异色瓢虫3个小分子热激蛋白序列及低温诱导表达分析

doi:10.3864/j.issn.0578-1752.2017.16.009

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，广泛应用于农林害虫的生物防治中。本研究旨在分析探索低温胁迫条件对3个小分子热激蛋白（small heat shock protein，sHSP）抗寒基因相对表达量的影响，为异色瓢虫低温冷藏及其抗寒机制研究提供科学依据。【方法】在转录组获得异色瓢虫基因序列的基础上，根据特异性引物获得HaHSP47.74（基因登录号：KX161871）、HaHSP21.53（基因登录号：KX161873）和HaHSP21.52（基因登录号：KX161874）基因cDNA的开放阅读框序列(open reading frame，ORF），采用实时荧光定量PCR(real-time fluorescent quantitative PCR，qRT-PCR）技术测定异色瓢虫3个sHSP基因在不同发育阶段、短时降温和短时降温后恢复处理、低温储存处理下以及不同色斑的表达水平。【结果】在不同发育阶段处理中，HaHSP47.74和HaHSP21.53在蛹期及成虫期高表达；HaHSP21.52在4龄幼虫第4天表达量显著高于对照组。在短时降温处理中，HaHSP47.74在降温至0℃和-5℃时表达量显著高于对照组；HaHSP21.53和HaHSP21.52在降温过程中表达量显著降低。在短时降温后恢复处理中，3个sHSP基因表达量无显著差异。在低温储藏条件下，实验种群：黑底雌瓢虫HaHSP47.74表达水平在储存第5天时显著升高，黄底雌瓢虫HaHSP47.74在储存5—15 d时表达量显著升高；黑底雌瓢虫HaHSP21.53在储存5—20 d时显著性高表达，黄底雌瓢虫HaHSP21.53在15 d时显著性高表达；黑底和黄底雌瓢虫HaHSP21.52无显著性高表达；越冬种群：黑底和黄底雌瓢虫HaHSP47.74、HaHSP21.53、HaHSP21.52在低温储存中均无显著性高表达。【结论】sHSP在异色瓢虫发育阶段可能发挥着作用。短时降温胁迫能够诱导sHSP基因高表达。实验种群在低温储存条件下HaHSP47.74和HaHSP21.53均出现了显著性高表达，表明这两个基因在瓢虫受到冷驯化时起到了作用。此外，不同色斑型异色瓢虫的抗寒机制和抗寒能力有所差异。

关键词: 异色瓢虫, 小分子热激蛋白, 抗寒基因, 低温储存, 实时荧光定量PCR, 基因表达

Abstract: 【Objective】 Harmonia axyridis is an important predator insect, and it is widely used in agricultural and forestry production. In this study, three small heat shock proteins (sHSP) were screened out and cloned using high throughput sequencing techniques. Also the expression levels of these three sHSP (sHPSs/sHSP genes) were detected under cold stress in order to explore the improvement of H. axyridis cold resistance role under low temperature stress conditions. 【Method】 Based on H. axyridis gene sequences in the transcriptome, several specific primers were designed to obtain the full-length open reading frame (ORF) sequence of the three sHSP genes. The expression of these sHSP genes was detected by quantitative real-time PCR (qRT-PCR) under short-time cooling or short-time cooling recover conditions, and between the black and yellow females of experimental and overwintering populations under low-temperature storage.【Result】 HaHSP47.74 and HaHSP21.53 were highly expressed at the pupal and adult stages during different developmental stages. And the expression of HaHSP21.52 in 4th instar larvae on the 4th day was significantly higher than that in control group. In short-term cooling treatment, the expression of HaHSP47.74 was significantly higher than that of the control group when the temperature was reduced to 0℃ and -5℃. The expression of HaHSP21.53 and HaHSP21.52 decreased significantly during cooling. There was no significant difference in the expression of the three sHSP genes in short-term heating treatment. The HaHSP47.74 expression of black female was significantly higher on the 5th day, while the yellow female was significantly higher from the 5th to 15th day in the experimental population under the condition of low temperature storage. The expression of HaHSP21.53 in black females was significantly higher from the 5th day to 20th day, while in the yellow females was significantly higher on the 15th day. Moreover, there was no significant expression of HaHSP21.52 in black or yellow female, and HaHSP47.74, HaHSP21.53, HaHSP21.52 in black females and yellow females were not significantly higher in the low temperature storage treatment in overwintering population. 【Conclusion】 sHSP may play a role in developmental stages of H. axyridis. HaHSP47.74 expression increased significantly under short-term cooling conditions, suggesting that sHSP in H. axyridis may play a key role under low temperature stress. While higher expression of HaHSP47.74 and HaHSP21.53 was found in the experimental population under low temperature storage, which indicated that these two genes played an important role in the cold acclimation. In addition, the expression level of black and yellow females of H. axyridis was different, it was speculated that the resistance and mechanism of different colors of H. axyridis are different.

Key words: Harmonia axyridis, small heat shock protein, cold-resistance gene, preservation in low temperature, qRT-PCR, gene expression

汪慧娟，赵静，施佐堃，邱玲玉，王甦，张帆，王世贵，唐斌. 异色瓢虫3个小分子热激蛋白序列及低温诱导表达分析[J]. 中国农业科学, 2017, 50(16): 3145-3154.

WANG HuiJuan, ZHAO Jing, SHI ZuoKun, QIU LingYu, WANG Su, ZHANG Fan, WANG ShiGui, TANG Bin. Sequence Analysis and Induced Expression of Three Novel Small Heat Shock Proteins Mediating Cold-Hardiness in Harmonia axyridis[J]. Scientia Agricultura Sinica, 2017, 50(16): 3145-3154.

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