华山松大小蠹幼虫耐寒生化与分子机制
发布时间：2019-06-09 18:20
【摘要】：冬季低温是决定寒、温带昆虫种群繁衍和种群数量的关键性因素,也决定着昆虫种群的扩散、分布和时空动态等一系列生命活动。本研究以秦岭林区持续发生的重大森林害虫——华山松大小蠹(Dendroctonus armandi Tsai and Li)为研究对象,旨在通过研究越冬期华山松大小蠹幼虫生理生化代谢规律,华山松大小蠹耐寒相关基因在不同低温和时间序列下的表达差异,华山松大小蠹越冬幼虫耐寒相关基因RNA干扰后的低温死亡率,以及越冬期华山松大小蠹幼虫肠道真菌和细菌群落变化等,揭示越冬期华山松大小蠹幼虫低温胁迫耐受性,低温胁迫下的生理生化响应,越冬幼虫肠道真菌和细菌群落结构多样性,以及华山松大小蠹耐寒相关基因抵御低温环境的作用,取得以下研究结果:1.通过研究华山松大小蠹越冬幼虫过冷却点(SCP)和低温胁迫死亡率,结果表明华山松大小蠹越冬幼虫过冷却点(SCP)与环境温度显著正相关,在最冷月(1月)华山松大小蠹越冬幼虫过冷却点(SCP)达到最低值;华山松大小蠹越冬幼虫的低温耐受性随环境温度的降低而逐渐增强,说明华山松大小蠹越冬幼虫具有较强的耐寒能力,且华山松大小蠹越冬幼虫的耐寒性可通过低温驯化得以提高。2.通过分析华山松大小蠹越冬幼虫生理生化指标和代谢酶活性,结果表明华山松大小蠹幼虫越冬期海藻糖、山梨糖醇、甘油含量变化与过冷却点(SCP)显著负相关,越冬期糖原含量大幅降低,越冬后期恢复至越冬初期水平,说明海藻糖、山梨糖醇、甘油为越冬期华山松大小蠹幼虫体内潜在的抗冻保护物质,糖原为越冬期华山松大小蠹幼虫体内重要的能源物质;越冬期间幼虫体内自由水转化为结合水,总水含率变幅不大,越冬后期结合水转化为自由水,表明华山松大小蠹越冬幼虫具有“节水”机制,以提高虫体耐寒性;10月~3月SOD、CAT、LDH和AchE酶活性持续减弱,POD、PK和MDH酶活性持续增强,SDH、TPS和GLK酶活性在越冬期间(10月~1月)减弱,春季活性增强后保持相对稳定,说明华山松大小蠹主要代谢酶具有调节耐寒能力的作用。3.采用实时荧光定量PCR(qRT-PCR)和RNA干扰技术研究华山松大小蠹越冬幼虫在不同低温和时间序列下海藻糖磷酸合酶(TPS)、山梨糖醇脱氢酶(SDH)、甘油激酶(GLK)基因表达差异,结果表明越冬期华山松大小蠹幼虫DarmTPS和DarmSDH表达量显著上调,DarmGLK表达量下调。12月华山松大小蠹越冬虫DarmTPS、DarmSDH和DarmGLK表达量显著高于5月。注射DarmTPS-dsRNA、DarmSDH-dsRNA和DarmGLK-dsRNA的华山松大小蠹幼虫基因表达量显著降低,RNA干扰后华山松大小蠹幼虫低温死亡率明显升高,表明华山松大小蠹DarmTPS、DarmSDH和DarmGLK在调控幼虫耐寒性方面发挥着十分重要的作用。4.采用实时荧光定量PCR(qRT-PCR)技术研究华山松大小蠹越冬幼虫在不同温度梯度和时间序列下热激蛋白(DaHSP90,DaHSP70和DasHSP)和冷休克蛋白(DarmCSP)基因表达差异,结果表明华山松大小蠹越冬幼虫DaHSP90基因对冷激反应更敏感,DaHSP70基因对热激反应更敏感;DaHSP90和DasHSP基因在长时温度刺激下表达量变化显著,DaHSP70基因在短时温度刺激下表达量变化显著。从华山松大小蠹cDNA转录组序列克隆得到1条华山松大小蠹冷休克蛋白基因序列DarmCSP,该基因在越冬期间表达量呈上调趋势,且低温胁迫能诱导DarmCSP表达;同时,越夏成虫在低温胁迫下DarmCSP也呈上调表达,但表达量低于越冬幼虫。5.利用高通量测序技术(Illumina MiSeq平台)分析越冬期华山松大小蠹幼虫肠道微生物群落结构、多样性和动态变化,结果表明华山松大小蠹肠道微生物群落结构相对简单,肠道微生物群落结构会随越冬期和环境温度的变化发生改变。华山松大小蠹越冬幼虫肠道细菌群落主要由变形菌门(主要是γ-变形菌)、放线菌门、厚壁菌门和拟杆菌门构成,采集于11、12月的华山松大小蠹幼虫肠道细菌群落比采集于10月、1月的幼虫肠道细菌群落更丰富;肠道真菌群落由接合菌门、担子菌门和子囊菌门构成,子囊菌门是越冬幼虫肠道内的优势真菌,占总真菌丰富度的81.11%。由此推测,越冬期华山松大小蠹幼虫肠道细菌和真菌对抵御低温环境胁迫起到了一定的保护作用。
[Abstract]:The low temperature in winter is a key factor for determining the population of cold and temperate insects, and also determines the spread, distribution and space-time dynamics of the insect population. This study is aimed at studying the physiological and biochemical metabolic regulation of the large-size and large-sized larvae of the Chinese mountain in the overwintering period by studying the major forest pest _ Hua-shan pine-size (Dendranctonus armand i Tsai and Li) in the forest region of the Qinling Mountains. the expression of the cold-resistant and cold-resistant related genes of the Chinese mountain pine is different in different low temperature and time sequences, the low-temperature death rate after the cold-resistant related gene RNA of the overwintering larvae of the Chinese mountain and the overwintering larvae, and the changes of the intestinal fungi and the bacterial communities of the large-size and large-sized larvae of the overwintering stage, The low-temperature stress tolerance, the physiological and biochemical response at low temperature stress, the structure diversity of the intestinal fungi and the bacterial community of the overwintering larvae, and the effects of the cold-resistant and cold-resistant related genes of the Chinese mountain pine on the low-temperature environment are disclosed, and the following research results are obtained:1. The results show that the overwintering larva overcooling point (SCP) is positively correlated with the ambient temperature by studying the overwintering larvae overcooling point (SCP) and the low-temperature stress and death rate. In the coldest month (January), the overwintering larvae overcooling point (SCP) reached the lowest value, and the low-temperature tolerance of the overwintering larvae of the Pinus armandii was gradually enhanced with the decrease of the ambient