利用木霉和RNAi技术提高烟草抗性和品质的研究
摘要
病毒病是全世界烟草栽培区发生普遍,危害严重的一大类病害。目前分布广、危害严重的有烟草普通花叶病毒(TMV)、马铃薯Y病毒(PVY)和烟草黄瓜花叶病毒(CMV)等。防治病毒病的措施主要有几个方面,其中培育抗病品种和施用抗病药剂最为直接有效。本文从这两个方面入手开展研究工作:一是研究木霉这种生防真菌对烟草抗病毒的作用及施用方法;二是通过RNAi技术获得抗病毒的RNAi烟。通过研究发现:
木霉孢子和康宁霉素分别与烟草花叶病毒混合后,能够引起病毒颗粒断裂、凝聚,极显著降低了病毒的侵染能力,钝化防效分别为85.81%和56.11%。木霉孢子与TMV混合后接种NC89,病毒RNA含量明显低于对照,仅为对照的2.05%,病情指数较对照极显著降低,说明木霉孢子能够体外钝化TMV,并且能够抑制病毒RNA在烟株内的积累。烟草NC89接种病毒6小时后,分别施用木霉孢子悬浮液和康宁霉素,48小时后研磨汁液接种枯斑三生烟苗,治疗防效分别为97.98%和70.26%,病情指数显著低于对照处理,病毒含量也明显降低,可见木霉及其产物对病毒病具有治疗效果,能够抑制病毒在植物体内的复制和积累,对于防治病毒的二次扩展十分重要。木霉孢子和康宁霉素均可诱导烟草产生抗性,抗性基因得到激活,防御酶活性增强,特别是在接种病毒后,防御酶活性进一步增强,这有利于对抗病毒,抑制病毒RNA复制,降低危害,MDA含量减少,最大叶长、宽极显著提高,病情指数明显降低,诱导防效分别为55.46%和57.68%。康宁霉素作为木霉产peptaibols,具有木霉孢子的部分功能,且诱导浓度较低。木霉孢子的盆栽和大田实验表明,浇灌木霉孢子后施用木霉专用肥处理烟叶外观质量好、化学成分相对协调、评吸质量优于其他处理,加之该处理烟叶病害显著降低、产量高、产值高,具有较高的推广应用价值。
从GenBank下载TMV、CMV的不同株系的移动蛋白氨基酸序列,PVY polyprotein酶氨基酸序列,运用DNAMAN进行对比分析,挑选高度重合序列,确定其编码碱基序列,经人工合成构建含有酶切位点的联合基因片段CTP,经过酶切得CTP、CT、CP、TP、C、T、P等7个片段。从GenBank下载TMV、CMV的不同株系的复制酶氨基酸序列,经对比分析确定其编码碱基序列后人工合成构建含有酶切位点的联合基因片段CrTr,经过酶切得CrTr、Cr、Tr 3个片段。将得到的10个片段分别插入RNAi-pUC中,再用双酶切反向连接至干涉载体,用PstⅠ酶切载体,回收片段,脱磷后,插入表达载体pUCC2300-35-ocs中,转化感受态农杆菌,获得富集质粒的农杆菌,经酶切验证,获得含hpDNA的农杆菌。用含有hpDNA的农杆菌感染事先准备好的无菌烟草叶片碎片中,MS培养基培养,待长出根系后,即初步得到RNAi烟苗,通过接毒实验、RT-PCR等手段检测其抗病性,得到抗病毒烟苗11株;通过Southern blot挑选单拷贝、高抗性RNAi烟苗2株进行单倍体育种,快速得到的双倍纯合转基因RNAi烟苗,烟苗抗病毒特性得到保留--能够同时抵抗三种病毒入侵,且化学成分与普通烟草基本一致。
Virus disease is a large class of diseases that widespread and severe damaged in the tobacco cultivation throughout the world. Tobacco mosaic virus (TMV), potato Y virus (PVY) and tobacco cucumber mosaic virus (CMV) are the most widespread and serious virus. There are several kinds of measures to control virus disease; cultivation resistant varieties and application of antiviral pharmaceutical are most direct and effective method. This article from these two-pronged approaches to carry out research work: one is study Trichoderma a biocontrol fungus anti-viral effect and application methods in tobacco; another is achievement tobacco seedlings by RNAi technology, which can resist three kinds of viruses at the same time. Through the study found that:
Trichoderma spores and Trichokonin mixed with tobacco mosaic virus respectively, can cause the virus particle fracture, cohesion, significantly reduced the ability of the virus infection, prevention of passivation are 85.81% and 56.11% respectively. Trichoderma spores mixed with TMV, then inoculate NC89, RNA of viral were significantly lower than the control, only 2.05%, disease index was significantly lower than the control. It is indicate that Trichoderma spores can passive TMV in vitro, and inhibit viral RNA accumulation in tobacco strain. Tobacco NC89 inoculation TMV first, 6 hours late, application of Trichoderma spore suspension and Trichokonin respectively, pestle into juice 48 hours later, then inoculated Samsun-NN with friction means, prevention effects were 97.98% and 70.26%, disease index was significantly lower than the control treatment, Virus levels also decreased, showing that Trichoderma and its product have therapeutic effect on viral disease, can inhibit the replication of the virus in plants and accumulation of the virus, that very important in prevention of virus secondary expansion. Trichoderma and Trichokonin can induce resistance of tobacco, genes of resistance are activated, defensive activity increased, especially in the tobacco inoculated virus, the defensive activity further enhanced, which will help to combat viruses, inhibit replication of viral RNA, reduce harm to tobacco, content of MDA was decreased. The maximum leaf length and width increased significantly, the disease index decreased significantly, induced control effects were 55.46% and 57.68% respectively. Trichokonin as Trichoderma product peptaibols, has some function of Trichoderma spores, can passive virus, inhibit the replication, induced resistance of tobacco; moreover the induced concentration is very lower. The pot and field experiments of Trichoderma spores show that leaves of tobacco treated by Trichoderma special fertilizer after water Trichoderma spores have best appearance quality, relative coordination chemical composition, best assessment of quality smoking, combined with this treatment significantly reduced leaf disease, high yield and value of output, has a high application value.
