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

doi:10.16819/j.1001-7216.2020.9034

Abstract

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

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

CLC Number:

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.

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.

Figures/Tables 8

References 30

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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

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

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

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

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