temperature. And the cold resistance of the overwintering larvae of the size of the Chinese mountain pine can be improved by the low-temperature domestication. The results showed that the changes of the content of trehalose, sorbitol and glycerol in the overwintering period were negatively correlated with the over-cooling point (SCP), and the content of glycogen in the overwintering period was significantly decreased. In the later stage of overwintering, it was found that trehalose, sorbitol and glycerol are the potential anti-freezing protective substances in the larvae of the overwintering period, and the glycogen is an important energy substance in the larva of the large-sized and large-sized larvae of the overwintering period. the free water in the larvae during the overwintering period is converted into the combined water, the total water content rate is not large, and the combined water in the late winter is converted into free water, so that the large-size and overwintering larvae of the Chinese mountain pine are provided with a "water-saving" mechanism to improve the cold resistance of the insect body, and the activity of the SOD, the CAT, the LDH and the AchE in the period of October to March is continuously reduced, The activities of POD, PK and MDH were continuously enhanced, and the activity of SDH, TPS and GLK decreased during the overwintering period (October-January), and the activity of the spring remained relatively stable after the spring activity was enhanced. The differences of the expression of trehalose phosphate synthase (TPS), sorbitol dehydrogenase (SDH) and glycerol kinase (GLK) gene were studied by real-time fluorescence quantitative PCR (qRT-PCR) and RNA interference technique. The results showed that the expression of DarmTPS and DarmSDH was up-regulated and the expression of DarmGLK was down-regulated during the overwintering period. The expression of DarmTPS, DarmSDH and DarmGLK was significantly higher than that in May. The expression of DarmTPS-dsRNA, DarmSDH-dsRNA and DarmGLK-dsRNA showed a significant decrease in the gene expression, and the low-temperature mortality of the large-sized and large-sized larvae of the Chinese pine was significantly higher after the RNA interference, indicating that the size of the Pinus armandii, DarmSDH and DarmGLK play a very important role in controlling the cold resistance of the larvae. The expression of heat shock protein (DHSP90, DaHSP70 and DasHSP) and cold shock protein (DarmCSP) was studied by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the DaHSP90 gene was more sensitive to the cold shock response than that of the overwintering larvae, DaHSP90. The expression of the DaHSP90 and the DasHSP gene in the long-term temperature stimulated by the DaHSP90 and the DasHSP gene is more sensitive, and the expression level of the DaHSP90 and the DasHSP gene under the short-time temperature stimulation is significant. The expression of DarmCSP was induced by low-temperature stress, and the expression of DarmCSP was induced by low-temperature stress, and the DarmCSP was also up-regulated at low temperature. But the expression is lower than that of the overwintering larvae. In this paper, the structure, diversity and dynamic change of the intestinal microflora in the large-size and small-sized larvae of the overwintering period were analyzed by using the high-throughput sequencing technology (Illumina MiSeq platform). The results showed that the structure of the microflora in the size of the Pinus armandii was relatively simple. The microbial community structure of the intestinal tract will change with the change of the overwintering period and the ambient temperature. the intestinal bacterial community