本文選題：禾谷縊管蚜 + 基因克隆　； 參考：《西北農(nóng)林科技大學》2016年博士論文

【摘要】：禾谷縊管蚜Rhopalosiphum padi(L.)屬于半翅目Hemiptera蚜科Aphididae,是世界性的重要小麥害蟲之一,其通過刺吸小麥汁液危害,分泌蜜露引起煤污病,影響小麥生長發(fā)育;而且還能傳播大麥黃矮病毒(Barley yellow dwarf virus,BYDV),使小麥葉色變黃,植物矮化,分蘗減少;該蟲危害給世界各地的小麥生產(chǎn)造成嚴重損失。禾谷縊管蚜在我國各小麥種植區(qū)均有分布,其較耐高溫,在長江流域和黃河流域麥區(qū)危害較重,但是近年來其嚴重危害區(qū)域逐漸向北方擴大,在我國北方許多小麥種植區(qū)的危害逐漸加重,這可能與氣溫變化等因素有關(guān)。全球氣候變暖增加了禾谷縊管蚜在北方以孤雌蚜越冬的可能,從而影響早春的蟲口基數(shù)和危害程度;全球變暖條件下,我國南北各地氣溫升高,經(jīng)常出現(xiàn)不正常的溫度驟升與驟降以及不正常的酷熱與嚴寒。昆蟲對溫度脅迫的響應和耐受性對其種群動態(tài)、發(fā)生規(guī)律以及分布和擴散能力有著重要的作用。昆蟲溫度適應性研究是當前昆蟲學研究中的一個重要分支,包括昆蟲對溫度脅迫的響應、溫度耐受性以及對環(huán)境的適應性進化,相關(guān)研究與昆蟲的種群動態(tài)、發(fā)生分布、地理擴散能力以及種群替代密切相關(guān),能夠為全球變暖趨勢下害蟲發(fā)生的預測預報、地理種群擴張以及合理防治策略的制定提供理論依據(jù)。本論文研究了高溫脅迫對禾谷縊管蚜存活率的影響以及短時高溫對禾谷縊管蚜存活、繁殖和后代生長發(fā)育的影響;克隆了7個禾谷縊管蚜熱激蛋白基因;分析了5個Hsp基因在禾谷縊管蚜不同翅型、生長發(fā)育以及生殖方式中的表達模式;研究了高溫脅迫后禾谷縊管蚜5個Hsp基因的表達變化;明確熱鍛煉和冷馴化對禾谷縊管蚜溫度耐受性和熱激蛋白基因表達的影響以及熱鍛煉對溫度耐受性、繁殖力和熱激蛋白基因表達的綜合影響;分析了高效氯氰菊酯誘導對熱激蛋白基因表達的影響。主要研究結(jié)果如下:一、短時高溫對禾谷縊管蚜存活及生殖的影響本研究結(jié)果顯示,無翅和有翅孤雌成蚜存活率隨脅迫溫度的升高和脅迫時間的延長而降低,并且無翅孤雌成蚜較有翅孤雌成蚜耐高溫。短時高溫處理后,禾谷縊管蚜當代成蚜的壽命顯著縮短,且產(chǎn)蚜量顯著降低;F1代成蚜的壽命無顯著變化,但36℃和38℃處理后F1代成蚜的產(chǎn)蚜量顯著少于對照和37℃處理。38℃處理后禾谷縊管蚜F1代種群的發(fā)育歷期顯著延長,凈增殖率r0、內(nèi)稟增長率rm和周限增長率λ顯著低于對照,而平均世代周期t則顯著高于對照組。本研究表明,短時高溫處理對禾谷縊管蚜當代種群具有顯著的不利影響,并且38℃短時高溫處理明顯抑制禾谷縊管蚜f1代種群增長。二、禾谷縊管蚜hsp基因的克隆及序列分析本研究成功從禾谷縊管蚜中克隆到7個hsp基因(rphsp60、rphsc70、rphsp70-1、rphsp70-2、rphsc70-3、rphsc70-5和rphsp90),氨基酸序列分析顯示,7個hsp基因分別具有hsp60家族,hsp70家族和hsp90家族的特征序列,其中rphsp60和rphsc70-5屬于線粒體熱激蛋白,rphsc70,rphsp70-1,rphsp70-2和rphsp90屬于細胞質(zhì)熱激蛋白,而rphsc70-3為內(nèi)質(zhì)網(wǎng)hsp70基因。序列相似性分析表明,7個禾谷縊管蚜熱激蛋白具有高度的保守性,并且與蚜科其他蚜蟲的氨基酸序列在進化上關(guān)系較近。三、禾谷縊管蚜hsp基因在不同翅型、發(fā)育階段和生殖方式的相對表達量分析禾谷縊管蚜5個hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)的相對表達量在不同翅型、生長發(fā)育階段、生殖方式和蚜型中均表達,但差異顯著。rphsp70-2表達量在不全周期有翅孤雌蚜中顯著高于無翅孤雌蚜,而5個hsp基因表達量在全周期型禾谷縊管蚜兩個翅型中無顯著性差異,但rphsp70-1、rphsp70-2和rphsp90表達量在全周期型無翅和有翅孤雌蚜中顯著高于不全周期型;rphsp60在若蚜期高表達;rphsc70在不同蚜型間表達量差異不顯著,rphsp70-1和rphsp70-2在禾谷縊管蚜雄蚜中表達量最高顯著高于其他蚜型,而rphsp90在禾谷縊管蚜卵的相對表達量最高。分析認為,5個hsp基因的表達與禾谷縊管蚜生長發(fā)育和生殖方式等密切相關(guān),hsp基因在維持生命活動中發(fā)揮不同的作用。四、熱脅迫下禾谷縊管蚜hsp基因的表達分析本研究表明,熱脅迫后5個hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)均上調(diào)表達。熱脅迫1h后,有翅孤雌蚜hsp基因在36℃處理后表達量最高,無翅孤雌蚜則在37℃或38℃處理后表達量達到最大值,而39℃和40℃脅迫下hsp基因表達量較37℃和38℃處理時下降,說明hsp基因?qū)囟让{迫的響應有限度;36℃處理不同時間后,5個hsp基因的表達量雖有不同程度的變化,但表達水平基本穩(wěn)定。此外,5個hsp基因?qū)崦{迫的響應強度不同:rphsp70-1和rphsp70-1對熱脅迫響應最強烈,rphsp90次之,rphsp60和rphsc70對熱脅迫響應較溫和。五、溫度預處理對禾谷縊管蚜hsp基因表達量的影響熱鍛煉后再經(jīng)歷高溫脅迫的存活率顯著提高,并且顯著上調(diào)hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)的表達量,但長時冷馴化后hsp基因表達模式不同。熱激處理后rphsp的表達量雖上調(diào)但與對照差異不顯著,熱鍛煉和熱鍛煉后熱激處理均顯著誘導5個hsp基因的上調(diào);冷馴化后禾谷縊管蚜兩個翅型rphsp60和rphsc70表達量較對照無顯著變化,對冷馴化不敏感,rphsp90表達量受冷馴化誘導表達,而冷馴化后RpHsp70-1和RpHsp70-2表達量顯著下調(diào),表明5個Hsp基因在低溫適應中發(fā)揮不同作用。六、禾谷縊管蚜耐熱能力和生殖力間的平衡本研究通過研究禾谷縊管蚜溫度處理后的溫度耐受性、生殖力和熱激蛋白基因的表達,結(jié)果表明,Hsp基因在增強禾谷縊管蚜溫度耐受性中具有重要作用。溫度馴化顯著提高禾谷縊管蚜無翅和有翅孤雌蚜的耐熱能力,但對抗寒能力無明顯提高;溫度處理后禾谷縊管蚜無翅和有翅孤雌成蚜的前10天總產(chǎn)蚜量顯著減少,說明溫度馴化明顯抑制了生殖能力;溫和的溫度馴化顯著誘導RpHsp70-1、RpHsp70-2和RpHsp90基因的表達,一方面提高了禾谷縊管蚜的耐熱能力,另一方面卻抑制了成蚜的生殖能力。七、亞致死濃度高效氯氰菊酯對禾谷縊管蚜Hsp基因表達量的影響高效氯氰菊酯亞致死濃度LC10(0.3987 mg/L)和LC30(0.9280 mg/L)誘導處理12h、24 h和36 h后,禾谷縊管蚜5個Hsp基因(Rp Hsp60,Rp Hsc70,Rp Hsp70-1,RpHsp70-2和Rp Hsp90)具有不同的響應模式。其中,RpHsp60在處理后表達量顯著下調(diào)且處理后不同時間表達量差異不顯著;高效氯氰菊酯脅迫誘導其余4個Hsp基因的上調(diào)表達,在處理后24 h表達量最高,且對不同濃度的響應程度不同。除RpHsp60外,5個Hsp基因?qū)Ω咝惹杈挣ッ{迫的響應具有時間和濃度的依賴性。這4個Hsp基因?qū)Ω咝惹杈挣サ捻憫獜姸让黠@低于高溫脅迫,推測Hsp基因?qū)Ω咝惹杈挣ズ蜏囟让{迫的響應機制不同。
