近零磁场下干扰磁响应关键基因对褐飞虱寿命的影响

doi:10.3864/j.issn.0578-1752.2019.01.005

摘要/Abstract

摘要：

【目的】隐花色素（cryptochrome, Cry）和铁硫簇蛋白IscA（iron-sulfur cluster assembly,即MagR）是生物体内潜在的磁受体蛋白,本研究通过RNA干扰（RNAi）技术,分别敲减褐飞虱（Nilaparvata lugens）体内的磁响应关键基因NlCry1、NlCry2和NlMagR,旨在探明近零磁场（near-zero magnetic field,NZMF）环境下,以上3种基因在褐飞虱寿命调节过程中的作用,从而间接探讨这3种基因对磁场的响应情况。【方法】采用RNAi技术,以实验室正常磁场环境下稳定饲养的短翅初羽化褐飞虱雌雄成虫为材料,通过向其体内注射双链RNA（dsRNA）分别抑制磁响应关键基因NlCry1、NlCry2和NlMagR,随后立即分别放入正常磁场（geomagnetic field,GMF）和近零磁场中,于每日相同时间观察记录试虫寿命。同时于注射后的1、2和3 d通过RNAiso Plus法提取GMF中褐飞虱雌成虫总RNA,反转录合成第一链DNA,后采用实时荧光定量PCR（RT-qPCR）技术检测该基因的表达情况,以确定基因干扰效率。【结果】注射dsNlCry1后,褐飞虱雌雄成虫寿命在近零磁场和正常磁场间均无显著差异。注射dsNlCry2后,近零磁场中褐飞虱雌雄成虫寿命比正常磁场分别显著延长27.78%和50.04%;此外,与注射dsGFP处理相比,正常磁场下注射dsNlCry2的雌成虫寿命缩短,而近零磁场下注射dsNlCry2的雌成虫寿命延长,但二者差异均不显著;近零磁场和正常磁场下注射dsNlCry2的雄成虫寿命均缩短（25.41%和10.73%）,且正常磁场下差异显著。近零磁场中,注射dsNlMagR的雌成虫寿命较注射dsGFP的寿命显著缩短了16.48%,而雄成虫寿命在磁场间、干扰处理间的差异均不显著。【结论】磁场变化下褐飞虱雌雄成虫体内3种磁响应关键基因对其寿命的调节功能存在差异。其中,NlCry2对磁场变化存在敏感响应,表现为敲减该基因与磁场变化的互作显著地影响雌雄成虫寿命,且表现出“性二型性”;NlMagR也可对磁场变化产生明显响应,但该响应只存在于雌成虫;此外,NlCry1对磁场变化无响应,该基因或与褐飞虱雌雄成虫寿命调节无关。

关键词: 磁场强度变化, 褐飞虱, 磁响应基因, RNA干扰, 成虫寿命, 磁生物学效应

Abstract:

【Objective】Cryptochrome (Cry) and iron-sulfur cluster protein IscA (iron-sulfur cluster assembly, MagR) are potential magnetic receptor proteins in organisms. In this study, key magnetic response genes of the brown planthopper (Nilaparvata lugens) were knocked-down by RNA interference (RNAi), including NlCry1, NlCry2 and NlMagR. The objective of this study is to investigate the role of these three magnetic response genes in the longevity mediation of N. lugens in near-zero magnetic field (NZMF). Thus, the response of these three genes to magnetic field could be studied indirectly.【Method】Newly emerged brachypterous female and male adults of N. lugens fed in the lab magnetic field were chosen as the experimental material, and RNAi technology was used to inhibit the key magnetic response genes’ (NlCry1, NlCry2 and NlMagR) expression by injection of double stranded RNA, respectively. Then the RNAi treated adults were immediately transformed into the geomagnetic field (GMF) and NZMF respectively to observe their longevity. The total RNA of the RNAi treated adults under GMF was extracted by using the RNAiso Plus method on the 1st, 2nd and 3rd day after the microinjection, respectively. And then the gene expressions of NlCry1, NlCry2 and NlMagR were measured by using the RT-qPCR (real-time quantitative polymerase chain reaction) after the reverse transcription synthesis of first strand DNA in order to test the efficiency of RNAi. 【Result】There was no significant difference in the longevity of female and male adults after the injection of dsNlCry1 between the treatments of NZMF and GMF, while after the injection of dsNlCry2, the longevity of female (27.78%) and male (50.04%) adults under NZMF was significantly longer than that of the individuals under GMF, respectively. Moreover, the longevity of female adults injected with dsNlCry2 was shorter under GMF while longer under NZMF than that of individuals injected with dsGFP, even if the difference was not significant. The longevity of male adults injected with dsNlCry2 was shorter than that of individuals injected with dsGFP under NZMF (25.41%) and GMF (10.73%), respectively, and the difference under GMF reached the significant level. Furthermore, the longevity of female adults injected with dsNlMagR was significantly shorter (16.48%) than that of individuals injected with dsGFP under the NZMF. 【Conclusion】There is a difference in the regulation of the key genes of magnetic susceptibility (NlCry1, NlCry2 and NlMagR) on the female and male longevity for N. lugens under the change of magnetific field. Hereinto, the NlCry2 susceptibly responses to the changes of magnetic fields, which shows that the gene knock-down and its interaction with magnetic field changes can significantly influence the longevity of female and male adults, and characterized as “sexual dimorphism”. Similarly, the NlMagR (IscA) also sensitively responds to magnetic field changes, but just for the female adults of N. lugens under the NZMF in contrast to the GMF. However, there is no response of NlCry1 to magnetic field changes, and this gene may not be involved in the regulation of female and male longevity for N. lugens.