of the overwintering larvae of the size of the Chinese mountain pine is mainly composed of a deformation fungus door (mainly of a strain-deformation bacterium), an actinomycete door, a thick-wall fungus door and a Bacteroides door, The intestinal bacterial community is more abundant than that collected in October and January, and the intestinal fungal community is composed of a joint fungus door, a basidiomycete door and a sub-sac fungus door, the sub-sac fungus door is an advantage fungus in the intestinal tract of the overwintering larvae, Which accounts for 81.11 percent of the total fungus richness. As a result, the intestinal bacteria and fungi of the large-sized larvae of the Chinese pine in the overwintering period play a certain protective role in the protection against the low-temperature environmental stress.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.38
，
本文编号：2495780
[Abstract]:The low temperature in winter is a key factor for determining the population of cold and temperate insects, and also determines the spread, distribution and space-time dynamics of the insect population. This study is aimed at studying the physiological and biochemical metabolic regulation of the large-size and large-sized larvae of the Chinese mountain in the overwintering period by studying the major forest pest _ Hua-shan pine-size (Dendranctonus armand i Tsai and Li) in the forest region of the Qinling Mountains. the expression of the cold-resistant and cold-resistant related genes of the Chinese mountain pine is different in different low temperature and time sequences, the low-temperature death rate after the cold-resistant related gene RNA of the overwintering larvae of the Chinese mountain and the overwintering larvae, and the changes of the intestinal fungi and the bacterial communities of the large-size and large-sized larvae of the overwintering stage, The low-temperature stress tolerance, the physiological and biochemical response at low temperature stress, the structure diversity of the intestinal fungi and the bacterial community of the overwintering larvae, and the effects of the cold-resistant and cold-resistant related genes of the Chinese mountain pine on the low-temperature environment are disclosed, and the following research results are obtained:1. The results show that the overwintering larva overcooling point (SCP) is positively correlated with the ambient temperature by studying the overwintering larvae overcooling point (SCP) and the low-temperature stress and death rate. In the coldest month (January), the overwintering larvae overcooling point (SCP) reached the lowest value, and the low-temperature tolerance of the overwintering larvae of the Pinus armandii was gradually enhanced with the decrease of the ambient temperature. And the cold resistance of the overwintering larvae of the size of the Chinese mountain pine can be improved by the low-temperature domestication. The results showed that the changes of the content of trehalose, sorbitol and glycerol in the overwintering period were negatively correlated with the over-cooling point (SCP), and the content of glycogen in the overwintering period was significantly decreased. In the later stage of overwintering, it was found that trehalose, sorbitol and glycerol are the potential anti-freezing protective substances in the larvae of the overwintering period, and the glycogen is an important energy substance in the larva of the large-sized and large-sized larvae of the overwintering period. the free water in the larvae during the overwintering period is converted into the combined water, the total water content rate is not large, and the combined water in the late winter is converted into free water, so