華山松大小蠹幼蟲耐寒生化與分子機制
發(fā)布時間：2019-06-09 18:20
【摘要】：冬季低溫是決定寒、溫帶昆蟲種群繁衍和種群數(shù)量的關鍵性因素,也決定著昆蟲種群的擴散、分布和時空動態(tài)等一系列生命活動。本研究以秦嶺林區(qū)持續(xù)發(fā)生的重大森林害蟲——華山松大小蠹(Dendroctonus armandi Tsai and Li)為研究對象,旨在通過研究越冬期華山松大小蠹幼蟲生理生化代謝規(guī)律,華山松大小蠹耐寒相關基因在不同低溫和時間序列下的表達差異,華山松大小蠹越冬幼蟲耐寒相關基因RNA干擾后的低溫死亡率,以及越冬期華山松大小蠹幼蟲腸道真菌和細菌群落變化等,揭示越冬期華山松大小蠹幼蟲低溫脅迫耐受性,低溫脅迫下的生理生化響應,越冬幼蟲腸道真菌和細菌群落結構多樣性,以及華山松大小蠹耐寒相關基因抵御低溫環(huán)境的作用,取得以下研究結果:1.通過研究華山松大小蠹越冬幼蟲過冷卻點(SCP)和低溫脅迫死亡率,結果表明華山松大小蠹越冬幼蟲過冷卻點(SCP)與環(huán)境溫度顯著正相關,在最冷月(1月)華山松大小蠹越冬幼蟲過冷卻點(SCP)達到最低值;華山松大小蠹越冬幼蟲的低溫耐受性隨環(huán)境溫度的降低而逐漸增強,說明華山松大小蠹越冬幼蟲具有較強的耐寒能力,且華山松大小蠹越冬幼蟲的耐寒性可通過低溫馴化得以提高。2.通過分析華山松大小蠹越冬幼蟲生理生化指標和代謝酶活性,結果表明華山松大小蠹幼蟲越冬期海藻糖、山梨糖醇、甘油含量變化與過冷卻點(SCP)顯著負相關,越冬期糖原含量大幅降低,越冬后期恢復至越冬初期水平,說明海藻糖、山梨糖醇、甘油為越冬期華山松大小蠹幼蟲體內潛在的抗凍保護物質,糖原為越冬期華山松大小蠹幼蟲體內重要的能源物質;越冬期間幼蟲體內自由水轉化為結合水,總水含率變幅不大,越冬后期結合水轉化為自由水,表明華山松大小蠹越冬幼蟲具有“節(jié)水”機制,以提高蟲體耐寒性;10月~3月SOD、CAT、LDH和AchE酶活性持續(xù)減弱,POD、PK和MDH酶活性持續(xù)增強,SDH、TPS和GLK酶活性在越冬期間(10月~1月)減弱,春季活性增強后保持相對穩(wěn)定,說明華山松大小蠹主要代謝酶具有調節(jié)耐寒能力的作用。3.采用實時熒光定量PCR(qRT-PCR)和RNA干擾技術研究華山松大小蠹越冬幼蟲在不同低溫和時間序列下海藻糖磷酸合酶(TPS)、山梨糖醇脫氫酶(SDH)、甘油激酶(GLK)基因表達差異,結果表明越冬期華山松大小蠹幼蟲DarmTPS和DarmSDH表達量顯著上調,DarmGLK表達量下調。12月華山松大小蠹越冬蟲DarmTPS、DarmSDH和DarmGLK表達量顯著高于5月。注射DarmTPS-dsRNA、DarmSDH-dsRNA和DarmGLK-dsRNA的華山松大小蠹幼蟲基因表達量顯著降低,RNA干擾后華山松大小蠹幼蟲低溫死亡率明顯升高,表明華山松大小蠹DarmTPS、DarmSDH和DarmGLK在調控幼蟲耐寒性方面發(fā)揮著十分重要的作用。4.采用實時熒光定量PCR(qRT-PCR)技術研究華山松大小蠹越冬幼蟲在不同溫度梯度和時間序列下熱激蛋白(DaHSP90,DaHSP70和DasHSP)和冷休克蛋白(DarmCSP)基因表達差異,結果表明華山松大小蠹越冬幼蟲DaHSP90基因對冷激反應更敏感,DaHSP70基因對熱激反應更敏感;DaHSP90和DasHSP基因在長時溫度刺激下表達量變化顯著,DaHSP70基因在短時溫度刺激下表達量變化顯著。從華山松大小蠹cDNA轉錄組序列克隆得到1條華山松大小蠹冷休克蛋白基因序列DarmCSP,該基因在越冬期間表達量呈上調趨勢,且低溫脅迫能誘導DarmCSP表達;同時,越夏成蟲在低溫脅迫下DarmCSP也呈上調表達,但表達量低于越冬幼蟲。5.利用高通量測序技術(Illumina MiSeq平臺)分析越冬期華山松大小蠹幼蟲腸道微生物群落結構、多樣性和動態(tài)變化,結果表明華山松大小蠹腸道微生物群落結構相對簡單,腸道微生物群落結構會隨越冬期和環(huán)境溫度的變化發(fā)生改變。華山松大小蠹越冬幼蟲腸道細菌群落主要由變形菌門(主要是γ-變形菌)、放線菌門、厚壁菌門和擬桿菌門構成,采集于11、12月的華山松大小蠹幼蟲腸道細菌群落比采集于10月、1月的幼蟲腸道細菌群落更豐富;腸道真菌群落由接合菌門、擔子菌門和子囊菌門構成,子囊菌門是越冬幼蟲腸道內的優(yōu)勢真菌,占總真菌豐富度的81.11%。由此推測,越冬期華山松大小蠹幼蟲腸道細菌和真菌對抵御低溫環(huán)境脅迫起到了一定的保護作用。
[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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S763.38
，
本文編號：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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S763.38
，
本文編號：2495780
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