蕙兰一个PEBP家族基因的克隆及生物信息学分析

doi:10.3969/j.issn.1004-1524.2022.08.12

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (8): 1679-1691.DOI: 10.3969/j.issn.1004-1524.2022.08.12

蕙兰一个PEBP家族基因的克隆及生物信息学分析

楚志刚¹(), 田云芳²^,³^,^*()

1.郑州师范学院信息科学与技术学院,河南郑州 450044
2.郑州师范学院生命科学学院,河南郑州 450044
3.郑州市观赏药用特色资源植物重点实验室,河南郑州 450044

收稿日期:2021-01-25 出版日期:2022-08-25 发布日期:2022-08-26
通讯作者: 田云芳
作者简介:*田云芳,E-mail: tianyunfang2011@163.com
楚志刚(1980—),男,河南荥阳人,硕士,讲师,研究方向为生物信息学。E-mail: chu_zhigang@163.com
基金资助:
河南省科技攻关计划(212102110184);河南省高等学校重点科研项目(22A210026);河南省高等学校青年骨干教师培养计划(2018GGJS162)

Cloning and bioinformatics analysis of a PEBP family gene from Cymbidium faberi

CHU Zhigang¹(), TIAN Yunfang²^,³^,^*()

1. College of Information Science and Technology, Zhengzhou Normal University, Zhengzhou 450044, China
2. College of Life Sciences, Zhengzhou Normal University, Zhengzhou 450044, China
3. Zhengzhou Key Laboratory of Ornamental and Medicinal Resources Plants, Zhengzhou 450044, China

Received:2021-01-25 Online:2022-08-25 Published:2022-08-26
Contact: TIAN Yunfang

摘要/Abstract

摘要：

为了初步探究蕙兰磷脂酰乙醇胺结合蛋白(phosphatidyl ethanolamine-binding proteins, PEBP)基因的特征与功能,以蕙兰花蕾期叶片为材料,利用反转录PCR(RT-PCR)结合cDNA末端快速扩增(RACE)技术克隆基因,使用在线生物信息学工具进行蛋白结构与功能预测,通过R语言和3个在线软件(CodonW、CHIPS和CUSP)分析该基因的密码子偏好性。结果表明,克隆得到的蕙兰PEBP基因包含531 bp长度的开放阅读框(ORF),对应编码氨基酸176个。该基因命名为CfPEBP(登录号MT795710)。生物信息学分析表明,CfPEBP分子式为C₈₈₂H₁₃₆₆N₂₅₄O₂₆₀S₅,分子量为19 848.39 u;等电点为 6.42;具有15个磷酸化位点。CfPEBP蛋白无信号肽结构域,具有PEBP功能域。进化树分析结果表明,CfPEBP蛋白与春兰FT进化距离最近,同源性为100%。分析显示,CfPEBP的密码子偏好性表现较弱;依据同义密码子相对使用度(RSCU)分析较强偏性密码子有GGC、AGA、AGU、AAG、CCA、CUC(RSCU≥2.00)。18个物种PEBP基因RSCU值分析表明,蕙兰HQ164434、文心兰KJ909968、文心兰EU583502、石斛MF063061、香蕉KF853468、牵牛AB154823的密码偏好性较强,均有25个以上RSCU值>1. 00的密码子。PEBP家族基因对于GGC、AGG、AGA、AGC、AGU、UGC、GAG、AAG、UAC、GCC、CCA、CUC的偏好性超过其他密码子。CfPEBP基因在蕙兰盛花期叶中相对表达量最高,花蕾期花蕾和盛花期花葶中表达量次之,其他组织几乎不表达。本研究为蕙兰PEBP家族基因功能的进一步研究提供了基础。

关键词: 蕙兰, PEBP, 生物信息学分析, 密码子偏好性, 表达

Abstract:

