细胞凋亡在麦长管蚜获取和传播大麦黄矮病毒过程中的调控作用

Regulatory role of apoptosis in the acquisition and transmission process of Barley yellow dwarf virus by Sitobion avenae(Hemiptera:Aphididae)

【目的】探明细胞凋亡在麦长管蚜Sitobion avenae传播大麦黄矮病毒(barley yellow dwarf virus,BYDV)GAV株系(BYDV-GAV)过程中的调控作用。【方法】采用qPCR法检测携带和未携带BYDV-GAV麦长管蚜成蚜体内细胞凋亡关键基因胱天蛋白酶家族基因Caspase-1和Caspase-3及凋亡抑制蛋白基因IAP-1和IAP-2的表达量,并采用原位末端标记法(TdT-mediated dUTP nick and labeling,TUNEL)检测上述麦长管蚜成蚜中肠和唾液腺组织细胞凋亡水平;分别在人工饲料中添加细胞凋亡抑制剂Ac-DEVD-CHO和Caspase-1特异性激活剂PAC-1饲喂携带和未携带BYDV-GAV麦长管蚜成蚜后,在获毒和传毒不同时间利用qPCR法检测成蚜体内Caspase-1的表达量和BYDV-GAV CP拷贝数,并利用RT-PCR法检测其传播BYDV-GAV的效率;通过在人工饲料中添加dsRNA沉默麦长管蚜成蚜Caspase-1和IAP-1后,测定不同时间沉默效率和基因沉默对麦长管蚜成蚜存活和繁殖的影响,同时测定麦长管蚜成蚜获毒和传毒时体内BYDV-GAV CP拷贝数和传毒效率。【结果】与未携带BYDV-GAV健康麦长管蚜成蚜相比,携带BYDV-GAV的麦长管蚜成蚜体内Caspase-1表达量显著提高,IAP-1和IAP-2表达量显著降低,且中肠组织中凋亡细胞百分率显著提高;与饲喂溶剂二甲基亚砜(dimethyl sulfoxide,DMSO)的对照相比,饲喂激活剂PAC-1的麦长管蚜成蚜在获毒和传毒过程中体内Caspase-1相对表达量显著升高,BYDV-GAV CP拷贝数均显著降低,且传毒效率也显著降低;与饲喂dsGFP的对照相比,饲喂dsCaspase-1和dsIAP-1的麦长管蚜成蚜体内Caspase-1和IAP-1分别在第3天时沉默效率达到最大值,分别为80.63%和71.91%,并对麦长管蚜成蚜存活和繁殖无显著影响,且饲喂dsCaspase-1的麦长管蚜成蚜获毒时体内BYDV-GAV CP拷贝数显著升高。【结论】麦长管蚜感染BYDV-GAV后,可促进其体内细胞凋亡水平,使其作为先天免疫抑制蚜虫获取和传播BYDV-GAV。本研究的结果为揭示介体-植物病毒的互作机制及通过干预介体细胞凋亡从而阻断植物病毒病流行这一新思路提供了理论依据。

【Aim】To clarify the regulatory role of apoptosis in the transmission of barley yellow dwarf virus(BYDV)GAV strain(BYDV-GAV)by Sitobion avenae.【Methods】The expression levels of the key apoptosis genes,including the caspase family genes Caspase-1 and Caspase-3,and the apoptosis inhibitor genes IAP-1 and IAP-2,were detected by the qPCR method,and the apoptosis levels of midgut and salivary gland tissues were also detected by TdT-mediated dUTP nick and labeling(TUNEL)assay in the BYDV-GAV-uninfected and-infected S.avenae adults.After adding the apoptosis inhibitor Ac-DEVD-CHO or the Caspase-1 specific activator PAC-1 to the artificial diet and feeding the BYDV-GAV-infected and-uninfected S.avencee adults,the expression levels of Caspase-1 and the copy numbers of BYDV-GAV CP in adults were examined at different virus acquisition and transmission time using the qPCR method,and the efficiency of transmitting BYDV-GAV was detected by RT-PCR.By incorporating dsRNA into artificial diets to silence Caspase-1 or IAP-1 in S.avenae adults,the silencing efficiency at different time and the corresponding effects of gene silencing on the survival and reproduction of S.avenae were determined,and the copy numbers of BYDV-GAV CP during the virus acquisition and transmission periods,and the virus transmission efficiency in S.avenae adults were also measured.【Results】Compared with the BYDV-GAV-uninfected healthy S.avenae adults,the BYDV-GAV-infected S.avenae adults showed significantly increased expression level of Caspase-1,and significantly decreased expression levels of IAP-1 and IAP-2.Additionally,the percentage of apoptotic cells in the midgut of the BYDV-GAV-infected S.avenae adults was significantly increased,as compared to that in the BYDV-GAV-uninfected healthy S.avenae adults.Compared with the control fed with dimethyl sulfoxide(DMSO),S.avenae adults fed with the activator PAC-1 exhibited enhanced expression level of Caspase-1 during the acquisition and transmission periods,and significantly decreased copy numbers of BYDV-GAV CP and the virus transmission efficiency.In S.avenae adults fed with dsCaspase-1 and dsIAP-1,the silencing efficiency of Caspase-1 and IAP-1 reached their maximum on the 3rd day(80.63%and 71.91%,respectively),as compared with that in the control fed with dsGFP.The silencing of Caspase-1 and IAP-1 had no significant effect on the survival and reproduction of S.avenae.Moreover,the copy number of BYDV-GAV CP in S.avenae adults fed with dsCaspase-1 significantly increased during the virus acquisition period as compared with that in the control fed with dsGFP.【Conclusion】BYDV-GAV could enhance the apoptosis level of S.avenae,which may serve as an innate immune system of S.avenae to suppress its capacity to acquire and transmit the virus.These findings provide theoretical bases for understanding the interaction mechanism between vectors and plant viruses,and a new intervention strategy to curtail the spread of plant viral diseases by targeting the apoptotic pathways in vectors.