褐飞虱小分子量热激蛋白基因表达特性和功能

doi:10.16819/j.1001-7216.2020.9034

中国水稻科学 ›› 2020, Vol. 34 ›› Issue (1): 37-45.DOI: 10.16819/j.1001-7216.2020.9034

褐飞虱小分子量热激蛋白基因表达特性和功能

潘磊^1,², 王利华², 朱凤³, 韩阳春², 王培², 方继朝^1,^2,^4,^*()

¹南京农业大学植物保护学院, 南京 210095
²江苏省农业科学院植物保护研究所, 南京 210014
³江苏省植物保护植物检疫站, 南京 210036
⁴江苏省区域现代农业与环境保护协同创新中心, 江苏淮安 223300

收稿日期:2019-03-27 修回日期:2019-06-14 出版日期:2020-01-10 发布日期:2020-01-10
通讯作者: 方继朝
作者简介:
作者简介:^#共同第一作者
基金资助:
国家重点研发计划资助项目（2016YFD0300706）;国家自然科学基金资助项目（31572004）;江苏省自然科学基金资助项目（BK20170072）;江苏省重点研发计划资助项目(BE2017366)

Expression Profiles and Functions of Small Heat Shock Proteins in Nilaparvata lugens

Lei PAN^1,², Lihua WANG², Feng ZHU³, Yangchun HAN², Pei WANG², Jichao FANG^1,^2,^4,^*()

¹College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
²Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
³Jiangsu Plant Protection and Plant Quarantine Station, Nanjing 210036, China
⁴Jiangsu Collaborative Innovation Center for Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huai’an 223300, China;

Received:2019-03-27 Revised:2019-06-14 Online:2020-01-10 Published:2020-01-10
Contact: Jichao FANG
About author:
About author:^#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】研究褐飞虱小分子量热激蛋白的表达特性和功能,明确其在褐飞虱温度胁迫适应中的作用。【方法】采用BLAST从转录组数据库中筛选褐飞虱小分子量热激蛋白基因序列;利用Bioedit、Mega等分子生物学软件进行序列分析;利用qPCR技术分析目的基因在不同处理下的表达特性;利用原核表达技术研究其功能。【结果】筛选到6个含有α-晶体结构的小分子量热激蛋白基因NlHsp20.9、NlHsp21.6、NlHsp21.9、NlHsp22.4、NlHsp23.1、NlHsp28.7,其ORF长度依次为561、531、570、570、588和735 bp,理论等电点为5.96、5.77、6.32、5.01、5.74和7.74。NlHsp28.7在3龄若虫中的表达量最高,而NlHsp21.9和NlHsp23.1在雌成虫中的表达量最高。雌虫在低温胁迫后所有小分量热激蛋白基因的表达量均下降,高温胁迫后除NlHsp22.4外的其他5个基因表达量不同程度上调;3龄若虫在低温胁迫后一半sHsps表达量下降,另一半上升,高温胁迫后全部上调。转化褐飞虱sHsps的重组大肠杆菌热激存活率显著上升。【结论】褐飞虱小分子量热激蛋白具有龄期和诱导表达特性及热胁迫保护功能,可能在其高温胁迫应激中具有重要作用,在低温胁迫应激中的作用与虫态有关。

关键词: 褐飞虱, 小分子量热激蛋白, 温度, 表达

Abstract:

