鸦胆子粗提物、臭椿粗提物和Y-D对TMV、CMV-RNA的抑制作用
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摘要
烟草花叶病毒(Tobacco mosaic virus,TMV)在世界范围内都有分布,可侵染十字花科、茄科、菊科等多种植物,是具有经济重要性的植物病毒之一。黄瓜花叶病毒(Cucumber mosaic virus,CMV)的寄主十分广泛,能侵染85科1000多种植物。TMV可以与CMV复合侵染,不仅造成植物品质的下降同时可以造成巨大的经济损失。TMV、CMV引起的病毒病不但危害严重,其防治也相当困难。
鸦胆子(Brucea javanica(L.)Merr)、臭椿(Ailanthus altissima Swingle)均系苦木科(Simaroubaceae)植物果实,为传统中药。在本实验室以前的研究中,已经得出,在接种TMV 6-8h后的普通烟K_(326),1.0 mg/mL的鸦胆子和臭椿粗提物处理的病毒增殖抑制率分别为90.9%和89.4%;分别利用半叶法和叶碟法测定了鸦胆子素D的抗TMV侵染和增殖作用,结果为,在0.025mg/mL浓度下抗TMV侵染效果达到75%以上,而在2×10~(-3)mg/mL的浓度下其抗TMV增殖作用仍达到60%。
Bruticine D(Y-D)、鸦胆子粗提物和臭椿粗提物对TMV有很好的抑制增殖的作用,为了研究Y-D、鸦胆子粗提物和臭椿粗提物对TMV核酸、CMV核酸在寄主体内的增殖情况的影响,本实验采用了semi-quanfitive RT-PCR的方法,对TMV-RNA的复制酶基因、外壳蛋白基因(CP)、运动蛋白基因(MP)、烟草内参基因(actin gene)和CMV-RNA3分别进行了退火温度优化、Mg~(2+)浓度优化、循环次数优化,进一步研究接种TMV 6h的普通烟K_(326)和接种CMV 12h的假酸浆(Nicandra physalodes(L.)Gaertner),用Y-D、鸦胆子粗提物和臭椿粗提物对其处理48h,检测三种药剂对TMV、CMV的RNA的抑制作用。同时采用quantitivereal-time PCR的方法,检测Y-D对TMV-RNA、CMV-RNA的抑制作用,对TMV基因组、TMV-CP亚基因组和TMV-MP亚基因组的抑制率分别为93.04%、94.79%和95.82%。两种方法的结果是相同的,不仅确定了Bruceine D、鸦胆子粗提物、臭椿粗提物对寄主体内的TMV-RNA的增殖有很强的抑制作用。同时证明了semi-quantitative RT-PCR实验结果的正确性,证明了semi-quantitative RT-PCR技术的可靠性,在相对定量的研究中,由于荧光实时定量PCR仪价格昂贵,当条件有限时,如果操作得当,半定量PCR法可以替代荧光定量PCR法,得到应用。但对CMV-RNA的抑制作用从实验结果看是抑制增殖作用不强,但并不能就此断定三种药剂对CMV没有抑制增殖的作用,也就不能做出Bruceine D、鸦胆子粗提物、臭椿粗提物三种药剂对+ssRNA病毒在寄主体内不具有广谱的抑制作用,还可以从蛋白等方面进一步研究药剂的抗病毒作用。
Tobacco mosaic virus is distributed in the world. It can infect many kinds of plants of Cruciferae、Solanaceae、Compositae, is one of economical important plant viruses. Cucumber mosaic virus's host is very widespread, can infect 85 families 1000 kind plants. TMV and CMV can compound infection. They not only cause the plant quality poor but create the huge economic loss. TMV、CMV has many kinds host, the strong resistance and the great harm in the production.
Brucea javanica (L.)Merr and Ailanthus altissima Swingle belongs simaroubaceae, is used in Chinese traditional medicine. In formerly research of our laboratory, we get to know, within 6-8 hours after inoculation, the inhibitory rate of Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract (1mg/mL) was 90.9% and 89.4%. Antiviral activity of Bruticine D against TMV infection and replication was screened by means of local lesion and leaf discs assay, the results demonstrated that the inhibitory TMV infection rate of Y-D (0.025mg/mL) was above 75%, when tested at a concentration of 2×10~(-3) mg/mL, the inhibitory TMV replication rate of Y-D was 60%.
Bruticine D、Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract can inhibit TMV infection. To research the effect of TMV RNA、CMV RNA replication in vivo, we using semi-quantitive RT-PCR, choose the suitable annealing temperature, Mg~(2+)concentration, the number of cycles of TMV replicase gene、coating protein gene(CP)、movement protein gene(MP)、tabacco actin gene and CMV-RNA3 . Further research that the K_(326) within 6h after inoculation TMV and Nicandra physalodes (L.) Gaertner inoculation within 12h after inoculation CMV, with Bruticine D、Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract soaking the inoculated leaves for 48h, detect the three medicine inhibitory TMV-RNA、CMV-RNA replication. We also use quantitive real-time PCR to detect Y-D inhibitory TMV-RNA、CMV-RNA replication, the inhibitory rate to TMV replicase gene, TMV-CP、TMV-MP is 93.04%, 94.79% and 95.82%. The results of the two methods are the same, They can effectively inhibit TMV-RNA replication. We also get to know the result of semi-quantitative RT-PCR is correct, and confirm semi-quantitative RT-PCR Reliability. Because of the quantitive real-time PCR instrument is expensive, when the condition is limited. If operation appropriated, semi-quantitative RT-PCR can displace quantitive real-time PCR. From the experiment result, the medicine almost can not inhibit CMV-RNA replication. But we can not get the conclusion the medicine can not inhibit CMV-RNA replication. And also can not conclude the three medicine can't generally inhibit +ssRNA virus replication. We can futher research inhibit virus protein.
