甜瓜PIN基因家族的鉴定及高温胁迫表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1196

摘要/Abstract

摘要：

生长素输出载体蛋白PIN（PIN-formed）是调控植物生长素极性运输的重要载体，在植物生长发育过程中发挥重要作用，但PIN蛋白在甜瓜基因组中的成员、性质、染色体分布、系统进化、启动子，以及家族成员在高温胁迫中的表达特性尚不明确。本研究利用生物信息学方法在甜瓜（Cucumis melo）全基因组数据库中筛选鉴定到18个CmPIN成员，编码氨基酸数量在51-642之间，分子量介于5.19-70.30 kD之间，不稳定指数在24.33-48.87之间；主要定位于细胞质膜上，除CmPIN6和CmPIN8无跨膜结构外，其他16个PIN蛋白均具有2-11个跨膜结构域；分布在9条染色体上，均含有1-9个Motif，CmPIN家族成员基因含有1-10个外显子，二级结构主要为α-螺旋和无规则卷曲结构。构建了CmPIN家族成员与拟南芥（Arabidopsis thaliana）、水稻（Oryza sativa）、番茄（Solanum lycopersicum）、玉米（Zea mays）的PIN蛋白进化树，将18个CmPINs聚类为7个亚族，其中亚族Ⅵ中数量最多。启动子顺式作用元件分析显示，甜瓜CmPIN启动子中存在大量的顺式作用基本元件，还含有与激素、光信号和干旱诱导等相关的顺式作用元件。对不同温度处理的甜瓜胚根进行荧光定量分析，结果表明，高温胁迫下Yucasin+IAA处理的CmPIN13和CmPIN18基因表达量显著高于Yucasin处理；NPA+IAA处理的CmPIN2、CmPIN13和CmPIN18基因表达量显著高于NPA处理，推测CmPIN基因家族参与了甜瓜发芽过程高温胁迫的响应调控。

关键词: 甜瓜, PIN基因家族, 基因特征, 生物信息学, 高温胁迫, 转录表达

Abstract:

Auxin efflux carrier protein PIN（PIN-formed）is an important carrier element for regulating polar auxin transport in plants, thus it plays an important role in plant growth and development. However，it is still unclear about the members, properties, chromosomal distribution, phylogeny, promoters of PIN proteins in the genome of melon and expression characteristics of family members under high temperature stress. In the present study, 18 CmPINs members were screened and identified in the whole genome database of Cucumis melo using bioinformatics methods. The number of amino acids encoded by CmPIN family members was 51-642, the molecular weight was 5.19-70.30 kD and the instability index was 24.33-48.87. The results of subcellular localization prediction showed that CmPINs members were mainly distributed in the plasma membrane. There were the transmembrane structures for rest 16 PIN proteins except no transmembrane structure in CmPIN6 and CmPIN8. CmPINs family members were distributed on 9 chromosomes and contained 1-9 Motif. The genes of 18 CmPINs family members contained 1-10 exons and their secondary structure was mainly α-spiral and irregular curly structure. Based on the PIN protein evolutionary tree of CmPIN family members and Arabidopsis（Arabidopsis thaliana）, rice（Oryza sativa）, tomato（Solanum lycopersicum）, corn（Zea mays）, results showed that 18 CmPINs were clustered into 7 subfamilies, with the largest number in subgroup VI. The analysis of cis-acting elements of the promoter revealed that there were a large number of cis-acting basic elements in the melon CmPINs promoters, as well as cis-acting elements related to hormones, light signals and drought induction. Fluorescence quantitative analysis of melon radicles treated with different temperatures showed that the transcriptional expression of CmPIN13 and CmPIN18 genes with Yucasin+IAA treatment were significantly higher than those with Yucasin treatment under high temperature stress; moreover, the transcriptional expressions of CmPIN2, CmPIN13 and CmPIN18 genes with NPA+IAA treatment were significantly higher than those with NPA treatment, which suggested that CmPINs gene family were involved in the tolerance regulation during melon germination under high temperature stress.

Key words: melon, PIN gene family, gene characteristic, bioinformatics, high temperature stress, transcriptional expression

李敬蕊, 王育博, 解紫薇, 李畅, 吴晓蕾, 宫彬彬, 高洪波. 甜瓜PIN基因家族的鉴定及高温胁迫表达分析[J]. 生物技术通报, 2023, 39(5): 192-204.

LI Jing-rui, WANG Yu-bo, XIE Zi-wei, LI Chang, WU Xiao-lei, GONG Bin-bin, GAO Hong-bo. Identification and Expression Analysis of PIN Gene Family in Melon Under High Temperature Stress[J]. Biotechnology Bulletin, 2023, 39(5): 192-204.