【Objective】To explore the adaptation mechanisms of brown planthoppers(BPH) to temperature stress, the expression profiles and functions of small heat shock proteins (sHSPs) in Nilaparvata lugens was studied. 【Method】The nucleotide sequence of small heat shock proteins in N. lugens was screened from transcriptome database by BLAST; The sequence analysis was carried out by molecular biological software such as Bioedit and Mega; the expression profiles were analyzed by qPCR; and the prokaryotic expression technology was used for functional research. 【Result】Six sHsps (NlHsp20.9, NlHsp21.6, NlHsp21.9, NlHsp22.4, NlHsp23.1 and NlHsp28.7) with conserved alpha crystal structure were screened. The ORF length of these genes was 561, 531, 570, 570, 588 and 735 bp, and the theoretical isoelectric point was 5.96, 5.77, 6.32, 5.01, 5.74 and 7.74, respectively. The peak expression of NlHsp28.7 was found in the third larvae and the peak of NlHsp21.9 and NlHsp23.1 in females. The expression of these genes decreased in females after exposure to low temperature, while increased after exposure to high temperature except NlHsp22.4. To the 3rd instar nymphs, the expression of about half of sHsps decreased after low temperature stress and half of sHsps increased, while all increased after high temperature stress. Moreover, the survival of transformed BL21 (DE3) was significantly increased after heat treatment. 【Conclusion】The small heat shock protein of N. lugens has stage-specific and inducible expression characteristics and show protection against heat stress. It may play an important role in response to high temperature stress, but its role in response to low temperature stress is related to insect developmental stages.

Key words: Nilaparvata lugens, small heat shock proteins, temperature, expression

中图分类号:

潘磊, 王利华, 朱凤, 韩阳春, 王培, 方继朝. 褐飞虱小分子量热激蛋白基因表达特性和功能[J]. 中国水稻科学, 2020, 34(1): 37-45.

Lei PAN, Lihua WANG, Feng ZHU, Yangchun HAN, Pei WANG, Jichao FANG. Expression Profiles and Functions of Small Heat Shock Proteins in Nilaparvata lugens[J]. Chinese Journal OF Rice Science, 2020, 34(1): 37-45.

图/表 8

表1 qPCR和原核表达引物

Table 1 Primer sequences for quantitative real-time PCR and recombinant expression in E. coli.

用途 Usage	基因 Gene	上游引物序列 Forward primer (5′-3′)	下游引物序列 Reverse primer(5′-3′)
实时荧光定量PCR qPCR	NlHsp20.9	AGCACGGCTTCATCTCTC	CCTCTCGCCTTTGGTTTC
	NlHsp21.6	GTCCTCCAGTATTCCGTTT	TTGTCCTGTTGTTCTTCGT
	NlHsp21.9	TGTTAGCCGCTCCACTCCATT	CCGTGATTGTCCTTGCGTTCT
	NlHsp22.4	TTTGGCGATTTCAGCGTTAT	CCACTGCCTCCTACATTCTT
	NlHsp23.1	GTTTACTCGTCGCTACACC	CTCCATCTTCTCCTGCTT
	NlHsp28.7	GAAATACAGATAAGCGGCACC	GAATACCGTCCTTGAAGTTGG
	Ref	TGTCTCTCACACAGTCCCCATCT	GTCAAGTCACGACCAGCCAAG
原核表达 Procaryotic expression	NlHsp20.9	ATGTCGCTAGTTCCGCTGCTGT	TTACTGTCCTTCTTTTTCAGCTGGC
	NlHsp21.6	ATGTCGTTGTTTCCGTAC	TTAGGCCTTTATCTTCTCC
	NlHsp21.9	ATGTCTCTGCTACCATATCTTTTTG	TCATGTTTCCATTTTATCCTGG
	NlHsp22.4	ATGGCTGAGAGCGGCGTGAGAC	TTATGTGTGATTGGAATCAGTTTTT
	NlHsp23.1	ATGTCTCTGCTACCGATTA	TTAGGTCTCCATCTTCTCC
	NlHsp28.7	ATGAACTCTTGCCGAAAATTG	TCAATTGATAACAATGCGGC

表2 褐飞虱小分子量热激蛋白序列特征

Table 2 Deduced protein sequences characteristics of NlHsps from N. lugens.