that the large-size and overwintering larvae of the Chinese mountain pine are provided with a "water-saving" mechanism to improve the cold resistance of the insect body, and the activity of the SOD, the CAT, the LDH and the AchE in the period of October to March is continuously reduced, The activities of POD, PK and MDH were continuously enhanced, and the activity of SDH, TPS and GLK decreased during the overwintering period (October-January), and the activity of the spring remained relatively stable after the spring activity was enhanced. The differences of the expression of trehalose phosphate synthase (TPS), sorbitol dehydrogenase (SDH) and glycerol kinase (GLK) gene were studied by real-time fluorescence quantitative PCR (qRT-PCR) and RNA interference technique. The results showed that the expression of DarmTPS and DarmSDH was up-regulated and the expression of DarmGLK was down-regulated during the overwintering period. The expression of DarmTPS, DarmSDH and DarmGLK was significantly higher than that in May. The expression of DarmTPS-dsRNA, DarmSDH-dsRNA and DarmGLK-dsRNA showed a significant decrease in the gene expression, and the low-temperature mortality of the large-sized and large-sized larvae of the Chinese pine was significantly higher after the RNA interference, indicating that the size of the Pinus armandii, DarmSDH and DarmGLK play a very important role in controlling the cold resistance of the larvae. The expression of heat shock protein (DHSP90, DaHSP70 and DasHSP) and cold shock protein (DarmCSP) was studied by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the DaHSP90 gene was more sensitive to the cold shock response than that of the overwintering larvae, DaHSP90. The expression of the DaHSP90 and the DasHSP gene in the long-term temperature stimulated by the DaHSP90 and the DasHSP gene is more sensitive, and the expression level of the DaHSP90 and the DasHSP gene under the short-time temperature stimulation is significant. The expression of DarmCSP was induced by low-temperature stress, and the expression of DarmCSP was induced by low-temperature stress, and the DarmCSP was also up-regulated at low temperature. But the expression is lower than that of the overwintering larvae. In this paper, the structure, diversity and dynamic change of the intestinal microflora in the large-size and small-sized larvae of the overwintering period were analyzed by using the high-throughput sequencing technology (Illumina MiSeq platform). The results showed that the structure of the microflora in the size of the Pinus armandii was relatively simple. The microbial community structure of the intestinal tract will change with the change of the overwintering period and the ambient temperature. the intestinal bacterial community of the overwintering larvae of the size of the Chinese mountain pine is mainly composed of a deformation fungus door (mainly of a strain-deformation bacterium), an actinomycete door, a thick-wall fungus door and a Bacteroides door, The intestinal bacterial community is more abundant than that collected in October and January, and the intestinal fungal community is composed of a joint fungus door, a basidiomycete door and a sub-sac fungus door, the sub-sac fungus door is an advantage fungus in the intestinal tract of the overwintering larvae, Which accounts for 81.11 percent of the total fungus richness. As a result, the intestinal bacteria and fungi of the large-sized larvae of the Chinese pine in the overwintering period play a certain protective role in the protection against the low-temperature environmental stress.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S763.38
，
本文编号：2495780
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