苹果HSP90家族基因鉴定及高温胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2020-0895

园艺学报 ›› 2021, Vol. 48 ›› Issue (5): 849-859.doi: 10.16420/j.issn.0513-353x.2020-0895

• 研究论文 • 下一篇

苹果HSP90家族基因鉴定及高温胁迫下的表达分析

姚富文^1,*, 王枚阁^1,*, 宋春晖¹, 宋尚伟¹, 焦健¹, 王苗苗¹, 王昆², 白团辉¹^,^**(), 郑先波¹^,^**()

¹河南农业大学园艺学院,郑州 450002
²中国农业科学院果树研究所,辽宁兴城 125100

收稿日期:2020-12-29 修回日期:2021-04-08 出版日期:2021-05-25 发布日期:2021-06-07
通讯作者: 白团辉,郑先波 E-mail:tuanhuibai88@163.com;xianboz@163.com
作者简介:*共同第一作者
基金资助:
国家重点研发计划项目(2018YFD1000300);国家重点研发计划项目(2019YFD1000100);国家自然科学基金项目(31872058);河南省高等学校青年骨干教师养计划项目(2018GGJS029);河南省大宗水果产业技术体系项目(Z2014-11-03)

Identification and Expression Analysis of HSP90 Gene Family Under High Temperature Stress in Apple

YAO Fuwen^1,*, WANG Meige^1,*, SONG Chunhui¹, SONG Shangwei¹, JIAO Jian¹, WANG Miaomiao¹, WANG Kun², BAI Tuanhui¹^,^**(), ZHENG Xianbo¹^,^**()

¹College of Horticulture,Henan Agricultural University,Zhengzhou 450002,China
²Institute of Pomology,Chinese Academy of Agricultural Sciences,Xingcheng,Liaoning 125100,China

Received:2020-12-29 Revised:2021-04-08 Online:2021-05-25 Published:2021-06-07
Contact: BAI Tuanhui,ZHENG Xianbo E-mail:tuanhuibai88@163.com;xianboz@163.com

摘要/Abstract

摘要：

为了探究热激蛋白(HSP90)基因家族成员在苹果(Malus × domesticaBorkh.)耐热中的作用,利用生物信息学方法对苹果HSP90基因家族成员进行鉴定,并分析其在高温胁迫下和不同组织中的表达规律。结果表明,苹果HSP90基因家族有11个成员,分布于10条染色体上,其氨基酸序列大小介于104 ~ 818 aa,蛋白质分子质量介于11.81 ~ 93.58 kD。转录组分析显示,11个MdHSP90家族成员中,有4个基因,MdHSP90-1、MdHSP90-3、MdHSP90-5和MdHSP90-11在高温胁迫下显著上调;亚细胞定位显示这4个基因中前三者定位在细胞膜与细胞核上,而后者定位在细胞核上;进一步通过qRT-PCR分析发现这4个基因在两种苹果砧木平邑甜茶(Malus hupehensis)和变叶海棠(Malus toringoides)中的表达趋势基本一致,其表达量在高温胁迫早期大幅升高,在中后期有所下降;不同苹果组织中均能检测到这4个基因的表达,高温处理可不同程度地激活这些基因的表达,表明它们可能在苹果耐热胁迫中具有重要作用。

关键词: 苹果, HSP90, 高温, 基因, 结构, 基因表达

Abstract:

In order to explore the role of heat shock protein(HSP90)gene family members in heat tolerance of apple(Malus × domestica Borkh.),bioinformatics method was used to identify the apple HSP90 gene family members,and their physicochemical properties,gene structure,cis elements,phylogenetic relationship and expression characteristics of HSP90 gene family under high temperature stress as well as in different tissues were analyzed. The results showed that a total of 11 HSP90 family members were identified in apple genome,which were distributed on 10 chromosomes. The amino acid sequence size was ranged from 104 to 818 aa,and molecular weight of protein ranged from 11.81 to 93.58 kD. Transcriptome analysis showed that four genes(MdHSP90-1,MdHSP90-3,MdHSP90-5and MdHSP90-11)of the 11 members were significantly up-regulated under high temperature stress. Subcellular localization analysis showed that MdHSP90-1,MdHSP90-3 and MdHSP90-5 proteins were located in the cell cytoplasm and nucleus,whereas MdHSP90-11 was targeted to the nucleus. Subsequent qRT-PCR analysis showed that the expression trend of the four genes in the two kinds of apple rootstocks were similar,the expression level increased greatly in the early stage of high temperature stress,and decreased in the middle and late stage. Four genes were also detected in different apple tissues,and the expressions of fourHSP90 genes were induced by heat stress to varying degrees,which indicated that the above four HSP90 genes may play an important role in the heat tolerance of apple.

Key words: apple, HSP90, high temperature stress, gene, structure, gene expression

中图分类号:

S661.1

姚富文, 王枚阁, 宋春晖, 宋尚伟, 焦健, 王苗苗, 王昆, 白团辉, 郑先波. 苹果HSP90家族基因鉴定及高温胁迫下的表达分析[J]. 园艺学报, 2021, 48(5): 849-859.

YAO Fuwen, WANG Meige, SONG Chunhui, SONG Shangwei, JIAO Jian, WANG Miaomiao, WANG Kun, BAI Tuanhui, ZHENG Xianbo. Identification and Expression Analysis of HSP90 Gene Family Under High Temperature Stress in Apple[J]. Acta Horticulturae Sinica, 2021, 48(5): 849-859.

