异色瓢虫抗寒关键基因筛选及其在诱导抗寒中的功能鉴定

菜单

摘要异色瓢虫是一种重要的捕食性天敌昆虫种质资源，东北及以北地区以成虫越冬，具有非常强的适应能力和抗寒能力。本研究应用转录组和数字表达谱（DGE）等高通量测序技术，测定常温和不同程度低温诱导处理条件下异色瓢虫转录组和差异基因mRNA水平变化情况，结果表明高达60个基因一直在调控低温诱导抗寒过程，其中7个基因起正调控作用，5个基因起负调控作用。通过进一步分析筛选获得6个非常关键的潜在抗寒基因，分别为hsp21.4、trehalase、transketolase、erythrocyte binding protein、ATP-grasp fold domain protein和dopa decarboxylase。以过冷却点作为瓢虫抗寒性能指标，将野外采集到的异色瓢虫通过0℃、5℃和10℃三种不同温度及2 h、12 h和24 h三种时间的低温冷驯化处理，结果显示5℃低温驯化12 h能够显著降低异色瓢虫的过冷却点，表明该驯化条件能够有效提高异色瓢虫的抗寒能力。以未冷驯化种群作为对照组，最佳冷驯化条件处理组种群为处理组，分别在5℃低温条件下储存10 d、20 d和30 d，结果表明未冷驯化组存活率和过冷点都显著高于冷驯化处理组。进一步通过实时荧光定量PCR检测两组异色瓢虫6个关键基因的相对表达量，研究结果显示hsp21.4、trehalase、transketolase、ATP-grasp fold domain protein和dopa decarboxylase在冷驯化过程中表达上调，而erythrocyte binding protein的表达下调；且hsp21.4在低温保存过程中都一直处于高表达状态，显著高于未冷驯化组，其余5个基因在低温保存过程中处于低表达状态，显著低于未冷驯化组。这些结果表明，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dopa decarboxylase通过在冷驯化过程中高表达，保护异色瓢虫抵御寒冷环境。47613

Abstract

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 Dongbei and other area. In this research, we measures the changes of transcriptome and mRNA level of H. axyridis in normal temperature and different treatment at low temperature, using high-throughput technology such as transcriptome and digital gene expression tag profiling (DGE). The result showed that it was sixty genes that regulated the whole cold-acclimation response, seven genes were always up-regulated and five genes down-regulated. After futher screening, six key cold-resistant genes including hsp21.4, trehalase, transketolase, erythrocyte binding protein, ATP-grasp fold domain proteinand dopa decarboxylase, was found very important. Measuring super cooing point as a index of cold-resistance ability, 0℃, 5℃, 10℃ three different temperatures and 2 h, 12 h, 24 h three different treatment time were set to acclimate H. axyridis which were wild population. It was found that its supercooling point was decreased significantly after acclimating 12 h in 5℃. It showed that acclimating 12 h in 5℃ can effectively improve the cold-resistance ability of H. axyridis. Setting treatment group which was acclimated 12 h in 5℃ and control group deposited for 10 d, 20 d, 30 d at 5℃, it found the survival rate and super cooling point of control group were all higher than treatment group. Meatime, we detected six genes’ relative expression thoughout real-time quantitative PCR. The 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 dow-regulated. Hsp21.4 was always highly expressed when cryopreservation, and treatment group was strikingly higher than control group. Others were on low expression when cryopreservation, and strikingly lower than control group. It showed that hsp21.4 and erythrocyte binding protein 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 ecpressed in cold acclimation to resist cold environment.