLAST搜索黄瓜基因组数据库(e值为1E−10)。两方面匹配所得的序列取并集(同一基因的不同转录本，仅择其一)当作CsAQP候选。所有候选AQP分别被提交至Interpro数据库及美国国家生物信息中心CDD数据库进行验证，剔除冗余和不匹配的氨基酸序列，最终获得CsAQP基因家族成员。

1.2. CsAQP基因家族系统进化分析

通过ClustalW对黄瓜、模式植物拟南芥和水稻的AQP家族成员以及番茄、马铃薯XIPs序列进行比对^[15]，采用MEGA-7的最大似然法(maximum likelihood, ML)，将自展法系数(Booststrap)设置为1 000次，进行重复实验，构建进化树。结合基因注释信息及物种进化结果，参考其他植物水通道蛋白命名法对CsAQP基因家族成员系统命名。

1.3. CsAQP家族成员的定位及基本性质分析

通过筛选到的CsAQP基因的序列信息，利用MG2C软件将其定位于染色体上。采用ExPASy-ProtParam在线预测CsAQP蛋白的分子量、电荷残基数、分子式、理论等电点、不稳定指数及脂肪系数；利用ExPASy-ProtScale在线分析其亲/疏水性；使用Plant-mPLoc 2.0预测亚细胞定位；使用SignalP-5.0预测其信号肽；采用TMHMM-2.0预测分析其跨膜结构。

1.4. CsAQP蛋白的二、三级结构预测

使用SOPMA对其二级结构进行预测分析，并基于SWISS-MODEL进行蛋白质三维结构建模。

1.5. CsAQP家族成员的基因结构及基序分析

通过GSDS 2.0可视化CsAQP基因家族成员的内含子-外显子的分布，并利用MEME 5.3.3在线工具对CsAQP家族蛋白的保守基序进行分析，限值为15。采用TBtools绘制示意图。

1.6. CsAQP的保守序列频率分析

利用DNAMAN对CsAQP蛋白成员进行多序列比对，并使用weblogo在线软件绘制seqlogo图，其特征高度反映相对的变化频率。

1.7. CsAQP基因家族上游的顺式元件预测

从黄瓜基因组中提取CsAQP家族成员起始密码子上游1 500 bp的序列，利用Plant CARE数据库分析启动子顺式作用元件；利用TBtools对其结果可视化。

1.8. CsAQP家族成员的重复及选择压力分析

提取转录本的CDS序列，建立目标序列数据库，多序列比对，构建索引，运行Perl程序，进一步筛选比对结果(筛选标准：序列两两之间的相似性大于75%，且2条序列比对上的长度大于较长序列的75%^[16])，最终获得串联重复基因对。采用ClustalW对各对串联重复基因进行全序列比对，并使用KaKs_Calculator 2.0计算其同义替换和非同义替换间的比率。

1.9. CsAQP蛋白成员的互作预测

使用STRING对黄瓜AQP蛋白成员进行互作预测，选取模式植物拟南芥作为参考，探究CsAQP蛋白之间的作用关系。

3. 讨论

本研究鉴定了黄瓜基因组中的33个CsAQP基因，CsAQP基因的数量与水稻及模式植物拟南芥的数量相似。这表明CsAQP基因在植物中有形成多基因家族的可能。基因进化的主要驱动力来源于基因扩增，而串联复制是基因扩增的方式之一。有研究表明：响应多种逆境胁迫的基因扩增与串联复制密切相关^[17]。这类基因通常是剂量不敏感基因或位于代谢途径两端。方璐等^[18]在白菜Brassica rapa中研究发现：与膜蛋白功能相关以及抗逆相关、新陈代谢相关的基因都发生了串联复制，这些相关基因在诱导防御反应机制中发挥作用。本研究发现：在黄瓜5号和6号染色体中共发现5对串联重复基因，这表明串联复制参与黄瓜CsAQP基因的扩增。