In order to explore the characteristics and function of PEBP gene family in Cymbidium faberi, the gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) combined with rapid-amplification of cDNA ends (RACE). R language, CodonW, CHIPS and CUSP software were used to analyze the codon preference, and the protein structure and function of the gene were predicted by using bioinformatics tools. The results showed that the cDNA sequence of PEBP gene contained 531 bp open reading frame (ORF) and encoded 176 amino acids. The gene was named as CfPEBP(Accession No. MT795710). The molecular formula of CfPEBP was C₈₈₂H₁₃₆₆N₂₅₄O₂₆₀S₅ with molecular weight of 19 848.39 u, pI of 6.42, and phosphorylation sites of 15. CfPEBP protein has no signal peptide domain and has PEBP functional domain. Phylogenetic tree analysis showed that CfPEBP protein had the closest evolutionary distance with FT gene of Cymbidium goeringii, and the homology was 100%. Codon preference analysis showed that the codon preference of CfPEBP was not obvious; based on the relative synonymous codon usage (RSCU) value, GGC, AGA, AGU, AAG, CCA, CUC (RSCU≥2) were the most preferred codons. The RSCU value analysis of PEBP gene in 18 species showed that there were more than 25 codons with RSCU value>1 in Cymbidium faberi, Oncidium hybrid(KJ909968), Oncidium hybrid(EU583502), Dendrobium hybrid(MF063061), Musa acuminata(KF853468) and Ipomoea nil(AB154823). PEBP family genes had more preference than other codons for GGC, AGG, AGA, AGC, AGU, UGC, GAG, AAG, UAC, GCC, CCA, CUC. The relative expression level of CfPEBP gene was the highest in the leaves of Cymbidium faberi at flowering stage, followed by flower bud at bud stage and inflorescence rachis at full-blossom stage. This study provided the basis for further study on the function of CfPEBP gene.

Key words: Cymbidium faberi, PEBP, bioinformatics analysis, codon usage bias, expression

中图分类号:

S682.31

楚志刚, 田云芳. 蕙兰一个PEBP家族基因的克隆及生物信息学分析[J]. 浙江农业学报, 2022, 34(8): 1679-1691.

CHU Zhigang, TIAN Yunfang. Cloning and bioinformatics analysis of a PEBP family gene from Cymbidium faberi[J]. Acta Agriculturae Zhejiangensis, 2022, 34(8): 1679-1691.

图/表 12

参考文献 44

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扩增方式 Amplification sort	引物序列 Primer sequences( 5'-3')
Specific amplification	PEBP-F: CCTTCAGCAGTAGTGGAGCAG PEBP-R: GGCGAAGTCACGGGTGTT
3'-RACE	3F1: TCAACACCCGTGACTTCG 3F2: TTTGGCAGCGAAATAGTGT
5'-RACE	5R1: ATCGGTGACCAACCAGTGTAAG 5R2: TGAAAGAGCACGAAGACG
RT-qPCR-PEBP	PEBP-QF: CATACACCGCTTCGTCTT PEBP-QR: ATCCTGCATCCTTCTTCC
RT-qPCR-ACT	ACT-QF: GCCATCCATGATTGGTAT ACT-QR: ATCTGCTGAAAGGTGCTG
RT-qPCR-UBQ3	UBQ3-QF: GGATGGGCGTACTTTAGCA UBQ3-QR: GTTCGGGCACTCCTTTCTG

扩增方式 Amplification sort	引物序列 Primer sequences( 5'-3')
Specific amplification	PEBP-F: CCTTCAGCAGTAGTGGAGCAG PEBP-R: GGCGAAGTCACGGGTGTT
3'-RACE	3F1: TCAACACCCGTGACTTCG 3F2: TTTGGCAGCGAAATAGTGT
5'-RACE	5R1: ATCGGTGACCAACCAGTGTAAG 5R2: TGAAAGAGCACGAAGACG
RT-qPCR-PEBP	PEBP-QF: CATACACCGCTTCGTCTT PEBP-QR: ATCCTGCATCCTTCTTCC
RT-qPCR-ACT	ACT-QF: GCCATCCATGATTGGTAT ACT-QR: ATCTGCTGAAAGGTGCTG
RT-qPCR-UBQ3	UBQ3-QF: GGATGGGCGTACTTTAGCA UBQ3-QR: GTTCGGGCACTCCTTTCTG