Key words: magnetic field changes, brown planthopper (Nilaparvata lugens), magnetic response gene, RNA interference (RNAi), adult longevity, magnetic bio-effect

贺静澜,张明,刘瑞莹,万贵钧,潘卫东,陈法军. 近零磁场下干扰磁响应关键基因对褐飞虱寿命的影响[J]. 中国农业科学, 2019, 52(1): 45-55.

HE JingLan,ZHANG Ming,LIU RuiYing,WAN GuiJun,PAN WeiDong,CHEN FaJun. Effects of the Interference of Key Magnetic Response Genes on the Longevity of Brown Planthopper (Nilaparvata lugens) Under Near-Zero Magnetic Field[J]. Scientia Agricultura Sinica, 2019, 52(1): 45-55.

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引物用途 Use of primers	引物名称 Primer name	引物序列 Primer sequence (5′-3′)
cDNA克隆 cDNA cloning	dsNlCry1-F	TAATACGACTCACTATAGGGTCAGACATGGGCTTCGATT
	dsNlCry1-R	TAATACGACTCACTATAGGGCATTTGGTAAGTTAGCGGTGGA
	dsNlCry2-F	TAATACGACTCACTATAGGGTGTCAGCATCAATAAGTGGAGG
	dsNlCry2-R	TAATACGACTCACTATAGGGGCACACCAAACTTGTCGTC
	dsNlMagR-F	TAATACGACTCACTATAGGGAGAAAGGAAAGTTTGACGAAG
	dsNlMagR-R	TAATACGACTCACTATAGGGAGCCCTAAATATTAACATCGT
	dsGFP-F	TAATACGACTCACTATAGGGACGTAAACGGCCACAAGTTC
	dsGFP-R	TAATACGACTCACTATAGGGTGTTCTGCTGGTAGTGGTCG
实时荧光定量PCR RT-qPCR	qNlCry1-F	CAGACATGGGCTTCGATTTCA
	qNlCry1-R	ACCAGCACTTTCTCCGTCAAAT
	qNlCry2-F	CGCATACTCTCTACAGACTTGAT
	qNlCry2-R	CACCGTCTGGAATTTGCGATAC
	qNlMagR-F	CGTTTAATACCTTCAAGAGCAGCAC
	qNlMagR-R	CCCTACTTTCAAGCCGATAGCAT
	qActin-F	CTTCTAAACGCCAACCACTCC
	qActin-R	TCACCCGAAATCACTCACGA
	q18S-F	TGTCTGCTTAATTGCGATAACGAAC
	q18S-R	CCTCAAACTTCCATCGGCTTG

影响因子 Impact factor	雌虫寿命 Female longevity		雄虫寿命 Male longevity
影响因子 Impact factor	F值 F value	P值 P value	F值 F value	P值 P value
RNAi	2.87	0.092	0.90	0.34
磁场 Magnetic?field (MF)	0.34	0.56	0.60	0.44
RNAi×磁场 RNAi×MF	0.54	0.47	1.88	0.17

影响因子 Impact factor	雌虫寿命 Female longevity		雄虫寿命 Male longevity
影响因子 Impact factor	F值 F value	P值 P value	F值 F value	P值 P value
RNAi	2.33	0.13	1.64	0.20
磁场 Magnetic?field (MF)	0.70	0.40	4.14	0.044*
RNAi×磁场 RNAi×MF	4.45	0.037*	7.08	0.009**

影响因子 Impact factor	雌虫寿命 Female longevity		雄虫寿命 Male longevity
影响因子 Impact factor	F值 F value	P值 P value	F值 F value	P值 P value
RNAi	3.97	0.048*	0.11	0.74
磁场 Magnetic?field (MF)	1.33	0.25	0.08	0.78
RNAi×磁场 RNAi×MF	0.68	0.41	2.59	0.11