褐飞虱热激蛋白70在不同温度胁迫下的差异表达特性研究

doi:10.16819/j.1001-7216.2017.6171 533

摘要/Abstract

摘要：

目的褐飞虱是我国主要水稻害虫之一,随着全球气候变暖,其越冬范围逐年扩大。为了明确褐飞虱对温度的适应机制,研究了其热激蛋白70基因家族的组成及温度诱导表达特性。方法利用转录组测序和GenBank数据库获取热激蛋白70基因序列,采用MEGA4.1邻接法构建进化树,利用实时荧光定量PCR研究其温度诱导表达特性。结果筛选出与热激蛋白70基因同源性较高且具有完整开放阅读框的序列共15个,其中8个在正常或温度胁迫下表达,可能分布在细胞质/细胞核、内质网、线粒体等器官,且本身表达丰度存在显著差异。30~44℃高温胁迫后KX976471、KX976473、KX976475、KX976476、KX976477和KX976478等6个基因的表达量不同程度上调,最大上调倍数为1.72~245.33;0~22℃低温胁迫后,除KX976475表达量上调2.38倍外,其余基因表达量变化不显著或下降。结论褐飞虱8个热激蛋白70基因在正常或温度胁迫下表达,可能分布于细胞质/核、线粒体、内质网;在其高温适应性中可能起重要作用,但在低温适应性中的作用有限。

关键词: 褐飞虱, 温度, 热激蛋白70, 诱导表达

Abstract:

【Objective】The brown planthopper (BPH), Nilaparvata lugens, is one of the major insect pests on rice in China. In recent years, the overwintering area of BPH expanded with global warming. In order to explore the response mechanisms of BPH to temperature, the hsp70s were cloned and the induced expression profiles of these genes were analyzed. 【Method】 The nucleotide sequences of hsp70s were gotten from transcriptome sequencing and GenBank database, the phylogenetic tree of HSP70 was based on neighbor-joining method by MEGA4.1, and the induced expression profiles were analyzed by real-time quantitative PCR. 【Result】 It was found that fifteen hsp70 homologous genes with complete open reading frame were gotten in BPH, and 8 out of 15 genes were expressed in normal conditions or temperature stress. These eight hsp70s were distributed in the cytoplasm/nucleus, endoplasmic reticulum or mitochondria, and had significantly different expression in normal conditions. After 30-44°C high temperature treatment, the expression levels of KX976471, KX976473, KX976475, KX976476, KX976477 and KX976478 were up-regulated, and the maximum increased times was 1.72-245.33. However, only the expression level of KX976475 increased by 2.38 times, the other seven were not changed or even significantly decreased in expression level after 0-22°C temperature treatment.【Conclusion】It was found that eight hsp70s were expressed in normal conditions or temperature stress in the cytoplasm/nucleus, endoplasmic reticulum or mitochondria. These hsp70s might play an important role in high temperature adaptation, but a limited role in cold adaptation in BPH.

Key words: Nilaparvata lugens, temperature, heat shock protein 70, induced expression

中图分类号:

单丹, 王利华, 张月亮, 韩阳春, 牛洪涛, 潘磊, 方继朝. 褐飞虱热激蛋白70在不同温度胁迫下的差异表达特性研究[J]. 中国水稻科学, 2017, 31(5): 533-541.

Dan SHAN, Lihua WANG, Yueliang ZHANG, Yangchun HAN, Hongtao NIU, Lei PAN, Jichao FANG. Induced Expression Profiles of Hsp70s in Brown Planthoppers, Nilaparvata lugens, Under Different Temperatures[J]. Chinese Journal OF Rice Science, 2017, 31(5): 533-541.

图/表 8

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基因登录号 Accession number	上游引物（5＇-3＇） Forward primer（5＇-3＇）	下游引物（5＇-3＇） Reverse primer（5＇-3＇）
KX976471	CTCAGCGTCAAGCAACCAAAG	AAGGTACCACCACCCAAATCG
KX976472	ATTCTACAGGTGTCAGCCG	CGCCTACTCACTCAAGAA
KX976473	TGAAAGAGCAAAGAGGACAC	TGATTGATAGGTTGAGAGAC
KX976474	TGAGGTGGATGTCAACGGTA	TTCTGCCTCCATAATCATCT
KX976475	GACCAAAGACAACAACCTGC	GGATGGTGATTTTCTCGGAT
KX976476	CACACGAAGACAATCAGCCT	CATTCAAAATACCATCCGCA
KX976477	GTCACTGATGTCCTCCTGCT	CCACCTGCGTCTGACCGTCG
KX976478	CCATTACCATTGCGTCTG	GGAAAGCGAGAAAAAGGA
Ref genes	TGTCTCTCACACAGTCCCCATCT	GTCAAGTCACGACCAGCCAAG