系统进化分析表明：CsAQP基因可以清晰地分为5个亚家族，相对于拟南芥、水稻，多1个新成员(CsXIP1;1)。有研究表明：植物中XIPs序列的分析及其功能方面的鉴定有助于更好地探究AQPs进化过程的分类^[19]。同时，外显子与内含子结构的分析也有益于探究植物基因家族内的进化关系^[20]。从预测结果中可以看出：基因间结构差别明显，但聚类较近的CsAQP基因其结构存在相似。CsNIP亚家族含有的外显子数量相对多于其他4个亚家族，这与JOHANSON等^[21]在拟南芥上的发现一致。基因家族成员在编码氨基酸数量、分子量、理论等电点等生理生化性质也随CsAQP基因编码序列的长短及碱基比例的不同而表现出一定差异。

MEME (multiple EM for motif elicitation)分析发现：基序组成总体保守，例如所有的CsAQP基因都有一个共同的保守基序(KAWDDHWIYWVGPFIGAAJAALYYQFILR)，但不同的亚家族之间也有其独特的保守基序，这为CsAQP基因家族的细分提供一定的依据。分别以限值10、15、20为条件，再次预测CsAQP基因家族的保守基序，在几个重复对之间均能发现某些Motif的得失，与ZHOU等^[22]在ClDof基因家族所得出的结论相似。初步分析这些Motif可能与CsAQP基因的功能分化相关。此外，CsAQP基因家族成员基因启动子的顺式调控元件种类丰富，包括黄瓜抗逆相关的元件以及黄瓜生长发育相关的作用元件，如所有CsAQP基因家族成员基因启动子区都含有多种与光响应相关的元件。因此，这33个基因可能参与黄瓜光反应调控。CsPIP1;2、CsNIP2;2、CsNIP3;1、CsTIP2;1、CsTIP3;1中含有HD-ZIP I元件，可能参与叶肉组织分化。

水通道蛋白在低等植物和高等植物中均有所研究^[23]，其中高等植物水通道蛋白研究相对较多。拟南芥作为模式植物，其AQP在低温、干旱等逆境下的相关研究已被陆续报道。许多植物AQP成员间的互作关联性也得到了验证，如番茄^[24]、水稻和玉米^[25]。与这些植物AQP相比，CsAQP基因功能的研究相对缓慢但却取得了一定进展。已有研究显示：CsAQP在胁迫反应中可能发挥重要作用。陈露倩等^[26]研究发现：CsPIP2;4于干旱胁迫处理下，其表达量在胁迫后2 h达到相对最高值，而随胁迫时间的延长，CsPIP2;4表达丰度显著下降。然而，目前对CsAQP基因的具体作用机制了解尚浅，尤其是不清楚CsAQP基因对非生物胁迫的调控机制。本研究通过对CsAQP基因结构、理化性质、保守基序、亚细胞定位与系统进化树等分析，将有助于鉴定其生物学功能，为进一步探究其功能特性提供了一定的理论依据。此外，对于CsAQP基因在整个生长发育中的调控网络以及与各种环境胁迫因子的关联性也有待探索。