图/表 8

表1 qRT-PCR和亚细胞定位引物序列

Table 1 Primer sequences of qRT-PCR and subcellular location

用途Useage	基因 Gene	引物(5′-3′)Primer
实时荧光定量qRT-PCR	MdHSP90-1	F:CGTCGATTCGGATGATTTGC;R:TGGCTCTGTTCTGGCTGTCC
	MdHSP90-3	F:GGAAATCAGCGACGACGAAG;R:CAAGGTGCTCCTCCCAGTCA
	MdHSP90-5	F:ATGTTGCTTCTGACAGCGTGAC;R:CCATATTCCTGCTTAATCTCCTTTT
	MdHSP90-11	F:CGTATTGCTCCGTTGCTTAG;R:AGTTTCTCCAGGAATGGTGTATT
	Actin	F:GGATTTGCTGGTGATGATGCT;R:AGTTGCTCACTATGCCGTGC
亚细胞定位 Subcellular location	GFPHSP90-1	F:GTGGATCCAAAGAATTCATGGCCGACGTTCAGATGCAC
		R:CTCCTTTACCCATGAATTCTCAAAATTATATTAACATTATCGAG
	GFPHSP90-3	F:GTGGATCCAAAGAATTCCCGATGTCAGCATGATTGG
		R:CTCCTTTACCCATGAATTCAAAAACCCGAAACATCCACAAACTC
	GFPHSP90-9	F:GTGGATCCAAAGAATTCATGCACAGGCTCCCAC
		R:CTCCTTTACCCATGAATTCTTTCTGGCTGCC
	GFPHSP90-11	F:GTGGATCCAAAGAATTCATGCACAGGCTCCCACGACGCTCCG
		R:CTCCTTTACCCATGAATTCTTTCTGACTGCCG

表2 苹果HSP90基因家族成员的理化性质

Table 2 Physical and chemical properties of HSP90 gene family in apple

基因名称 Gene name	序列ID Sequence ID	染色体 Chr	基因组位置 Genomic location	开放阅读框/bp ORF	氨基酸/aa Amino acid	蛋白质分子质量/kD mw	等电点 pI
MdHSP90-1	MD01G1208700	1	30 315 406 ~ 30 318 680	2 112	703	80.77	5.00
MdHSP90-2	MD03G1036600	3	2 907 372 ~ 2 910 570	2 100	699	80.21	4.95
MdHSP90-3	MD07G1279200	7	34 367 607 ~ 34 369 506	1 749	582	67.39	5.06
MdHSP90-4	MD08G1011200	8	864 457 ~ 869 106	2 547	818	93.59	4.85
MdHSP90-5	MD09G1122200	9	9 421 880 ~ 9 427 733	2 430	809	91.89	5.29
MdHSP90-6	MD11G1037400	11	3 204 284 ~ 3 207 461	2 100	699	80.04	4.95
MdHSP90-7	MD11G1152300	11	14 614 376 ~ 14 614 690	315	104	11.81	4.16
MdHSP90-8	MD13G1055300	13	3 867 643 ~ 3 871 210	1 143	380	43.56	4.89
MdHSP90-9	MD14G1110600	14	17 566 378 ~ 17 567 467	930	309	36.35	9.40
MdHSP90-10	MD16G1054100	16	3 825 148 ~ 3 830 828	2 385	794	89.67	5.02
MdHSP90-11	MD17G1113200	17	9 690 370 ~ 9 696 630	2 421	806	91.69	5.34

图1 苹果HSP90基因家族成员在染色体的定位和共线性分析

Fig. 1 Chromosome location and collinearity analysis of apple HSP90 gene family

图2 苹果HSP90基因启动子的顺式作用元件分析

Fig. 2 cis-Element analysis of apple HSP90 gene promoters

图 3 苹果HSP90基因在高温40 ℃胁迫4 h转录组热图分析

Fig. 3 The heat map of apple HSP90 gene family under 4 h high temperature stress

图4 苹果HSP90基因在高温胁迫下的表达分析 PCK:平邑甜茶对照;PH:平邑甜茶高温;BCK:变叶海棠对照;BH:变叶海棠高温。不同小写字母表示同一种砧木在不同胁迫时间表达量存在显著差异(P < 0.05)。

Fig. 4 Expression analysis of HSP90 genes in apple under high temperature stress PCK:Malus hupehensis(control);PH:Malus hupehensis(high temperature);BCK:Malus toringoides(Control);BH:Malus toringoides(high temperature). Different lowercase letters in each bar indicate significant differences at different stress times in the same rootstock(P < 0.05).

图 5 苹果HSP90基因的组织表达分析不同小写字母表示同一种砧木不同组织之间表达量存在显著差异(P < 0.05)。

Fig. 5 Tissue expression analysis of HSP90 genes in apple In the same apple rootstock,different lowercase letters indicated significant differences in the expression levels among different tissues(P < 0.05).

图 6 MdHSP90-1、MdHSP90-3、MdHSP90-5和MdHSP90-11蛋白在烟草细胞中的亚细胞定位

Fig. 6 Subcellular localization of MdHSP90-1、MdHSP90-3、MdHSP90-5 and MdHSP90-11proteins in tobacco

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苹果HSP90家族基因鉴定及高温胁迫下的表达分析

Identification and Expression Analysis of HSP90 Gene Family Under High Temperature Stress in Apple

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苹果HSP90家族基因鉴定及高温胁迫下的表达分析

Identification and Expression Analysis of HSP90 Gene Family Under High Temperature Stress in Apple

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