基因登录号 Accession number	上游引物（5＇-3＇） Forward primer（5＇-3＇）	下游引物（5＇-3＇） Reverse primer（5＇-3＇）
KX976471	CTCAGCGTCAAGCAACCAAAG	AAGGTACCACCACCCAAATCG
KX976472	ATTCTACAGGTGTCAGCCG	CGCCTACTCACTCAAGAA
KX976473	TGAAAGAGCAAAGAGGACAC	TGATTGATAGGTTGAGAGAC
KX976474	TGAGGTGGATGTCAACGGTA	TTCTGCCTCCATAATCATCT
KX976475	GACCAAAGACAACAACCTGC	GGATGGTGATTTTCTCGGAT
KX976476	CACACGAAGACAATCAGCCT	CATTCAAAATACCATCCGCA
KX976477	GTCACTGATGTCCTCCTGCT	CCACCTGCGTCTGACCGTCG
KX976478	CCATTACCATTGCGTCTG	GGAAAGCGAGAAAAAGGA
Ref genes	TGTCTCTCACACAGTCCCCATCT	GTCAAGTCACGACCAGCCAAG

GenBank登录号 Accession number	开放阅读框碱基数 Nucleotide base number of ORF	热激蛋白70来源 Organisms of Hsp70s	相似性 Identity/%
GANM01000978.1	1965	Nilaparvata lugens	99
GAYF02039790.1	2028	Laodelphax striatella	99
GAYF02024329.1	2031	Aschersonia aleyrodis	86
GAYF02024558.1	2013	Purpureocillium lilacinum	97
GAYF02029723.1	2007	Conidiobolus coronatus	99
GAYF02024777.1	2001	Hirsutella minnesotensis	87
GANM01002476.1	1875	Bemisa tabaci	81
GAYF02029340.1	1962	Rhopalosiphum padi	100
GAYF02028552.1	1848	Mortierella verticillata	76
GAYF02028886.1	1968	Spizellomyces punctatus	87
GANM01005448.1	1896	N. lugens	99
GAYF02024579.1	1884	Amyelois transitella	70
GANM01021225.1	2070	L. striatella	95
GAYF02031278.1	1932	Lygus Hesperus	84
N1400	1845	Hirsutella minnesotensis	95

GenBank登录号 Accession number	开放阅读框碱基数 Nucleotide base number of ORF	热激蛋白70来源 Organisms of Hsp70s	相似性 Identity/%
GANM01000978.1	1965	Nilaparvata lugens	99
GAYF02039790.1	2028	Laodelphax striatella	99
GAYF02024329.1	2031	Aschersonia aleyrodis	86
GAYF02024558.1	2013	Purpureocillium lilacinum	97
GAYF02029723.1	2007	Conidiobolus coronatus	99
GAYF02024777.1	2001	Hirsutella minnesotensis	87
GANM01002476.1	1875	Bemisa tabaci	81
GAYF02029340.1	1962	Rhopalosiphum padi	100
GAYF02028552.1	1848	Mortierella verticillata	76
GAYF02028886.1	1968	Spizellomyces punctatus	87
GANM01005448.1	1896	N. lugens	99
GAYF02024579.1	1884	Amyelois transitella	70
GANM01021225.1	2070	L. striatella	95
GAYF02031278.1	1932	Lygus Hesperus	84
N1400	1845	Hirsutella minnesotensis	95

GenBank登录号 Accession number	NCBI转录组同源基因 Homologues in transcriptomic submitted to NCBI	相似性 Identity/%	理论等电点 Theoretical pI of encoded protein	分子量 Molecular weight of Encoded protein	ATPase位置 The location of ATPase domain/bp
KX976471	GANM01000978.1	99.5	5.46	71422.69	6-383
KX976472	GAYF02039790.1	98.3	5.45	75283.42	28-405
KX976473	GAYF02031278.1	95.3	5.63	70661.89	7-384
KX976474	-	-	5.39	66801.97	9-386
KX976475	GANM01005448.1	98.8	5.84	69010.35	6-384
KX976476	GAYF02024579.1	99.7	5.23	69705.43	6-384
KX976477	GANM01021225.1	99.5	5.78	74812.09	59-433
KX976478	GAYF02024329.1	99.8	6.15	73462.19	50-423