Download TMVand CMV different strains’movement protein amino acid sequence and PVY polyprotein enzyme amino acid sequence from GenBank, use DNAMAN systerm to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CTP which contains restriction enzyme, after digestion got gene chips such as CTP, CT, CP, TP, C, T, P, etc. Download TMV, CMV of different strains’replicase amino acid sequence from GenBank, use DNAMAN to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CrTr which contains restriction enzyme, after digestion got gene chips such as CrTr, Cr, Tr. 10 fragments are plugged in RNAi-pUC respectively, connect to the carrier reversely by Double Digests, pieces are got after carrier dgested by PstⅠ, after removal of phosphorus, insert to pUCC2300-35-ocs a expression vector dgested by PstⅠfirst, transform competent Agrobacterium, obtain plasmid enrichment Agrobacterium, via enzyme validation,obtain Agrobacterium containing hpDNA. aseptic tobacco leaf fragments are infected by Agrobacterium, cultivate in MS media culture to grow roots, that is, RNAi tobacco seedlings are got initial, by toxicity, RT-PCR and other means to detect their resistance, get 11 anti-virus tobacco seedlings; 2 RNAi tobacco strains which have single copies and high resistance are picked out by Southern blot studies, get double homozygous transgenic RNAi tobacco seedlings quickly through haploid breeding, tobacco seedlings anti-virus features retained--can resist three virus intrusion at the same time, they and ordinary tobacco are subject to essentially the same chemical composition.
木霉孢子和康宁霉素分别与烟草花叶病毒混合后,能够引起病毒颗粒断裂、凝聚,极显著降低了病毒的侵染能力,钝化防效分别为85.81%和56.11%。木霉孢子与TMV混合后接种NC89,病毒RNA含量明显低于对照,仅为对照的2.05%,病情指数较对照极显著降低,说明木霉孢子能够体外钝化TMV,并且能够抑制病毒RNA在烟株内的积累。烟草NC89接种病毒6小时后,分别施用木霉孢子悬浮液和康宁霉素,48小时后研磨汁液接种枯斑三生烟苗,治疗防效分别为97.98%和70.26%,病情指数显著低于对照处理,病毒含量也明显降低,可见木霉及其产物对病毒病具有治疗效果,能够抑制病毒在植物体内的复制和积累,对于防治病毒的二次扩展十分重要。木霉孢子和康宁霉素均可诱导烟草产生抗性,抗性基因得到激活,防御酶活性增强,特别是在接种病毒后,防御酶活性进一步增强,这有利于对抗病毒,抑制病毒RNA复制,降低危害,MDA含量减少,最大叶长、宽极显著提高,病情指数明显降低,诱导防效分别为55.46%和57.68%。康宁霉素作为木霉产peptaibols,具有木霉孢子的部分功能,且诱导浓度较低。木霉孢子的盆栽和大田实验表明,浇灌木霉孢子后施用木霉专用肥处理烟叶外观质量好、化学成分相对协调、评吸质量优于其他处理,加之该处理烟叶病害显著降低、产量高、产值高,具有较高的推广应用价值。
从GenBank下载TMV、CMV的不同株系的移动蛋白氨基酸序列,PVY polyprotein酶氨基酸序列,运用DNAMAN进行对比分析,挑选高度重合序列,确定其编码碱基序列,经人工合成构建含有酶切位点的联合基因片段CTP,经过酶切得CTP、CT、CP、TP、C、T、P等7个片段。从GenBank下载TMV、CMV的不同株系的复制酶氨基酸序列,经对比分析确定其编码碱基序列后人工合成构建含有酶切位点的联合基因片段CrTr,经过酶切得CrTr、Cr、Tr 3个片段。将得到的10个片段分别插入RNAi-pUC中,再用双酶切反向连接至干涉载体,用PstⅠ酶切载体,回收片段,脱磷后,插入表达载体pUCC2300-35-ocs中,转化感受态农杆菌,获得富集质粒的农杆菌,经酶切验证,获得含hpDNA的农杆菌。用含有hpDNA的农杆菌感染事先准备好的无菌烟草叶片碎片中,MS培养基培养,待长出根系后,即初步得到RNAi烟苗,通过接毒实验、RT-PCR等手段检测其抗病性,得到抗病毒烟苗11株;通过Southern blot挑选单拷贝、高抗性RNAi烟苗2株进行单倍体育种,快速得到的双倍纯合转基因RNAi烟苗,烟苗抗病毒特性得到保留--能够同时抵抗三种病毒入侵,且化学成分与普通烟草基本一致。
Virus disease is a large class of diseases that widespread and severe damaged in the tobacco cultivation throughout the world. Tobacco mosaic virus (TMV), potato Y virus (PVY) and tobacco cucumber mosaic virus (CMV) are the most widespread and serious virus. There are several kinds of measures to control virus disease; cultivation resistant varieties and application of antiviral pharmaceutical are most direct and effective method. This article from these two-pronged approaches to carry out research work: one is study Trichoderma a biocontrol fungus anti-viral effect and application methods in tobacco; another is achievement tobacco seedlings by RNAi technology, which can resist three kinds of viruses at the same time. Through the study found that:
