抗蜜蜂病毒天然产物的初步筛选研究

doi:10.3969/j.issn.1004-1524.20230190

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (1): 67-74.DOI: 10.3969/j.issn.1004-1524.20230190

抗蜜蜂病毒天然产物的初步筛选研究

黄知楚(), 周倪红, 刘瑶, 郑火青()

浙江大学动物科学学院,浙江杭州 310058

收稿日期:2023-02-17 出版日期:2024-01-25 发布日期:2024-02-18
作者简介:黄知楚(1999—),女,江西赣州人,硕士研究生,研究方向为蜜蜂科学。E-mail:zhichu@zju.edu.cn
通讯作者: * 郑火青,E-mail:hqzheng@zju.edu.cn
基金资助:
国家蜂产业技术体系专项(CARS-44);浙江省重大科技专项(2021C02068-8)

Natural products screening with resistance to honeybee viruses

HUANG Zhichu(), ZHOU Nihong, LIU Yao, ZHENG Huoqing()

College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

Received:2023-02-17 Online:2024-01-25 Published:2024-02-18

摘要/Abstract

摘要：

为筛选出具有抗蜜蜂病毒作用效果的天然产物,给感染以色列急性麻痹病毒(IAPV)的蜜蜂分别饲喂含有9种天然产物(百里酚、青蒿素、梓醇、胡椒碱、可可碱、苦杏仁苷、咖啡酸、橙皮苷和ρ-香豆酸)各两种浓度的蔗糖溶液,采用RT-PCR法检测9种天然产物饲喂7 d后对蜜蜂体内病毒的抑制效果,同时测定可能有效的天然产物对蜜蜂免疫相关基因表达的影响。结果表明:外源接种IAPV病毒实验条件下,10 μmol·L^-1百里酚饲喂7 d后IAPV病毒载量比对照组显著降低;未外源接种病毒的条件下,10 μmol·L^-1百里酚饲喂7 d后蜜蜂携带的IAPV和蜜蜂囊状幼虫病毒(SBV)病毒载量比对照组显著降低,卵黄原蛋白基因在转录水平显著提高。经过筛选发现,蜜蜂饲喂10 μmol·L^-1百里酚7 d后能达到较好的抗病毒效果,百里酚可成为抗蜜蜂病毒药物研发的潜力成分。

关键词: 蜜蜂, 天然产物, 病毒, 免疫

Abstract:

In order to identify natural products with anti-honeybee virus effects, 9 natural products (thymol, artemisinin, catalpol, piperine, theobromine, amygdalin, caffeic acid, hesperidin and ρ-coumaric acid) were tested in lab conditions. RT-PCR was used to measure the inhibitory effect of the 9 natural products on honeybee viruses after 7 days. Additionally, the effects of the natural products on the expression of honeybee immunity-related genes were also determined. The results of the exogenous inoculation of Israeli acute paralysis virus (IAPV) virus suggest that feeding honeybees with 10 μmol·L^-1 thymol for 7 days can significantly reduce the viral load of IAPV and Sacbrood virus (SBV). Without external vaccination of the virus, after feeding with 10 μmol · L^-1 thymol for 7 days, the viral load of IAPV and SBV carried by honeybees was significantly reduced compared with the control group, and the transcription level of vitellogenin gene was significantly increased. After screening, it was found that honeybees fed with 10 μmol · L^-1 thymol can achieve good antiviral effects after 7 days, and thymol can become a potential component in the development of anti-honeybee virus drugs.

Key words: honeybee, natural product, viral disease, immunity

中图分类号:

S895

黄知楚, 周倪红, 刘瑶, 郑火青. 抗蜜蜂病毒天然产物的初步筛选研究[J]. 浙江农业学报, 2024, 36(1): 67-74.

HUANG Zhichu, ZHOU Nihong, LIU Yao, ZHENG Huoqing. Natural products screening with resistance to honeybee viruses[J]. Acta Agriculturae Zhejiangensis, 2024, 36(1): 67-74.

图/表 7

参考文献 41

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物质名称 Product	化学物质分类 Classification of chemical substances	参考文献 Reference	试验浓度 Experimental concentration/ (μmol·L^-1)	购买货号 Item number
百里酚Thymol	萜类Terpenes	[7,9-11]	10,100	T6316
青蒿素Artemisinin	萜类Terpenes	[12]	10,100	A800831
梓醇Catalpol	萜类(环烯醚萜苷)Terpenes (Iridoid glycoside)	[7]	500,1 000	C805146
胡椒碱Piperine	生物碱Alkaloid	[13]	10,100	P886278
可可碱Theobromine	生物碱Alkaloid	[14]	50,500	T819117
苦杏仁苷Amygdalin	生氰糖苷Cyanogenic glycoside	[7,15]	10,100	A800517
咖啡酸Caffeic acid	苯丙酸类Phenylpropanoids	[16]	5,50	C804975
橙皮苷Hesperidin	黄酮类Flavonoids	[7]	50,500	H810939
ρ-香豆酸ρ-Coumaric acid	苯丙酸类Phenylpropanoids	[8,16]	50,500	C804995