基因 Gene				理论等电点 Theoretical isoelectric point	理论分子量 Theoretical molecular weight/kDa	Pfam编号 Pfam number
NlHsp20.9	561	186	致死蛋白Protein lethal(2) essential for life	5.96	20.9	PF00011
NlHsp21.6	531	176	α-晶体蛋白α-crystallin A chain-like	5.77	21.6	PF00011
NlHsp21.9	570	189	α-晶体蛋白α-crystallin A chain-like	6.32	21.9	PF00011
NlHsp22.4	570	189	α-晶体蛋白α-crystallin A chain-like	5.01	22.4	PF00011
NlHsp23.1	588	195	小分子热激蛋白22.0 Small heat shock protein 22.0	5.74	23.1	PF00011
NlHsp28.7	735	244	30 kDa小分子热激蛋白30 kDa small heat shock protein	7.74	28.7	PF00011

表3 褐飞虱小分子量热激蛋白氨基酸序列间的一致性

Table 3 Identities of six NlHSPs from N. lugens.

蛋白质 Protein	NlHSP20.9	NlHSP21.6	NlHSP21.9	NlHSP22.4	NlHSP23.1	NlHSP28.7
NlHSP20.9	100.0
NlHSP21.6	33.5	100.0
NlHSP21.9	42.4	49.4	100.0
NlHSP22.4	24.1	17.2	19.3	100.0
NlHSP23.1	39.1	50.0	68.2	20.5	100.0
NlHSP28.7	6.9	9.8	9.8	8.2	10.6	100.0

图1 邻接法构建褐飞虱、黑腹果蝇和人类小分子量热激蛋白开放阅读框DNA序列进化树 HSPB开头的基因来自人类,Dm前缀的基因来自于黑腹果蝇,Nl前缀的基因来自褐飞虱。

Fig. 1. Molecular Phylogenetic tree of ORF DNA sequences of sHsps from Nilaparvata lugens, Drosophila melanogaster and Homo sapiens by Neighbor-Joining method. The genes with HSPB starts from Homo sapiens, the genes prefixed with Dm from Drosophila melanogaster, and prefixed with Nl from N. lugens.

图2 褐飞虱小分子量热激蛋白基因在不同龄期间的相对表达量柱上标相同小写字母者表示材料间差异未达0.05显著水平。

Fig. 2. Relative expression level of NlHsps in different stage of N. lugens. The same lowercase letters above the bars indicate no significant difference among the materials at the 0.05 level.

图3 高温热激后褐飞虱雌成虫和3龄若虫小分子量热激蛋白基因的相对表达量 A-雌成虫中的相对表达量; B-3龄若虫中的相对表达量。柱上标相同小写字母者表示处理间差异未达0.05显著水平。

Fig. 3. The diagram showed the relative expression level of sHsps of N. lugens after heat treatment. A, Relative expression level of sHsps of female; B, Relative expression level of sHsps of the 3rd larvae. The same lowercase letters above the bars indicate no significant difference among the treatments at the 0.05 level.

图4 低温处理后褐飞虱雌成虫和3龄若虫小分子量热激蛋白基因的相对表达量 A-雌成虫中的相对表达量; B-3龄若虫中的相对表达量。柱上标相同小写字母者表示处理间差异未达0.05显著水平。

Fig. 4. Relative expression levels of sHsps of N. lugens after cold treatment. A, Relative expression level of sHsps of female after cold shock; B, Relative expression level of sHsps of the 3rd larvae after cold shock. The same lowercase letters above the bars indicate no significant difference among the treatments at the 0.05 level.

图5 重组大肠杆菌高温热激存活率柱上标相同小写字母者表示材料间差异未达0.05显著水平。

Fig. 5. Survival of recombinant BL21(DE3) transformed sHsps after heat treatment. The same lowercase letters above the bars indicate no significant difference among the materials at the 0.05 level.

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褐飞虱小分子量热激蛋白基因表达特性和功能

Expression Profiles and Functions of Small Heat Shock Proteins in Nilaparvata lugens

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