Trichoderma spores and Trichokonin mixed with tobacco mosaic virus respectively, can cause the virus particle fracture, cohesion, significantly reduced the ability of the virus infection, prevention of passivation are 85.81% and 56.11% respectively. Trichoderma spores mixed with TMV, then inoculate NC89, RNA of viral were significantly lower than the control, only 2.05%, disease index was significantly lower than the control. It is indicate that Trichoderma spores can passive TMV in vitro, and inhibit viral RNA accumulation in tobacco strain. Tobacco NC89 inoculation TMV first, 6 hours late, application of Trichoderma spore suspension and Trichokonin respectively, pestle into juice 48 hours later, then inoculated Samsun-NN with friction means, prevention effects were 97.98% and 70.26%, disease index was significantly lower than the control treatment, Virus levels also decreased, showing that Trichoderma and its product have therapeutic effect on viral disease, can inhibit the replication of the virus in plants and accumulation of the virus, that very important in prevention of virus secondary expansion. Trichoderma and Trichokonin can induce resistance of tobacco, genes of resistance are activated, defensive activity increased, especially in the tobacco inoculated virus, the defensive activity further enhanced, which will help to combat viruses, inhibit replication of viral RNA, reduce harm to tobacco, content of MDA was decreased. The maximum leaf length and width increased significantly, the disease index decreased significantly, induced control effects were 55.46% and 57.68% respectively. Trichokonin as Trichoderma product peptaibols, has some function of Trichoderma spores, can passive virus, inhibit the replication, induced resistance of tobacco; moreover the induced concentration is very lower. The pot and field experiments of Trichoderma spores show that leaves of tobacco treated by Trichoderma special fertilizer after water Trichoderma spores have best appearance quality, relative coordination chemical composition, best assessment of quality smoking, combined with this treatment significantly reduced leaf disease, high yield and value of output, has a high application value.
Download TMVand CMV different strains’movement protein amino acid sequence and PVY polyprotein enzyme amino acid sequence from GenBank, use DNAMAN systerm to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CTP which contains restriction enzyme, after digestion got gene chips such as CTP, CT, CP, TP, C, T, P, etc. Download TMV, CMV of different strains’replicase amino acid sequence from GenBank, use DNAMAN to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CrTr which contains restriction enzyme, after digestion got gene chips such as CrTr, Cr, Tr. 10 fragments are plugged in RNAi-pUC respectively, connect to the carrier reversely by Double Digests, pieces are got after carrier dgested by PstⅠ, after removal of phosphorus, insert to pUCC2300-35-ocs a expression vector dgested by PstⅠfirst, transform competent Agrobacterium, obtain plasmid enrichment Agrobacterium, via enzyme validation,obtain Agrobacterium containing hpDNA. aseptic tobacco leaf fragments are infected by Agrobacterium, cultivate in MS media culture to grow roots, that is, RNAi tobacco seedlings are got initial, by toxicity, RT-PCR and other means to detect their resistance, get 11 anti-virus tobacco seedlings; 2 RNAi tobacco strains which have single copies and high resistance are picked out by Southern blot studies, get double homozygous transgenic RNAi tobacco seedlings quickly through haploid breeding, tobacco seedlings anti-virus features retained--can resist three virus intrusion at the same time, they and ordinary tobacco are subject to essentially the same chemical composition.
引文
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