[Abstract]:Rhopalosiphum PADI (L.), which belongs to the family of Hemiptera, Hemiptera aphid Aphididae, is one of the most important wheat pests in the world. It is harmful to wheat juice by sucking the juice of wheat, secreting honeydew causes coal pollution and affects the growth and development of wheat, and it can also spread the barley yellow dwarf virus (Barley yellow dwarf virus, BYDV), and make the wheat leaf color yellow and plant. Dwarf and tiller decrease; the insect damage to wheat production in all parts of the world is serious loss. The cereal constriction aphid is distributed in all wheat planting areas in China. It is more endanger in high temperature and in the wheat area of the Yangtze River Basin and the the Yellow River basin. But in recent years, the serious harm region has gradually expanded to the north in the north of China, and in many wheat planting areas in the north of China. Global warming increases the number and extent of the insect's mouth in the north, which affects the number of insects in the north and the degree of harm in the early spring. The response and tolerance of insects to temperature stress and tolerance play an important role in its population dynamics, regularity, distribution and diffusion. The study of insect temperature adaptation is an important branch of entomological studies, including response to temperature stress, temperature tolerance and adaptation to the environment. Sexual evolution, related studies are closely related to insect population dynamics, distribution, geographical diffusion and population substitution. It can provide a theoretical basis for predicting the occurrence of pests in the trend of global warming, geographical population expansion and the formulation of reasonable control strategies. Effects of short time high temperature on the survival, reproduction and growth of offspring of the aphid, cloned 7 cereal constrictions heat shock protein gene, and analyzed the expression patterns of 5 Hsp genes in the different wings, growth and reproductive mode of the cereal aphid, and the expression of 5 Hsp genes in the cereal aphid after high temperature stress. The effects of heat exercise and cold acclimatization on the temperature tolerance and heat shock protein gene expression of cereal constrictions and the effect of heat exercise on temperature tolerance, fecundity and gene expression of heat shock protein were determined. The effects of Cypermethrin induced gene expression on heat shock protein gene expression were analyzed. The results of the study on the survival and reproduction of the aphids showed that the survival rate of wingless and winged female aphids decreased with the increase of the stress temperature and the prolongation of the stress time, and the aphis Aphis was more resistant to high temperature than that of the aphis APHIS. There was no significant change in the life span of F1 aphid, but the aphid yield of aphids of F1 generation aphid was significantly less than that of control at 36 and 38 C, and the growth period of F1 generation of cereal constriction aphid was significantly prolonged, the