华山松大小蠹冷休克结合蛋白的鉴定与表达

doi:10.11707/j.1001-7488.20180407

摘要/Abstract

摘要： [目的]研究华山松大小蠹冷休克结合蛋白基因和表达特性，为揭示冷休克结合蛋白基因在华山松大小蠹适应性、耐寒性分子机制奠定基础。[方法]从华山松大小蠹cDNA转录组中克隆获得的扩增序列经Blast同源比对鉴定基因，应用MEGA软件构建系统发育树和实时荧光定量PCR分析冷休克结合蛋白基因在不同低温环境与时间序列的表达特性。[结果]从华山松大小蠹cDNA转录组序列克隆出1条华山松大小蠹冷休克结合蛋白基因序列，命名为DarmCSP。Blast比对结果显示，华山松大小蠹DarmCSP与黑山大小蠹CSP最为相似，相似性达92%。经实时荧光定量PCR分析华山松大小蠹冷休克结合蛋白基因DarmCSP在越冬期间（9月-次年3月）的表达特性，发现DarmCSP在越冬期间相对表达量呈上调趋势，DarmCSP在1月的相对表达量是9月的4.26倍，3月的2.90倍。越夏成虫（7月）和越冬幼虫（1月）DarmCSP相对表达量均呈现上调表达。华山松大小蠹幼虫在-10℃ 1~24 h低温胁迫下，DarmCSP的相对表达量随处理时间的延长而升高，24 h时相对表达量达到最大值，而在-2、-4、-6和-8℃低温下胁迫12 h，相对表达量达到最大值，这说明0℃~-10℃低温胁迫均能诱导华山松大小蠹DarmCSP的表达。同时，越夏成虫在4℃~-6℃低温胁迫下DarmCSP也呈上调表达，但相对表达量小于越冬幼虫。[结论]华山松大小蠹冷休克结合蛋白基因DarmCSP的表达受低温诱导，参与了华山松大小蠹对低温适应过程，这对进一步研究华山松大小蠹冷休克结合蛋白信号转导途径和作用机制具有重要的价值。

关键词: 华山松大小蠹, 冷休克结合蛋白, 低温, 基因表达, RT-qPCR

Abstract: [Objective] We cloned and characterized the cold shock domain-containing protein (CSP) gene in Chinese white pine beetle, Dendroctonus armandi in order to reveal the molecular mechanism in adaptability and cold-tolerance of D. armandi.[Method] The DarmCSP cDNA fragment of D. armandi was cloned. The phylogynetic tree was constructed with MEGA. The expression profiles of DarmCSP in D. armandi exposed to different low temperatures for different time series were detected by using real-time quantitative PCR.[Result]The cloned gene was named as DarmCSP. The result showed that the sequence from DarmCSP gene shared the highest level of amino acid sequence identity with CSP of the bark beetle species (Dendroctonus ponderosae) (92%). Real-time PCR analysis was used to detect the transcript levels of DarmCSP in D. armandi. The DarmCSP expression was up-regulated during the overwintering stage. The DarmCSP expression level of overwintering larvae (Jan. 2015) was 4.26 higher more than that early winter stage (Sep. 2014) and 2.90 higher than late winter stage (Mar. 2015). The DarmCSP expressions of overwintering larvae (Jan. 2015) and oversummering adults (Jul. 2015) were both up-regulated when beetles were exposed at different low temperatures for different duration. DarmCSP expression level of D. armandi increased with the exposure time increasing from 1 h to 24 h at -10℃, reaching its highest expression level at the time of 24 h. The DarmCSP reached the highest expression levels all for 12 h when larvae were exposed among -2, -4, -6 and -8℃. The DarmCSP can be induced from 0℃ to -10℃. The DarmCSP expression of oversummering adults was also up-regulated when beetles were exposed from 4℃ to -6℃. However, the expression level was lower than that of overwintering larvae.[Conclusion] The result of this study demonstrated that the DarmCSP expression in D. armandi can be induced by low temperature. The molecular changes participate in the adaptation process of D. armandi to low temperature. Our result can contribute more to further study on CSP and its physiological biochemical mechanism of Chinese white pine beetle.

Key words: Dendroctonus armandi, cold shock domain-containing protein, low temperature, gene expression, RT-qPCR

中图分类号:

S718.7

王娟, 陈辉. 华山松大小蠹冷休克结合蛋白的鉴定与表达[J]. 林业科学, 2018, 54(4): 58-66.

Wang Juan, Chen Hui. Identification and Expression of Cold Shock Domain-containing Protein Gene in Dendroctonus armandi[J]. Scientia Silvae Sinicae, 2018, 54(4): 58-66.

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