物质名称 Product	化学物质分类 Classification of chemical substances	参考文献 Reference	试验浓度 Experimental concentration/ (μmol·L^-1)	购买货号 Item number
百里酚Thymol	萜类Terpenes	[7,9-11]	10,100	T6316
青蒿素Artemisinin	萜类Terpenes	[12]	10,100	A800831
梓醇Catalpol	萜类(环烯醚萜苷)Terpenes (Iridoid glycoside)	[7]	500,1 000	C805146
胡椒碱Piperine	生物碱Alkaloid	[13]	10,100	P886278
可可碱Theobromine	生物碱Alkaloid	[14]	50,500	T819117
苦杏仁苷Amygdalin	生氰糖苷Cyanogenic glycoside	[7,15]	10,100	A800517
咖啡酸Caffeic acid	苯丙酸类Phenylpropanoids	[16]	5,50	C804975
橙皮苷Hesperidin	黄酮类Flavonoids	[7]	50,500	H810939
ρ-香豆酸ρ-Coumaric acid	苯丙酸类Phenylpropanoids	[8,16]	50,500	C804995

病毒名称Virus	引物Primer	序列Sequence(5'-3')	参考文献Reference
急性蜜蜂麻痹病毒Acute bee paralysis virus	ABPV-F	GAAACGGCTACCACATCTAAGGA	[18]
	ABPV-R	TGACATAGGGAGGTAGTGACAAGAAA
黑蜂王台病毒Black queen cell virus	BQCV-F	GGAGTCGCAGAGTTCCAAAT	[19]
	BQCV-R	GTGGGAGGTGAAGTGGCTAT
慢性蜜蜂麻痹病毒Chronic bee paralysis virus	CBPV-F	GGCACCTCAAGATCGTCCAAGTTAC	[20]
	CBPV-R	ACGGAGATGGTGACCTGGTATGG
残翅病毒Deformed wing virus	DWV-F	ACCTGGAACATCAGGTAAGCG	[21]
	DWV-R	TTGAATCTCGAGTTCGGGACG
以色列急性麻痹病毒Israeli acute paralysis virus	IAPV-F	TCGCTGAAGGCATGTATTTC	[22]
	IAPV-R	ATTACCACTGCTCCGACACA
囊状幼虫病毒Sacbrood virus	SBV-F	AACGTCCACTACACCGAAATGTC	[23]
	SBV-R	ACACTGCGCGTCTAACATTCC
克什米尔蜜蜂病毒Kashmir bee virus	KBV-F	CCGGACATTTGACTGGATTC	[19]
	KBV-R	TCCTTGGATTCACCAAGAGC

病毒名称Virus	引物Primer	序列Sequence(5'-3')	参考文献Reference
急性蜜蜂麻痹病毒Acute bee paralysis virus	ABPV-F	GAAACGGCTACCACATCTAAGGA	[18]
	ABPV-R	TGACATAGGGAGGTAGTGACAAGAAA
黑蜂王台病毒Black queen cell virus	BQCV-F	GGAGTCGCAGAGTTCCAAAT	[19]
	BQCV-R	GTGGGAGGTGAAGTGGCTAT
慢性蜜蜂麻痹病毒Chronic bee paralysis virus	CBPV-F	GGCACCTCAAGATCGTCCAAGTTAC	[20]
	CBPV-R	ACGGAGATGGTGACCTGGTATGG
残翅病毒Deformed wing virus	DWV-F	ACCTGGAACATCAGGTAAGCG	[21]
	DWV-R	TTGAATCTCGAGTTCGGGACG
以色列急性麻痹病毒Israeli acute paralysis virus	IAPV-F	TCGCTGAAGGCATGTATTTC	[22]
	IAPV-R	ATTACCACTGCTCCGACACA
囊状幼虫病毒Sacbrood virus	SBV-F	AACGTCCACTACACCGAAATGTC	[23]
	SBV-R	ACACTGCGCGTCTAACATTCC
克什米尔蜜蜂病毒Kashmir bee virus	KBV-F	CCGGACATTTGACTGGATTC	[19]
	KBV-R	TCCTTGGATTCACCAAGAGC

基因名称Gene name	引物Primer	序列Sequence(5'-3')	参考文献Reference
β-actin	β-actin-F	TTGTATGCCAACACTGTCCTTT	[24]
	β-actin-R	TGGCGCGATGATCTTAATTT
vitellogenin	Vg-F	TCGACAACTGCGATCAAAGGA	[25]
	Vg-R	TGGTCACCGACGATTGGATG
hymenoptaecin	hymenoptaecin-F	ATATCCCGACTCGTTTCCGA	[26]
	hymenoptaecin-R	TCCCAAACTCGAATCCTGCA
abaecin	abaecin-F	AGATCTGCACACTCGAGGTCTG	[26]
	abaecin-R	TCGGATTGAATGGTCCCTGA
defensin	defensin-F	TGTCGGCCTTCTCTTCATGG	[26]
	defensin-R	TGACCTCCAGCTTTACCCAAA
dicer	dicer-F	TGCAGAATGAATCAAAGAACCGA	[27]
	dicer-R	TGAGCCAATACAAAGCTGGA
protein lethal (2) essential for life-like	pl2-F	ATTTGGATCGTCCACATCGT	[28]
	pl2-R	CGGACAATGGCCGATAGTAG
heat shock 70-ku protein cognate	hsc70-F	CTTGCGTGGGTGTTTTCCAG	[29]
	hsc70-R	TATCATCGAAGCGACGACCG
heat shock protein 90	hsp90-F	CATGGCTAATGCCGGAGAGG	[29]
	hsp90-R	CTGCACCAGCTTGAAGAGC

抗蜜蜂病毒天然产物的初步筛选研究

Natural products screening with resistance to honeybee viruses

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