net proliferation rate was R0, the intrinsic growth rate RM and the circumferential growth rate were significantly lower than those of the control, while the average generation period T was significantly higher than that of the control. The average generation period T was significant. Compared with the control group, this study showed that short time high temperature treatment had significant adverse effects on the contemporary population of cereal constriction aphid, and the high temperature treatment at 38 C significantly inhibited the growth of F1 generation of cereal aphid. Two, the cloning and sequence analysis of the hsp gene of cereal constriction aphid was cloned to 7 HSP genes from the cereal constriction aphid (rphsp6 0, rphsc70, rphsp70-1, rphsp70-2, rphsc70-3, rphsc70-5 and rphsp90). The analysis of amino acid sequences showed that the 7 HSP genes were characterized by the Hsp60 family, the Hsp70 family and the Hsp90 family, and rphsp60 and rphsc70-5 belong to the mitochondrial heat shock protein. The sequence similarity analysis of the endoplasmic reticulum HSP70 gene showed that the heat shock protein of 7 cereal constrictions was highly conserved and closely related to the amino acid sequence of other aphid aphids. Three, the relative expression of the hsp gene of the cereal constriction aphid in the different wings, developmental stages and reproductive formula was 5 HSP bases of the cereal constriction aphid. The relative expressions of (rphsp60, rphsc70, rphsp70-1, rphsp70-2 and rphsp90) were expressed in the different wings, growth stages, reproductive mode and aphid type, but the difference of the significant.Rphsp70-2 expression was significantly higher than that of the apapid Aphis Aphis in the incomplete period of the aphis aphis, and the 5 HSP gene expression was two wings of the full cycle grasses. There was no significant difference, but the expression of rphsp70-1, rphsp70-2 and rphsp90 was significantly higher in the full cycle type of wingless and winged female aphids, and the expression of rphsc70 was not significant in the aphid stage, and the expression of rphsc70 in different aphids was not significant. The highest expression of rphsp70-1 and rphsp70-2 in the male aphids of the cereal aphid was significantly higher than that of other aphids. The relative expression of rphsp90 in the oocytes of cereal constrictions was the highest. The analysis suggested that the expression of the 5 HSP genes was closely related to the growth and reproductive mode of the cereal constriction aphid, and the hsp gene played a different role in the maintenance of life activities. Four, the expression analysis of the HSP base of the cereal constriction aphid under heat stress showed that 5 HSP after heat stress Genes (rphsp60, rphsc70, rphsp70-1, rphsp70-2 and rphsp90) were all up-regulated. After heat stress 1H, the expression of HSP gene of winged female aphid was the highest after treatment at 36. No winged female aphid had reached the maximum value after treatment at 37 or 38 C, while the expression of HSP gene was decreased at 39 and 40 degrees centigrade than at 37 and 38 C, indicating the hsp gene. The response to temperature stress was limited. The expression levels