真核表达产物Y3诱导烟草对烟草花叶病毒的抗性研究

doi:10.16409/j.cnki.2095-039x.2021.02.006

摘要/Abstract

摘要： 从毛头鬼伞Coprinus comatus中提取的碱性糖蛋白Y3可以降低烟草花叶病毒（TMV）的侵染。克隆获得Y3蛋白cDNA后与真核表达载体pPIC-9k连接，重组载体pPIC-9k-Y3成功电转化入毕赤酵母Pichia pastoris后，转化子在28℃、250 r/min培养条件下，使用1.0%甲醇诱导表达6 d，成功实现了Y3蛋白的真核表达。300 μg/mL浓度的Y3蛋白对普通烟进行诱导处理后摩擦接种TMV结果表明，诱导处理后烟草体内除过氧化物酶POD外，多酚氧化酶PPO、苯丙氨酸解氨酶PAL和β-1，3葡聚糖酶活性都有不同程度提高。蛋白处理24 h后植株中PPO活性达到最大，为对照组的2倍。利用实时荧光定量PCR （qRT-PCR）研究抗性相关基因表达情况，Y3诱导后烟草植株碱性病程相关基因1（PR1-b）、病程相关基因非表达子1（NPR1）、PAL在转录水平上均显著上调（P<0.05）。推测真核表达产物Y3蛋白可通过提高水杨酸信号途径相关防御酶活性以及抗病基因转录来诱导烟草对TMV产生系统抗性，为Y3蛋白作为生物农药开发提供理论基础。

关键词: Y3蛋白, 真核表达, 烟草花叶病毒, 诱导抗性

Abstract: Protein Y3, an alkaline glycoprotein extracted from Coprinus comatus, could reduce TMV infection to tobacco. Vector pPIC-9k-Y3 was constructed based on the cDNA of Y3 and the expression plasmid pPIC-9k, and then transformed into Pichia pastoris GS115 by eletroporation successfully. The positive transformants was induced for 6 d by 1.0% methanol at the condition of 28℃ and 250 r/min. After sprayed with 300 μg/mL Y3 protein, tobacco plants were inoculated by TMV, then suffered with the activity detection of the disease-resistant defense enzymes such as polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL) and β-1,3 glucanase. The results showed that they were enhanced, and the maximum activity of PPO reached to 2 times compared with control after 24 h of protein treatment. Analysis of resistance-related genes expression by Quantitative real-time PCR (qRT-PCR) showed that basic pathogenesis-related gene 1 (PR1-b), nonexpressor of pathogenesis-related gene 1 (NPR1) and PAL gene were significantly up regulated after Y3 treatment (P<0.05). Thus, we can infer that the recombinant Y3 induce systemic disease resistance of tobacco by increasing the activity of defense enzyme and transcription of resistance genes related to salicylic acid signal transduction pathway.

Key words: protein Y3, eukaryotic expression, tobacco mosaic virus, induced resistance

中图分类号:

Q945.8

张志云, 边叶雨, 黄航, 韩潇潇, 吴兰, 吴丽萍. 真核表达产物Y3诱导烟草对烟草花叶病毒的抗性研究[J]. 中国生物防治学报, 2021, 37(2): 323-330.

ZHANG Zhiyun, BIAN Yeyu, HUANG Hang, HAN Xiaoxiao, WU Lan, WU Liping. Resistance to TMV in Tobacco Induced by Eukaryotic Expression Production of Y3[J]. Chinese Journal of Biological Control, 2021, 37(2): 323-330.

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