图/表 10

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基因 Gene	序列 Sequence（5'-3'）		退火温度 Annealing temperature Tm/℃	GC含量 GC content/%	产物长度 Product length/bp
Actin3	F	GGCAGTGGTGGTGAACATG	59.04	57.89	149
Actin3	R	TTCTGGTGATGGTGTGAGTC	57.16	50.00	149
CmPIN3	F	CTTCTCCTGTTTCTGATGTGT	51.40	42.90	123
CmPIN3	R	GTTCTGTCTTGGTGTCTCTTC	52.00	47.60	123
CmPIN13	F	CAGGTGATGAAAACTAAACGCCA	59.75	43.48	172
CmPIN13	R	CCGGTGCTCAAAATGTCAGG	59.48	55.00	172
CmPIN18	F	GAGTAGGAGGAACAGAGAAAG	50.10	47.60	155
CmPIN18	R	GACTAAGGACCAAGTGAGACC	52.70	52.40	155
CmPIN2	F	GGGTTTGGTGGTGGGTAAGA	59.52	55.00	172
CmPIN2	R	TTTTGAGTTTGACCGATGCAGG	59.71	45.45	172

基因 Gene	序列 Sequence（5'-3'）		退火温度 Annealing temperature Tm/℃	GC含量 GC content/%	产物长度 Product length/bp
Actin3	F	GGCAGTGGTGGTGAACATG	59.04	57.89	149
Actin3	R	TTCTGGTGATGGTGTGAGTC	57.16	50.00	149
CmPIN3	F	CTTCTCCTGTTTCTGATGTGT	51.40	42.90	123
CmPIN3	R	GTTCTGTCTTGGTGTCTCTTC	52.00	47.60	123
CmPIN13	F	CAGGTGATGAAAACTAAACGCCA	59.75	43.48	172
CmPIN13	R	CCGGTGCTCAAAATGTCAGG	59.48	55.00	172
CmPIN18	F	GAGTAGGAGGAACAGAGAAAG	50.10	47.60	155
CmPIN18	R	GACTAAGGACCAAGTGAGACC	52.70	52.40	155
CmPIN2	F	GGGTTTGGTGGTGGGTAAGA	59.52	55.00	172
CmPIN2	R	TTTTGAGTTTGACCGATGCAGG	59.71	45.45	172

基因 Gene	基因ID Gene ID	氨基酸数量 Number of amino acids	分子量 Molecular weight/kD	理论等电点 Theoretical pI	不稳定指数 Instability index	脂溶指数 Aliphatic index	总平均疏水指数 Grand average of hydropathicity	跨膜结构域数目 Transmembrane domain number	亚细胞定位 Subcellular location
CmPIN1	MELO3C001206.2	135	15.15	6.69	24.33	111.93	0.795	4	液泡膜 Tonoplast membrane
CmPIN2	MELO3C017414.2	623	68.44	9.22	41.57	85.47	-0.012	10	细胞质膜Cytomembrane
CmPIN3	MELO3C017357.2	591	64.52	8.5	43.16	88.76	0.064	10	细胞质膜Cytomembrane
CmPIN4	MELO3C009892.2	417	46.35	9.76	48.87	118.63	0.454	11	细胞质膜Cytomembrane
CmPIN5	MELO3C008726.2	402	43.62	6.23	34.38	113.26	0.501	9	液泡膜Tonoplast membrane
CmPIN6	MELO3C008744.2	51	5.25	6.51	30.81	139.61	1.208	0	细胞质Cytoplasm
CmPIN7	MELO3C031448.2	166	17.73	9.26	40.24	107.65	0.489	2	叶绿体Chloroplast
CmPIN8	MELO3C031243.2	51	5.19	7.98	30.81	141.57	1.275	0	细胞外基质Extracellular matrix
CmPIN9	MELO3C031244.2	109	12.42	7.65	27.69	102.02	0.693	3	液泡膜Tonoplast membrane
CmPIN10	MELO3C013710.2	297	32.35	7.11	45.14	114.81	0.405	6	内质网Endoplasmic reticulum
CmPIN11	MELO3C013711.2	428	46.24	5.02	40.85	117.78	0.576	10	细胞质膜Cytomembrane
CmPIN12	MELO3C017060.2	421	45.46	7.66	36.48	128.5	0.704	10	液泡膜Tonoplast membrane
CmPIN13	MELO3C019102.2	611	65.37	8.75	37.4	96.73	0.265	10	细胞质膜Cytomembrane
CmPIN14	MELO3C025264.2	417	45.67	8.86	34.71	123.72	0.748	10	细胞质膜Cytomembrane
CmPIN15	MELO3C005311.2	456	50.01	5.25	33.04	123.53	0.594	10	细胞质膜Cytomembrane
CmPIN16	MELO3C005326.2	636	69.50	7.15	36.26	91.42	0.111	10	细胞质膜Cytomembrane
CmPIN17	MELO3C018353.2	628	68.55	8.69	36.15	92.77	0.142	10	细胞质膜Cytomembrane
CmPIN18	MELO3C002132.2	642	70.30	9.01	36.31	83.99	-0.029	10	细胞质膜Cytomembrane