密码子 Codon	氨基酸 AA	比例 Fraction	频率 Frequency	数目 Number	密码子 Codon	氨基酸 AA	比例 Fraction	频率 Frequency	数目 Number
GCA	A	0.246	13.354	43	CCA	P	0.407	29.193	94
GCC	A	0.457	24.845	80	CCC	P	0.173	12.422	40
GCG	A	0.131	7.143	23	CCG	P	0.26	18.634	60
GCT	A	0.166	9.006	29	CCT	P	0.16	11.491	37
TGC	C	0.844	11.801	38	CAA	Q	0.242	9.006	29
TGT	C	0.156	2.174	7	CAG	Q	0.758	28.261	91
GAC	D	0.360	21.118	68	AGA	R	0.360	34.783	112
GAT	D	0.640	37.578	121	AGG	R	0.267	25.776	83
GAA	E	0.200	9.317	30	CGA	R	0.042	4.037	13
GAG	E	0.800	37.267	120	CGC	R	0.113	10.87	5
TTC	F	0.719	35.714	115	CGG	R	0.122	11.801	38
TTT	F	0.281	13.975	45	CGT	R	0.096	9.317	30
GGA	G	0.217	18.012	58	AGC	S	0.263	16.149	52
GGC	G	0.517	42.857	138	AGT	S	0.283	17.391	56
GGG	G	0.135	11.180	36	TCA	S	0.116	7.143	3
GGT	G	0.131	10.870	35	TCC	S	0.116	7.143	23
CAC	H	0.656	13.043	42	TCG	S	0.096	5.901	19
CAT	H	0.344	6.832	22	TCT	S	0.126	7.764	25
ATA	I	0.333	9.938	32	ACA	T	0.267	16.77	54
ATC	I	0.344	10.248	33	ACC	T	0.282	17.702	57
ATT	I	0.323	9.627	31	ACG	T	0.163	10.248	3
AAA	K	0.130	0.932	3	ACT	T	0.287	18.012	58
AAG	K	0.870	6.211	20	GTA	V	0.128	13.975	45
CTA	L	0.093	7.453	24	GTC	V	0.322	35.093	113
CTC	L	0.323	25.776	83	GTG	V	0.319	34.783	112
CTG	L	0.144	11.491	37	GTT	V	0.231	25.155	1
CTT	L	0.210	16.77	54	TGG	W	1.000	11.18	36
TTA	L	0.105	8.385	27	TAC	Y	0.729	29.193	94
TTG	L	0.125	9.938	32	TAT	Y	0.271	10.87	35
ATG	M	1.000	20.807	67	TAA	*	0.056	0.311	1
AAC	N	0.473	19.255	62	TAG	*	0.222	1.242	4
AAT	N	0.527	21.429	69	TGA	*	0.722	4.037	13

密码子 Codon	氨基酸 AA	比例 Fraction	频率 Frequency	数目 Number	密码子 Codon	氨基酸 AA	比例 Fraction	频率 Frequency	数目 Number
GCA	A	0.246	13.354	43	CCA	P	0.407	29.193	94
GCC	A	0.457	24.845	80	CCC	P	0.173	12.422	40
GCG	A	0.131	7.143	23	CCG	P	0.26	18.634	60
GCT	A	0.166	9.006	29	CCT	P	0.16	11.491	37
TGC	C	0.844	11.801	38	CAA	Q	0.242	9.006	29
TGT	C	0.156	2.174	7	CAG	Q	0.758	28.261	91
GAC	D	0.360	21.118	68	AGA	R	0.360	34.783	112
GAT	D	0.640	37.578	121	AGG	R	0.267	25.776	83
GAA	E	0.200	9.317	30	CGA	R	0.042	4.037	13
GAG	E	0.800	37.267	120	CGC	R	0.113	10.87	5
TTC	F	0.719	35.714	115	CGG	R	0.122	11.801	38
TTT	F	0.281	13.975	45	CGT	R	0.096	9.317	30
GGA	G	0.217	18.012	58	AGC	S	0.263	16.149	52
GGC	G	0.517	42.857	138	AGT	S	0.283	17.391	56
GGG	G	0.135	11.180	36	TCA	S	0.116	7.143	3
GGT	G	0.131	10.870	35	TCC	S	0.116	7.143	23
CAC	H	0.656	13.043	42	TCG	S	0.096	5.901	19
CAT	H	0.344	6.832	22	TCT	S	0.126	7.764	25
ATA	I	0.333	9.938	32	ACA	T	0.267	16.77	54
ATC	I	0.344	10.248	33	ACC	T	0.282	17.702	57
ATT	I	0.323	9.627	31	ACG	T	0.163	10.248	3
AAA	K	0.130	0.932	3	ACT	T	0.287	18.012	58
AAG	K	0.870	6.211	20	GTA	V	0.128	13.975	45
CTA	L	0.093	7.453	24	GTC	V	0.322	35.093	113
CTC	L	0.323	25.776	83	GTG	V	0.319	34.783	112
CTG	L	0.144	11.491	37	GTT	V	0.231	25.155	1
CTT	L	0.210	16.77	54	TGG	W	1.000	11.18	36
TTA	L	0.105	8.385	27	TAC	Y	0.729	29.193	94
TTG	L	0.125	9.938	32	TAT	Y	0.271	10.87	35
ATG	M	1.000	20.807	67	TAA	*	0.056	0.311	1
AAC	N	0.473	19.255	62	TAG	*	0.222	1.242	4
AAT	N	0.527	21.429	69	TGA	*	0.722	4.037	13

