转录因子NbNAC062及其纳米药物对PVY侵染的抑制作用  

NbNAC062 and Its Nanomedicine Confer Inhibition to Potato Virus Y

作　　者：曲潇玲 宋丽云[1] 张道顺 丁程瀛 沈广材 张友臣 张晓亮 焦裕冰 李莹 杨金广[1] 申莉莉[1] QU Xiaoling;SONG Liyun;ZHANG Daoshun;DING Chengying;SHEN Guangcai;ZHANG Youchen;ZHANG Xiaoliang;JIAO Yubing;LI Ying;YANG Jinguang;SHEN Lili(Tobacco Research Institute of CAAS,Qingdao 266101;Changjing University,Jingzhou,Hubei 434200;Yunnan Baoshan Tobacco Co.,Baoshan,Yunnan 678000;Shiyan Municipal Tobacco Company of Hubei Province,Shiyan,Hubei 442000,China)

机构地区：[1]中国农业科学院烟草研究所,青岛266101 [2]长江大学,湖北荆州434200 [3]云南省烟草公司保山市公司,云南保山678000 [4]湖北省烟草公司十堰市公司,湖北十堰442000

出　　处：《中国烟草科学》2023年第2期35-42,共8页Chinese Tobacco Science

基　　金：山东省自然科学基金(ZR2021MC058);中国烟草总公司重大专项(110202101045-LS-05);烟草病虫害监测与综合治理重点实验室揭榜挂帅项目(KLTPMIMT2022-02);云南省烟草公司保山市公司科技项目(2022530000242003)。

摘　　要：前期研究表明NbNAC062能够抑制PVY早期侵染,本研究通过构建NbNAC062敲除突变体和过表达植株进一步明确NbNAC062的抗病毒功能,并利用异硫氰酸荧光素(fluorescein-5-isothiocyanate,FITC)标记的壳聚糖季铵盐(chitosan quaternary ammonium salt,HACC)包被NbNAC062质粒,制备HACC-NbNAC062纳米药物。激光共聚焦显微镜对纳米药物进行示踪;透射电镜和激光粒子分析仪对其表征进行分析。通过GFP荧光差异、qRT-PCR和蛋白免疫印迹检测病毒含量来探明纳米药物对PVY侵染的影响。结果显示,敲除组病毒GFP荧光增强,而过表达组病毒GFP荧光减弱,PVY CP含量与上述结果一致。纳米药物粒径集中分布在18~32 nm之间;Zeta电位为+41.8 mV。浸润纳米药物HACC-NbNAC062后48 h,在细胞内观察到FITC-HACC(绿色荧光)与RFP-NbNAC062(红色荧光);接种PVY-GFP后5、7、9 d,NbNAC062-HACC施药组的PVY CP mRNA水平较对照组分别下调17.41%、47.81%、13.03%;第7天施药组PVY CP蛋白水平明显低于对照组,病毒荧光强度显著暗于对照组。上述研究结果说明HACC-NbNAC062纳米药物成功递送了NbNAC062,并发挥了其对PVY初期侵染的抑制作用。Previous studies have shown that NbNAC062 inhibits early PVY infection.In this study,NbNAC062 knockout mutants and overexpression plants were used to further clarify the antiviral function of NbNAC062.The NbNAC062 plasmid was coated with fluorescein isothiocyanate(FITC)-labeled chitosan quaternary ammonium salt(HACC)to prepare HACC-NbNAC062 nanomedicine.Laser confocal microscopy was used to trace the nanomedicine and its characteristics were analyzed by transmission electron microscopy and laser particle analyzer.The viral content was determined GFP fluorescence difference,qRT-PCR and Western blot to explore the effects of nanomedicine on PVY infection.The results showed that the viral GFP fluorescence in the knockout group was enhanced,while the viral fluorescence in the overexpression group was weakened.The PVY CP content was consistent with the above results.The diameter of HACC-NbNAC062 nanomedicine was 18~32 nm;the Zeta potential was+41.8 mV.FITC-HACC(green fluorescence)and RFP-NbNAC062(red fluorescence)were observed in the plant cells at 48 h after infiltrated with the HACC-NbNAC062 nanomedicine.Comparing with the control group,the PVY CP mRNA levels in the NbNAC062-HACC group were down-regulated by 17.41%,47.81%,and 13.03%respectively 5,7,and 9 days after PVY-GFP inoculation.The protein level was significantly lower than that of the control group at 7 days after viral inoculation,and the GFP fluorescence intensity was significantly darker than that of the control group.The above research results indicated that the HACC-NbNAC062 nanomedicine successfully delivered NbNAC062 and exerted its inhibitory effect on the initial infection of PVY.

关 键 词：NbNAC062 马铃薯Y病毒 敲除 过表达 HACC-NbNAC062纳米药物 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]