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基因名称	编码蛋白数量/个	分子量/kD	电荷残基数/个		分子式	理论等电点
基因名称	编码蛋白数量/个	分子量/kD	(−)	(+)	分子式	理论等电点
CsNIP1;1	276	29.45	14	20	C₁₃₅₇H₂₁₂₆N₃₄₆O₃₇₃S₆	9.58
CsNIP2;1	288	30.55	15	22	C₁₃₇₇H₂₁₇₀N₃₆₈O₃₉₁S₁₃	9.40
CsNIP2;2	261	27.62	20	17	C₁₂₄₂H₁₉₅₆N₃₂₄O₃₅₉S₁₄	6.05
CsNIP3;1	249	26.01	12	15	C₁₁₈₃H₁₈₇₇N₃₁₁O₃₂₇S₁₀	8.86
CsNIP4;1	269	28.88	16	17	C₁₃₃₁H₂₁₀₀N₃₂₆O₃₅₈S₁₅	7.67
CsNIP5;1	298	30.80	15	18	C₁₃₉₉H₂₂₀₀N₃₆₆O₃₉₄S₁₁	8.63
CsNIP6;1	304	31.52	18	20	C₁₄₂₂H₂₂₆₃N₃₆₉O₄₁₃S₁₂	8.26
CsNIP7;1	268	28.45	8	8	C₁₃₁₂H₂₀₁₈N₃₂₀O₃₆₀S₁₃	6.88
CsPIP1;1	292	31.43	24	25	C₁₄₅₁H₂₂₂₈N₃₇₀O₃₉₅S₈	7.67
CsPIP1;2	292	31.37	23	24	C₁₄₅₁H₂₂₂₅N₃₆₇O₃₉₂S₉	7.68
CsPIP1;3	286	30.76	17	24	C₁₄₂₃H₂₁₈₈N₃₆₄O₃₈₀S₉	9.23
CsPIP1;4	292	31.40	24	25	C₁₄₅₀H₂₂₂₅N₃₆₉O₃₉₅S₈	7.67
CsPIP2;1	284	29.92	17	17	C₁₃₉₀H₂₁₀₇N₃₄₇O₃₇₄S₈	7.04
CsPIP2;2	284	30.24	14	18	C₁₄₀₈H₂₁₃₂N₃₅₄O₃₇₁S₉	8.78
CsPIP2;3	284	29.98	17	18	C₁₃₈₅H₂₁₃₂N₃₄₈O₃₇₇S₉	7.64
CsPIP2;4	283	30.24	19	20	C₁₄₀₀H₂₁₄₃N₃₅₃O₃₇₇S₉	7.63
CsPIP2;5	276	29.37	14	21	C₁₃₅₉H₂₁₀₁N₃₅₁O₃₅₆S₁₀	9.36
CsPIP2;6	279	29.72	13	18	C₁₃₇₂H₂₁₃₁N₃₅₁O₃₆₈S₉	8.97
CsPIP2;7	287	30.52	16	22	C₁₄₁₅H₂₁₅₂N₃₆₂O₃₇₇S₈	9.11
CsPIP2;8	280	29.85	15	21	C₁₃₇₉H₂₁₂₃N₃₅₇O₃₆₈S₈	9.24
CsPIP2;9	191	20.26	9	16	C₉₃₉H₁₄₃₆N₂₄₂O₂₄₅S₇	9.51
CsPIP2;10	290	31.25	15	21	C₁₄₃₉H₂₂₂₆N₃₇₂O₃₈₁S₁₃	9.10
CsSIP1;1	243	25.59	7	12	C₁₂₀₆H₁₈₆₅N₂₈₇O₃₀₇S₉	9.41
CsSIP2;1	238	25.93	11	21	C₁₂₁₃H₁₉₀₁N₃₀₃O₃₀₉S₈	10.00
CsTIP1;1	250	25.72	12	8	C₁₂₀₂H₁₈₃₆N₂₉₂O₃₂₆S₄	5.64
CsTIP1;2	253	26.29	11	8	C₁₂₄₈H₁₉₀₈N₂₉₈O₃₁₉S₃	6.03
CsTIP1;3	254	26.53	14	8	C₁₂₃₈H₁₈₅₆N₂₉₆O₃₃₇S₈	5.30
CsTIP2;1	248	25.44	10	7	C₁₁₉₅H₁₈₁₃N₂₈₁O₃₂₃S₅	5.66
CsTIP2;2	250	25.09	10	7	C₁₁₇₇H₁₈₁₅N₂₇₇O₃₂₀S₄	5.39
CsTIP3;1	289	30.66	18	18	C₁₄₂₅H₂₁₅₅N₃₆₉O₃₇₈S₅	7.17
CsTIP4;1	247	25.72	12	8	C₁₂₀₆H₁₈₅₈N₂₉₆O₃₁₆S₅	6.01
CsTIP5;1	255	26.12	9	9	C₁₁₉₃H₁₈₄₇N₃₀₃O₃₃₁S₁₂	6.88
CsXIP1;1	319	34.47	20	23	C₁₅₈₁H₂₄₈₇N₃₉₃O₄₂₅S₂₁	8.27