鸦胆子(Brucea javanica(L.)Merr)、臭椿(Ailanthus altissima Swingle)均系苦木科(Simaroubaceae)植物果实,为传统中药。在本实验室以前的研究中,已经得出,在接种TMV 6-8h后的普通烟K_(326),1.0 mg/mL的鸦胆子和臭椿粗提物处理的病毒增殖抑制率分别为90.9%和89.4%;分别利用半叶法和叶碟法测定了鸦胆子素D的抗TMV侵染和增殖作用,结果为,在0.025mg/mL浓度下抗TMV侵染效果达到75%以上,而在2×10~(-3)mg/mL的浓度下其抗TMV增殖作用仍达到60%。
Bruticine D(Y-D)、鸦胆子粗提物和臭椿粗提物对TMV有很好的抑制增殖的作用,为了研究Y-D、鸦胆子粗提物和臭椿粗提物对TMV核酸、CMV核酸在寄主体内的增殖情况的影响,本实验采用了semi-quanfitive RT-PCR的方法,对TMV-RNA的复制酶基因、外壳蛋白基因(CP)、运动蛋白基因(MP)、烟草内参基因(actin gene)和CMV-RNA3分别进行了退火温度优化、Mg~(2+)浓度优化、循环次数优化,进一步研究接种TMV 6h的普通烟K_(326)和接种CMV 12h的假酸浆(Nicandra physalodes(L.)Gaertner),用Y-D、鸦胆子粗提物和臭椿粗提物对其处理48h,检测三种药剂对TMV、CMV的RNA的抑制作用。同时采用quantitivereal-time PCR的方法,检测Y-D对TMV-RNA、CMV-RNA的抑制作用,对TMV基因组、TMV-CP亚基因组和TMV-MP亚基因组的抑制率分别为93.04%、94.79%和95.82%。两种方法的结果是相同的,不仅确定了Bruceine D、鸦胆子粗提物、臭椿粗提物对寄主体内的TMV-RNA的增殖有很强的抑制作用。同时证明了semi-quantitative RT-PCR实验结果的正确性,证明了semi-quantitative RT-PCR技术的可靠性,在相对定量的研究中,由于荧光实时定量PCR仪价格昂贵,当条件有限时,如果操作得当,半定量PCR法可以替代荧光定量PCR法,得到应用。但对CMV-RNA的抑制作用从实验结果看是抑制增殖作用不强,但并不能就此断定三种药剂对CMV没有抑制增殖的作用,也就不能做出Bruceine D、鸦胆子粗提物、臭椿粗提物三种药剂对+ssRNA病毒在寄主体内不具有广谱的抑制作用,还可以从蛋白等方面进一步研究药剂的抗病毒作用。
Tobacco mosaic virus is distributed in the world. It can infect many kinds of plants of Cruciferae、Solanaceae、Compositae, is one of economical important plant viruses. Cucumber mosaic virus's host is very widespread, can infect 85 families 1000 kind plants. TMV and CMV can compound infection. They not only cause the plant quality poor but create the huge economic loss. TMV、CMV has many kinds host, the strong resistance and the great harm in the production.
Brucea javanica (L.)Merr and Ailanthus altissima Swingle belongs simaroubaceae, is used in Chinese traditional medicine. In formerly research of our laboratory, we get to know, within 6-8 hours after inoculation, the inhibitory rate of Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract (1mg/mL) was 90.9% and 89.4%. Antiviral activity of Bruticine D against TMV infection and replication was screened by means of local lesion and leaf discs assay, the results demonstrated that the inhibitory TMV infection rate of Y-D (0.025mg/mL) was above 75%, when tested at a concentration of 2×10~(-3) mg/mL, the inhibitory TMV replication rate of Y-D was 60%.
Bruticine D、Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract can inhibit TMV infection. To research the effect of TMV RNA、CMV RNA replication in vivo, we using semi-quantitive RT-PCR, choose the suitable annealing temperature, Mg~(2+)concentration, the number of cycles of TMV replicase gene、coating protein gene(CP)、movement protein gene(MP)、tabacco actin gene and CMV-RNA3 . Further research that the K_(326) within 6h after inoculation TMV and Nicandra physalodes (L.) Gaertner inoculation within 12h after inoculation CMV, with Bruticine D、Brucea javanica (L.)Merr extract and Ailanthus altissima Swingle extract soaking the inoculated leaves for 48h, detect the three medicine inhibitory TMV-RNA、CMV-RNA replication. We also use quantitive real-time PCR to detect Y-D inhibitory TMV-RNA、CMV-RNA replication, the inhibitory rate to TMV replicase gene, TMV-CP、TMV-MP is 93.04%, 94.79% and 95.82%. The results of the two methods are the same, They can effectively inhibit TMV-RNA replication. We also get to know the result of semi-quantitative RT-PCR is correct, and confirm semi-quantitative RT-PCR Reliability. Because of the quantitive real-time PCR instrument is expensive, when the condition is limited. If operation appropriated, semi-quantitative RT-PCR can displace quantitive real-time PCR. From the experiment result, the medicine almost can not inhibit CMV-RNA replication. But we can not get the conclusion the medicine can not inhibit CMV-RNA replication. And also can not conclude the three medicine can't generally inhibit +ssRNA virus replication. We can futher research inhibit virus protein.
引文
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