物种(核酸登录号)	U3s	C3s	A3s	G3s	CAI	FOP	ENC	GC3s	GC	RSCU>1	RSCU>1.5
蕙兰Cymbidium faberi PEBP (MT795710)	0.2968	0.3548	0.3053	0.2381	0.161	0.363	57.37	0.497	0.513	22	10
建兰Cymbidium ensifolium FT(HM803115)	0.2968	0.3548	0.2901	0.2540	0.163	0.368	55.83	0.509	0.517	23	10
水稻Oryza sativa RFT1(AB809564)	0.1753	0.5065	0.1069	0.4048	0.223	0.476	41.22	0.759	0.605	24	17
山白竹Sasa veitchii SvFT1(LC020117)	0.1569	0.4837	0.1250	0.4355	0.195	0.476	47.59	0.762	0.610	24	16
蝴蝶兰Phalaenopsis aphrodite FT1(KJ609179)	0.3419	0.2903	0.3939	0.1760	0.138	0.308	48.53	0.390	0.468	22	12
春兰Cymbidium goeringii FT(HM106985)	0.2968	0.3548	0.3053	0.2381	0.161	0.363	57.37	0.497	0.513	22	10
春兰Cymbidium goeringii FT(HM120863)	0.2968	0.3548	0.2977	0.2460	0.161	0.363	57.14	0.503	0.515	23	10
蕙兰Cymbidium faberi FT(HQ164434)	0.2839	0.3613	0.3130	0.2381	0.161	0.363	55.37	0.503	0.517	25	12
文心兰Oncidium hybrid OnFT(KJ909968)	0.3247	0.3052	0.3106	0.2619	0.181	0.404	51.93	0.468	0.492	25	12
墨兰Cymbidium sinense FT (HM120862)	0.2968	0.3548	0.2977	0.2460	0.163	0.368	56.06	0.503	0.515	22	10
文心兰Oncidium hybrid FT(EU583502)	0.3247	0.2857	0.3182	0.2778	0.172	0.392	54.53	0.462	0.491	28	11
石斛Dendrobium hybrid FT (MF063061)	0.3807	0.2841	0.2819	0.2695	0.158	0.350	52.47	0.447	0.481	27	13
香蕉Musa acuminata FT6 (KF853468)	0.2133	0.4200	0.2214	0.3651	0.162	0.400	50.17	0.641	0.560	25	14
野生稻Oryza rufipogon RFT1(KR423010)	0.1765	0.5098	0.1061	0.4016	0.228	0.476	41.23	0.759	0.607	24	16
蝴蝶兰Phalaenopsis hybrid FT (JX162558)	0.3312	0.2922	0.4015	0.1760	0.139	0.316	49.10	0.392	0.473	22	12
毛竹Phyllostachys edulis FTL1(KX714711)	0.0943	0.5094	0.0741	0.5038	0.217	0.517	38.22	0.855	0.670	21	19
绿竹Bambusa oldhamii FTL1 (KX714693)	0.0818	0.5220	0.0741	0.5038	0.217	0.523	37.60	0.866	0.674	21	19
牵牛Ipomoea nil PnFTA (AB154823)	0.3355	0.3484	0.2143	0.2975	0.241	0.497	55.12	0.533	0.538	26	11

蕙兰一个PEBP家族基因的克隆及生物信息学分析

Cloning and bioinformatics analysis of a PEBP family gene from Cymbidium faberi

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