利用植物抗体中和真菌毒素DON及提高赤霉病抗性的研究
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摘要
小麦赤霉病是影响我国南方麦区、东北麦区以及黄淮流域麦区的重要真菌病害，主要是由禾谷镰刀菌Fusarium graminearum引起的。赤霉病菌引起小麦的穗腐，不但使小麦产量降低、品质变劣，而且病麦粒中含有镰刀菌代谢产生的真菌毒素，如脱氧雪腐镰刀菌烯醇(Deoxynivalenol，DON)，食用污染毒素的病麦及其制成品后会引起人畜呕吐、中毒甚至致癌。因此，中和DON毒素的研究成为控制其毒性和减轻赤霉病对植物为害的重要研究方向。近十多年来，植物抗体研究取得了巨大进展。Hiatt等首次证明在植物体内可以表达和装配抗体蛋白以来，已有很多在这方面的研究。随着抗体技术的发展，已报道在多种植物中表达了单链抗体，且可以减轻植物病害。本研究以中和DON的毒性，减轻小麦赤霉病的发生为目标，探索出一条中和毒素之毒性、提高植物抗病性的新途径。
本研究将抗脱氧雪腐镰刀菌烯醇的单链抗体(DONscFv)基因导入烟草，获得高效表达植物抗体的转基因烟草，并研究了表达单链抗体的转基因烟草中和毒素DON的作用，结果表明与野生型相比，转基因烟草的种子在含毒素(8μM／L)的培养基中萌发时，能表现出更耐毒素的毒性。
此外，本研究构建了DONscFv基因的小麦表达载体，以培养4天的幼胚愈伤组织为材料，用农杆菌介导法转化小麦，经PCR和ELISA证实获得了高效表达DONscFv的4株转基因小麦，对T_1代转基因小麦进行赤霉病抗性鉴定，接种8天后观察发病情况，野生型对照植株在接种的中部发病严重，并长满了菌丝，然而32株T_1代转基因植株中25株的穗子中部均表现出较轻的发病症状。但是，随着时间的推移，接种10天后转基因小麦也表现出了比较明显的发病症状。这表明转DONscFv基因小麦只是在一定程度上延迟了发病症状的出现。DONScFv起到中和毒素DON的作用，由于病菌代谢产生毒素的含量降低，影响了病原菌在组织中的定殖时间和其在组织中的扩展，所以在一定程度上延迟并减轻了病害症状。
迄今为止，对小麦成熟胚再生体系的研究很少报道。本研究以晋麦2148成熟胚为外植体，进行再生体系的研究。在此基础上，用基因枪转化法将GUS基因导入小麦，以便初步建立小麦成熟胚的基因枪转化体系。
Fusarium head blight or wheat scab,caused by Fusarium graminearum(teleo-morph Gibberella zeae(Schew)Petch),is one of the most important diseases of wheat,barley and corn in China. Head blight not only causes the severe loss of grain yield,but also causes the harvested grain contaminated with several mycotoxin,such as deoxynivalenol(DON).The consumption of food product made from grains contaminated with the toxin DON is a potential healthy problem for human and farm animals,which is known to cause carcinogenic,genotoxic acute or chronic diseases and to directly target the kidney,liver or immune system. So it is very important to study how to neutralize the mycotoxin DON so as to alliveate the severity of wheat scab.Researchers has achieved great progress in the study of plantibody since a decade years ago.There has been many studies about plantibody since Hiatt first testified antibody protein could be expressed and assemblied in plants.With the development of antibody engeering,it has been reported the scFvs(single chain variable fragment) are expressed in many kinds of plants, and it can specially resist against some plant pathogens.This study takes aim at neutralizing the toxicity of DON, alliveating the severity of wheat scab and exploring a new pathway of resistance against wheat scab.
In this paper, the DONscFv gene was cloned,transferred and expressed in tobacoo plants,and we studied that the scFv functioned to neutralize the toxicity of DON.The study showed that the seeds of transgenic tobacoo lines were more tolerant than wild type when they germinated in the MS media containing 8μM/L DON.
Moreover,we constructed the expression vector used in wheat plant,and we got transgenic wheat plants by Agrobacterium-mediated method. The transformed plants were screened by PCR and ELISA for the presence and expression of the scFv, respectively.And we got four transgenic To plants.We used T transgenic wheat plants to indentify the resistance against wheat scab by inoculating Fusarium spores. The wild types showed typical symptoms of wheat head scab,while most of the transgenic T plants showed less symptoms 8 days after inoculation. But the transgenic T plants show as typical symptoms as the wild types 10 days after inoculation.These results showed that, in inoculating experiment, transgenic wheat plants only delayed symptoms of wheat head scab to some extent,for DON play a key role in symptom development as well as in pathogen spreading within host tissues, and the scFv expressed in transgenic wheat plants may neutralize the mycotocity of DON,induced by Fusarium metabolizing.