基因 Gene	基因ID Gene ID	氨基酸数量 Number of amino acids	分子量 Molecular weight/kD	理论等电点 Theoretical pI	不稳定指数 Instability index	脂溶指数 Aliphatic index	总平均疏水指数 Grand average of hydropathicity	跨膜结构域数目 Transmembrane domain number	亚细胞定位 Subcellular location
CmPIN1	MELO3C001206.2	135	15.15	6.69	24.33	111.93	0.795	4	液泡膜 Tonoplast membrane
CmPIN2	MELO3C017414.2	623	68.44	9.22	41.57	85.47	-0.012	10	细胞质膜Cytomembrane
CmPIN3	MELO3C017357.2	591	64.52	8.5	43.16	88.76	0.064	10	细胞质膜Cytomembrane
CmPIN4	MELO3C009892.2	417	46.35	9.76	48.87	118.63	0.454	11	细胞质膜Cytomembrane
CmPIN5	MELO3C008726.2	402	43.62	6.23	34.38	113.26	0.501	9	液泡膜Tonoplast membrane
CmPIN6	MELO3C008744.2	51	5.25	6.51	30.81	139.61	1.208	0	细胞质Cytoplasm
CmPIN7	MELO3C031448.2	166	17.73	9.26	40.24	107.65	0.489	2	叶绿体Chloroplast
CmPIN8	MELO3C031243.2	51	5.19	7.98	30.81	141.57	1.275	0	细胞外基质Extracellular matrix
CmPIN9	MELO3C031244.2	109	12.42	7.65	27.69	102.02	0.693	3	液泡膜Tonoplast membrane
CmPIN10	MELO3C013710.2	297	32.35	7.11	45.14	114.81	0.405	6	内质网Endoplasmic reticulum
CmPIN11	MELO3C013711.2	428	46.24	5.02	40.85	117.78	0.576	10	细胞质膜Cytomembrane
CmPIN12	MELO3C017060.2	421	45.46	7.66	36.48	128.5	0.704	10	液泡膜Tonoplast membrane
CmPIN13	MELO3C019102.2	611	65.37	8.75	37.4	96.73	0.265	10	细胞质膜Cytomembrane
CmPIN14	MELO3C025264.2	417	45.67	8.86	34.71	123.72	0.748	10	细胞质膜Cytomembrane
CmPIN15	MELO3C005311.2	456	50.01	5.25	33.04	123.53	0.594	10	细胞质膜Cytomembrane
CmPIN16	MELO3C005326.2	636	69.50	7.15	36.26	91.42	0.111	10	细胞质膜Cytomembrane
CmPIN17	MELO3C018353.2	628	68.55	8.69	36.15	92.77	0.142	10	细胞质膜Cytomembrane
CmPIN18	MELO3C002132.2	642	70.30	9.01	36.31	83.99	-0.029	10	细胞质膜Cytomembrane

基因 Gene	α-螺旋 Alpha helix/%	延伸链 Extended strand/%	无规则卷曲 Random coil/%	β-折叠 Beta turn/%
CmPIN1	51.11	22.22	25.19	1.48
CmPIN2	31.14	16.05	48.48	4.33
CmPIN3	30.12	15.91	49.24	4.74
CmPIN4	52.52	15.35	25.66	6.47
CmPIN5	46.02	16.67	31.59	5.72
CmPIN6	50.98	25.49	13.73	9.80
CmPIN7	24.70	27.71	39.76	7.83
CmPIN8	49.02	21.57	19.61	9.80
CmPIN9	46.79	25.69	26.61	0.92
CmPIN10	39.39	15.49	42.76	2.36
CmPIN11	40.42	18.93	36.45	4.21
CmPIN12	38.00	20.19	37.29	4.51
CmPIN13	31.75	14.24	49.75	4.26
CmPIN14	40.29	18.94	36.69	4.08
CmPIN15	41.45	17.11	38.38	3.07
CmPIN16	32.08	15.41	47.80	4.72
CmPIN17	30.89	16.40	47.13	5.57
CmPIN18	29.75	15.42	49.53	5.30