异色瓢虫低温胁迫下过冷却点变化及抗寒基因表达分析

doi:10.3864/j.issn.0578-1752.2016.04.007

摘要/Abstract

摘要： 【目的】异色瓢虫(Harmonia axyridis)是一种重要的捕食性天敌昆虫资源，在东北地区以成虫越冬，具有非常强的抗寒能力。以过冷却点和抗寒基因的mRNA水平变化作为瓢虫抗寒性能指标，探索低温胁迫提高瓢虫抗寒的作用。【方法】将野外采集到的异色瓢虫通过0、5和10℃ 3种不同温度，进行2、12和24 h 3种时间的低温胁迫处理，测定过冷却点的变化，得到最佳低温胁迫条件。以室内饲养的异色瓢虫作为对照组，分别在5℃低温条件下储存10、20和30 d，测定过冷却点变化和存活情况。根据转录组和数字表达谱（digital gene expression，DGE）数据分析确定6个重要抗寒基因，选择18S作为内参基因，采用实时荧光定量PCR检测异色瓢虫6个重要抗寒基因在低温胁迫和保存时mRNA水平上的相对表达量，分析其在抗寒过程中所起的作用。【结果】5℃低温胁迫12 h能够显著降低异色瓢虫的过冷却点。在低温保存过程中，低温胁迫组异色瓢虫过冷却点和存活率低于未进行低温胁迫组。实时荧光定量PCR检测发现HSP21.4、trehalase、transketolase、ATP-grasp fold domain protein和dopa decarboxylase在低温胁迫过程中表达量上调，而erythrocyte binding protein的表达量下调。且在低温保存过程中HSP21.4一直处于高表达状态，显著高于未进行低温胁迫组，其余5个基因在低温保存过程中处于低表达状态，显著低于未进行低温胁迫组。【结论】低温胁迫能够降低异色瓢虫的过冷却点，但不一定能够提高夏季野外耐高温异色瓢虫的存活能力。HSP21.4和erythrocyte binding protein分别通过高表达和低表达来提高异色瓢虫的抗寒能力，而trehalase、transketolase、ATP-grasp fold domain protein和dopadecarboxylase通过在低温胁迫过程中高表达，保护异色瓢虫抵御寒冷环境。

关键词: 异色瓢虫, 低温胁迫, 抗寒基因, 定量表达, 低温储存

Abstract: 【Objective】Harmonia axyridis is an important predatory lady beetle as natural enemies attacking agricultural and forestry pests. H. axyridis has strong cold-hardiness as it can overwinter as disapausing adults in Northeast population of China. The objective of this study is to use the super cooing point and the changes of cold resistance gene mRNA level as the index of cold-resistance ability, and to define the effect of low temperature stress on improving cold resistance of H. axyridis.【Method】The wild H. axyridis adults were collected and treated for 2, 12 and 24 h by three different low temperatures of 0, 5 and 10℃ to get the best low temperature stress. The changes of super cooling point were detected all the time during the period of low temperature processing. The H. axyridis adults of experimental populations and wild adults were used as the control groups and experimental groups, respectively. All of these adults were treated under the condition of 5℃ low temperature and stored for 10, 20 and 30 d, and the changes of super cooling point and survival rate were measured after the treatment. Six important cold hardiness genes were sure by transcriptome and digital gene expression tag profiling (DGE). The relative expressions of six important cold hardiness genes at mRNA level were detected by real-time fluorescent quantitative PCR. 【Result】The super cooling point was decreased significantly after acclimation for 12 h at 5℃. Acclimation for 12 h at 5℃ could effectively decrease the super cooling point and other indexes of cold resistance in H. axyridis. Setting treatment group which was acclimated for 12 h at 5℃ and control group deposited for 10 , 20, and 30 d at 5℃, the survival rate and super cooling point of control group were all higher than the treatment group. In the same time, the 6 genes’ relative expression of mRNA level results showed HSP21.4, trehalase, transketolase, ATP-grasp fold domain protein and dopa decarboxylase were up-regulated when cold acclimation, but erythrocyte binding protein was down-regulated. HSP21.4 was always highly expressed in cryopreservation, and the treatment group was strikingly higher than the control group. Others were on low expression in cryopreservation, and strikingly lower than the control group. 【Conclusion】The low temperature stress can reduce the super cooing point of the H. axyridis, but it can’t improve the survival ability of summer high temperature resistant population. HSP21.4 and erythrocyte binding protein can help H. axyridis to improve the cold-resistance ability respectively by high expression and low expression. Trehalase, transketolase, ATP-grasp fold domain protein and dopa decarboxylase are highly expressed in cold acclimation to resist cold environment.

Key words: Harmonia axyridis, low temperature stress, cold-resistant gene, quantitative expression, preservation in low temperature

邬梦静，徐青叶，刘雅，施兴荣，沈祺达，杨萌萌，王世贵，唐斌. 异色瓢虫低温胁迫下过冷却点变化及抗寒基因表达分析[J]. 中国农业科学, 2016, 49(4): 677-685.

WU Meng-jing, XU Qing-ye, LIU Ya, SHI Xing-rong, SHEN Qi-da, YANG Meng-meng, WANG Shi-gui, TANG Bin. The Super Cooling Point Change of Harmonia axyridis Under Low Temperature Stress and Its Cold-Resistance Genes’ Expression Analysis[J]. Scientia Agricultura Sinica, 2016, 49(4): 677-685.

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