家蚕胸腺素对蚕体抗核型多角体病毒感染能力的影响
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摘要
胸腺素(THY)在动物进化过程中高度保守,并在许多生命活动中发挥重要作用。将家蚕核型多角体病毒(Bm NPV)感染家蚕5龄幼虫后,分别提取蚕体及不同组织的RNA和蛋白质,检测Bm THY基因的转录及蛋白质表达的变化,并探究利用重组胸腺素r Bm THY增强家蚕抗病毒能力的可行性。qRT-PCR检测结果表明,感染Bm NPV后的家蚕5龄幼虫,其血淋巴、脂肪体、丝腺和气管中的Bm THY基因mRNA转录水平不同程度上调,而马氏管、中肠和头部组织中Bm THY基因mRNA转录水平则呈下调趋势;Western blotting检测家蚕5龄幼虫感染Bm NPV后72 h,蚕体内Bm THY蛋白的表达水平下降42.3%。家蚕5龄幼虫接种Bm NPV后,分别添食质量浓度为340μg/m L(高剂量)、34μg/m L(中剂量)和3.4μg/m L(低剂量)的r Bm THY溶液,调查3组幼虫的发病率为43.8%、54.0%和57.6%,而对照组幼虫分发病率为62.2%。研究结果初步证明Bm NPV感染可降低蚕体及部分组织中Bm THY的基因转录与蛋白质表达水平,高剂量r Bm THY添食处理可以在一定程度上提高家蚕抗Bm NPV病毒感染的能力。
Thymosin( THY) is highly conserved during animal evolution and plays an important role in many life activities. After being infected by Bombyx mori nuclopolyhedrovirus( Bm NPV),silkworm larvae of 5 th instar were used to extract RNAs and proteins from various larval body and various tissues for measuring changes of Bm THY gene transcription and protein expression,so as to explore the feasibility of using recombinant silkworm thymosin( r Bm THY) to enhance the antiviral ability of silkworm. The results of qRT-PCR showed that Bm THY transcription in hemolymph,fat body,silk gland and trachea of the 5 th instar larvae infected with Bm NPV was up-regulated in different degrees,while that in Malpighian tubule,midgut and head tissues was down-regulated. Western blotting showed that the expression of Bm THY in larval body decreased by 42. 3% at 72 h after Bm NPV infection. After Bm NPV inoculation,the 5 th instar larvae were administered with r Bm THY solution at concentration of 340 μg/m L( high dose),34 μg/m L( middle dose) and3. 4 μg/m L( low dose). Disease incidence of the above three treatment groups was 43. 8%, 54. 0% and 57. 6%respectively,while that of the control group was 62. 2%.These results showed that Bm NPV infection could reduce the transcription and protein expression of Bm THY in larval body and in certain larval tissues of silkworm,and high dose r Bm THY treatment could improve antiviral ability of silkworm larvae against Bm NPV to certain extent.
Thymosin( THY) is highly conserved during animal evolution and plays an important role in many life activities. After being infected by Bombyx mori nuclopolyhedrovirus( Bm NPV),silkworm larvae of 5 th instar were used to extract RNAs and proteins from various larval body and various tissues for measuring changes of Bm THY gene transcription and protein expression,so as to explore the feasibility of using recombinant silkworm thymosin( r Bm THY) to enhance the antiviral ability of silkworm. The results of qRT-PCR showed that Bm THY transcription in hemolymph,fat body,silk gland and trachea of the 5 th instar larvae infected with Bm NPV was up-regulated in different degrees,while that in Malpighian tubule,midgut and head tissues was down-regulated. Western blotting showed that the expression of Bm THY in larval body decreased by 42. 3% at 72 h after Bm NPV infection. After Bm NPV inoculation,the 5 th instar larvae were administered with r Bm THY solution at concentration of 340 μg/m L( high dose),34 μg/m L( middle dose) and3. 4 μg/m L( low dose). Disease incidence of the above three treatment groups was 43. 8%, 54. 0% and 57. 6%respectively,while that of the control group was 62. 2%.These results showed that Bm NPV infection could reduce the transcription and protein expression of Bm THY in larval body and in certain larval tissues of silkworm,and high dose r Bm THY treatment could improve antiviral ability of silkworm larvae against Bm NPV to certain extent.
引文
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[2]余文英.农村蚕病高发因素分析及防治措施[J].安徽农学通报,2013(16):138;146
[3]段金河,张晓彤.浅谈蚕病发生与综合防治[J].云南农业,2014(12):30-31
[4]赵远.家蚕抗核型多角体病毒病的微卫星分子标记筛选、定位及其病毒侵染家蚕中肠组织的差异蛋白质表达图谱研究[D].镇江:江苏大学,2007
[5]孙波,周洪英,吴宏丽.家蚕核型多角体病毒基因与抗病育种[J].湖北农业科学,2011,50(16):3249-3252
[6]叶明强,杨琼,吴福泉,等.家蚕抗核型多角体病毒病育种研究进展[J].应用昆虫学报,2013,50(5):1447-1452
[7]吕鹏,潘晔,王鹏,等.家蚕抗核型多角体病毒的研究进展[J].科学通报,2015,60(14):1285-1297
[8]吴传华,范涛.家蚕脓病的防治技术[J].安徽农业科学,2007,35(7):2021-2022;2029
[9]刘爱玲.家蚕新品种9805×9806、华康1号农村春季试养初报[J].蚕桑茶叶通讯,2015(1):1-3
[10]李有江,钟国新,姚炳英,等.家蚕新品种“华康2号”农村试养初报[J].蚕桑通报,2015,46(1):32-34;53
[11]GOLDSTEIN A L,SLATER F D,WHITE A.Preparation,assay,and partial purification of a thymic lymphocytopoietic factor(thymosin)[J].Proc Natl Acad Sci USA,1966,56(3):1010-1017
[12]ZHANG W,ZHANG C,LYU Z,et al.Molecular characterization,tissue distribution,subcellular localization and actin-sequestering function of a thymo-sin protein from silkworm[J/OL].PLo S ONE,2012,7(2):e31040[2017-09-15].https://doi.org/10.1371/journal.pone.0031040.DOI:10.1371/journal.pone.0031040
[13]ZHANG C,WANG Y D,FANG Q,et al.Thymosin from Bombyx mori is down-regulated in expression by Bm NPV exhibiting antiviral activity[J].J Insect Sci,2016,16(1):1-8
[14]GOMEZ-MARQUEZ J,ANADON R.The beta-thymosins,small actin-binding peptides widely expressed in the developing and adult cerebellum[J].Cerebellum,2002,1(2):95-102
[15]DUBE K N,BOLLINI S,SMART N,et al.Thymosinβ4 protein therapy for cardiac repair[J].Curr Pharm Des,2012,18(6):799-806
[16]郭丽花.胸腺五肽治疗单纯疱疹病毒性角膜炎复发疗效观察[J].中国误诊学杂志,2011,11(24):5881-5882
[17]SHI X Z,SHI L J,ZHAO Y R,et al.beta-Thymosins participate in antiviral immunity of red swamp crayfish(Procambarus clarkii)[J].Dev Comp Immunol,2015,51(2):213-225
[18]黄志君,邓小娟,陈芳艳,等.家蚕中肠组织在变态发育不同时期的蛋白表达差异分析[J].蚕业科学,2007,33(2):207-215
[19]陈慧芳,王鑫,谢康,等.家蚕V型ATP酶B亚基的克隆及表达特征[J].生物工程学报,2016,32(4):487-496