of the 5 HSP genes were changed at different times at 36 degrees, but the expression level was basically stable. In addition, the response intensity of 5 HSP genes to heat stress was different: rphsp70-1 and rphsp70-1 had the strongest response to heat stress, rphsp90 times, and rphsp60 and rphsc70 responses to heat stress. Mild. Five, the effect of temperature pretreatment on the expression of HSP gene in cereal constriction aphid, the survival rate of high temperature stress increased significantly after heat exercise, and the expression of HSP gene (rphsp60, rphsc70, rphsp70-1, rphsp70-2 and rphsp90) was significantly up-regulated, but the expression pattern of HSP gene was different after long time cold acclimation. The expression of rphsp after heat shock treatment was observed. Although there was no significant difference between the 5 HSP genes after hot exercise and hot exercise, the expression of rphsp60 and rphsc70 in rphsp60 and rphsc70 was not significantly changed after cold acclimatization, and was insensitive to cold acclimatization. The expression of rphsp90 was induced by cold acclimation, and RpHsp70-1 and RpHsp70- after cold acclimation. The 2 expression decreased significantly, indicating that the 5 Hsp genes played different roles in the low temperature adaptation. Six, the balance between the heat tolerance and the reproductive capacity of the cereal constriction aphid was studied by the study of the temperature tolerance, the reproductive capacity and the expression of the heat shock protein gene after the temperature treatment of the cereal constriction aphid, and the results showed that the Hsp gene was more tolerant to the temperature of the cereal aphid. The temperature domestication significantly improved the heat resistance of the wingless and winged aphid aphids, but had no obvious improvement in the cold resistance; the total yield of the aphids without wingless and wing parthenogenetic aphids decreased significantly in the first 10 days after temperature treatment, indicating that temperature domestication inhibited reproductive capacity and temperate taming of temperature. The expression of RpHsp70-1, RpHsp70-2 and RpHsp90 genes significantly increased the heat tolerance of cereal constrictions, on the other hand, inhibition of the reproductive capacity of aphids. Seven, the sublethal concentration of Cypermethrin on the Hsp gene expression of cereal constricon (0.3987 mg/L) and LC30 (0.92) (0.92) 80 mg/L) after induction treatment of 12h, 24 h and 36 h, 5 Hsp genes (Rp Hsp60, Rp Hsc70, Rp Hsp70-1) had different response patterns. The expression of 24 h was highest after treatment, and the response degree to different concentrations was different. Except RpHsp60, the response of 5 Hsp genes to cypermethrin stress was dependent on time and concentration. The response intensity of the 4 Hsp genes to cypermethrin was significantly lower than that of high temperature stress, and the Hsp gene was conjectured to the cypermethrin and temperature of high performance cypermethrin. The response mechanism of degree stress is different.
【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】：2016
【分類號】：S435.122

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