Moreover,because there are few studies on the regeneration system of wheat mature embryos.We also used wheat variety JM 2148 mature embryos as explants to study the regeneration. Moreover,we transferred GUS gene into JM 2148 mature embryos by particle bombardment so as to establish the transformational system of mature embryos by particle bombardment.The results indicated that it is a pratical way for using the wheat mature embryos for gene transferring.
本研究将抗脱氧雪腐镰刀菌烯醇的单链抗体(DONscFv)基因导入烟草，获得高效表达植物抗体的转基因烟草，并研究了表达单链抗体的转基因烟草中和毒素DON的作用，结果表明与野生型相比，转基因烟草的种子在含毒素(8μM／L)的培养基中萌发时，能表现出更耐毒素的毒性。
此外，本研究构建了DONscFv基因的小麦表达载体，以培养4天的幼胚愈伤组织为材料，用农杆菌介导法转化小麦，经PCR和ELISA证实获得了高效表达DONscFv的4株转基因小麦，对T_1代转基因小麦进行赤霉病抗性鉴定，接种8天后观察发病情况，野生型对照植株在接种的中部发病严重，并长满了菌丝，然而32株T_1代转基因植株中25株的穗子中部均表现出较轻的发病症状。但是，随着时间的推移，接种10天后转基因小麦也表现出了比较明显的发病症状。这表明转DONscFv基因小麦只是在一定程度上延迟了发病症状的出现。DONScFv起到中和毒素DON的作用，由于病菌代谢产生毒素的含量降低，影响了病原菌在组织中的定殖时间和其在组织中的扩展，所以在一定程度上延迟并减轻了病害症状。
迄今为止，对小麦成熟胚再生体系的研究很少报道。本研究以晋麦2148成熟胚为外植体，进行再生体系的研究。在此基础上，用基因枪转化法将GUS基因导入小麦，以便初步建立小麦成熟胚的基因枪转化体系。
Fusarium head blight or wheat scab,caused by Fusarium graminearum(teleo-morph Gibberella zeae(Schew)Petch),is one of the most important diseases of wheat,barley and corn in China. Head blight not only causes the severe loss of grain yield,but also causes the harvested grain contaminated with several mycotoxin,such as deoxynivalenol(DON).The consumption of food product made from grains contaminated with the toxin DON is a potential healthy problem for human and farm animals,which is known to cause carcinogenic,genotoxic acute or chronic diseases and to directly target the kidney,liver or immune system. So it is very important to study how to neutralize the mycotoxin DON so as to alliveate the severity of wheat scab.Researchers has achieved great progress in the study of plantibody since a decade years ago.There has been many studies about plantibody since Hiatt first testified antibody protein could be expressed and assemblied in plants.With the development of antibody engeering,it has been reported the scFvs(single chain variable fragment) are expressed in many kinds of plants, and it can specially resist against some plant pathogens.This study takes aim at neutralizing the toxicity of DON, alliveating the severity of wheat scab and exploring a new pathway of resistance against wheat scab.
In this paper, the DONscFv gene was cloned,transferred and expressed in tobacoo plants,and we studied that the scFv functioned to neutralize the toxicity of DON.The study showed that the seeds of transgenic tobacoo lines were more tolerant than wild type when they germinated in the MS media containing 8μM/L DON.
Moreover,we constructed the expression vector used in wheat plant,and we got transgenic wheat plants by Agrobacterium-mediated method. The transformed plants were screened by PCR and ELISA for the presence and expression of the scFv, respectively.And we got four transgenic To plants.We used T transgenic wheat plants to indentify the resistance against wheat scab by inoculating Fusarium spores. The wild types showed typical symptoms of wheat head scab,while most of the transgenic T plants showed less symptoms 8 days after inoculation. But the transgenic T plants show as typical symptoms as the wild types 10 days after inoculation.These results showed that, in inoculating experiment, transgenic wheat plants only delayed symptoms of wheat head scab to some extent,for DON play a key role in symptom development as well as in pathogen spreading within host tissues, and the scFv expressed in transgenic wheat plants may neutralize the mycotocity of DON,induced by Fusarium metabolizing.
Moreover,because there are few studies on the regeneration system of wheat mature embryos.We also used wheat variety JM 2148 mature embryos as explants to study the regeneration. Moreover,we transferred GUS gene into JM 2148 mature embryos by particle bombardment so as to establish the transformational system of mature embryos by particle bombardment.The results indicated that it is a pratical way for using the wheat mature embryos for gene transferring.
引文
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