基因名称	不稳定指数	脂肪系数	跨膜结构域	亚细胞定位	基因名称	不稳定指数	脂肪系数	跨膜结构域	亚细胞定位
CsNIP1;1	28.94	106.67	6	细胞膜	CsPIP2;6	33.09	111.61	6	细胞膜
CsNIP2;1	32.02	93.44	6	细胞膜	CsPIP2;7	29.96	94.91	6	细胞膜
CsNIP2;2	37.94	97.59	6	细胞膜	CsPIP2;8	33.56	101.11	6	细胞膜
CsNIP3;1	37.36	108.55	6	细胞膜	CsPIP2;9	26.40	93.04	4	细胞膜
CsNIP4;1	32.33	113.64	5	细胞膜	CsPIP2;10	35.24	103.90	6	细胞膜
CsNIP5;1	35.91	97.92	5	细胞膜	CsSIP1;1	32.96	113.74	6	细胞膜
CsNIP6;1	26.82	99.24	5	细胞膜	CsSIP2;1	26.14	113.91	5	细胞膜/液泡
CsNIP7;1	39.31	101.98	6	细胞膜	CsTIP1;1	24.91	110.52	7	液泡
CsPIP1;1	30.99	94.62	6	细胞膜	CsTIP1;2	25.33	120.36	7	液泡
CsPIP1;2	33.49	95.62	6	细胞膜	CsTIP1;3	27.98	99.13	6	液泡
CsPIP1;3	29.78	96.26	6	细胞膜	CsTIP2;1	31.99	111.33	6	液泡
CsPIP1;4	30.85	94.28	5	细胞膜	CsTIP2;2	30.49	118.32	6	液泡
CsPIP2;1	31.99	97.64	6	细胞膜	CsTIP3;1	29.98	102.39	5	液泡
CsPIP2;2	33.61	97.96	6	细胞膜	CsTIP4;1	23.72	123.24	7	液泡
CsPIP2;3	30.51	103.10	7	细胞膜	CsTIP5;1	34.10	103.37	6	细胞膜/液泡
CsPIP2;4	26.21	102.12	6	细胞膜	CsXIP1;1	46.41	108.15	7	细胞膜
CsPIP2;5	29.51	103.51	6	细胞膜

基序	基序序列	基序长度/个
Motif 1	GISGGHJNPAVTFGLFLARKISLVRAILYIIAQCLGAICAC	41
Motif 2	KRNARDSHVPVLAPJPIGFAVFLVHLATGPITGTSMNPARS	41
Motif 3	KDYKDPPPAPLIDPEELTKWSFYRAIIAEFVATLLFLYVTVLTVIGYNRQ	50
Motif 4	KAWDDHWIYWVGPFIGAAJAALYYQFILR	29
Motif 5	LVKAFQKAYYNRYGGGANSLADGYSKGTGLAAEIIGTFVLVYTVFSATDP	50
Motif 6	DGNTCGGVGILGIAWAFGGMIFVLVYCTA	29
Motif 7	RFEEATSPSAJRALLAEFISTFJLVFAGVGS	31
Motif 8	FGTAPGVSALQAFVLEIIITFGLVYVVY	28
Motif 9	MEGKEEDVRLGANKFNERQPI	21
Motif 10	AVKALGSFRSS	11
Motif 11	NQKYNGVVTLIGIAAVAGLIVMVMIYSVG	29
Motif 12	FGAAVIFN	8
Motif 13	KLTSDGAATPAGLVVAAIAHAFALFVAVS	29
Motif 14	TAAQSQDD	8
Motif 15	FTLKGVFHPIM	8

串联重复基因对	K_a	K_s	K_a/K_s	串联重复基因对	K_a	K_s	K_a/K_s
CsPIP1;2&CsPIP1;1	0.040	0.610	0.659	CsPIP2;6&CsPIP2;3	0.135	0.692	0.196
CsPIP1;2&CsPIP1;4	0.360	0.656	0.055	CsPIP2;2&CsPIP2;3	0.073	1.046	0.070
CsPIP1;1&CsPIP1;4	0.005	0.053	0.096