禾谷缢管蚜对温度胁迫的响应及分子机制

发布时间：2018-06-29 05:47

本文选题：禾谷缢管蚜 + 基因克隆　；参考：《西北农林科技大学》2016年博士论文

【摘要】：禾谷缢管蚜Rhopalosiphum padi(L.)属于半翅目Hemiptera蚜科Aphididae,是世界性的重要小麦害虫之一,其通过刺吸小麦汁液危害,分泌蜜露引起煤污病,影响小麦生长发育;而且还能传播大麦黄矮病毒(Barley yellow dwarf virus,BYDV),使小麦叶色变黄,植物矮化,分蘖减少;该虫危害给世界各地的小麦生产造成严重损失。禾谷缢管蚜在我国各小麦种植区均有分布,其较耐高温,在长江流域和黄河流域麦区危害较重,但是近年来其严重危害区域逐渐向北方扩大,在我国北方许多小麦种植区的危害逐渐加重,这可能与气温变化等因素有关。全球气候变暖增加了禾谷缢管蚜在北方以孤雌蚜越冬的可能,从而影响早春的虫口基数和危害程度;全球变暖条件下,我国南北各地气温升高,经常出现不正常的温度骤升与骤降以及不正常的酷热与严寒。昆虫对温度胁迫的响应和耐受性对其种群动态、发生规律以及分布和扩散能力有着重要的作用。昆虫温度适应性研究是当前昆虫学研究中的一个重要分支,包括昆虫对温度胁迫的响应、温度耐受性以及对环境的适应性进化,相关研究与昆虫的种群动态、发生分布、地理扩散能力以及种群替代密切相关,能够为全球变暖趋势下害虫发生的预测预报、地理种群扩张以及合理防治策略的制定提供理论依据。本论文研究了高温胁迫对禾谷缢管蚜存活率的影响以及短时高温对禾谷缢管蚜存活、繁殖和后代生长发育的影响;克隆了7个禾谷缢管蚜热激蛋白基因;分析了5个Hsp基因在禾谷缢管蚜不同翅型、生长发育以及生殖方式中的表达模式;研究了高温胁迫后禾谷缢管蚜5个Hsp基因的表达变化;明确热锻炼和冷驯化对禾谷缢管蚜温度耐受性和热激蛋白基因表达的影响以及热锻炼对温度耐受性、繁殖力和热激蛋白基因表达的综合影响;分析了高效氯氰菊酯诱导对热激蛋白基因表达的影响。主要研究结果如下:一、短时高温对禾谷缢管蚜存活及生殖的影响本研究结果显示,无翅和有翅孤雌成蚜存活率随胁迫温度的升高和胁迫时间的延长而降低,并且无翅孤雌成蚜较有翅孤雌成蚜耐高温。短时高温处理后,禾谷缢管蚜当代成蚜的寿命显著缩短,且产蚜量显著降低;F1代成蚜的寿命无显著变化,但36℃和38℃处理后F1代成蚜的产蚜量显著少于对照和37℃处理。38℃处理后禾谷缢管蚜F1代种群的发育历期显著延长,净增殖率r0、内禀增长率rm和周限增长率λ显著低于对照,而平均世代周期t则显著高于对照组。本研究表明,短时高温处理对禾谷缢管蚜当代种群具有显著的不利影响,并且38℃短时高温处理明显抑制禾谷缢管蚜f1代种群增长。二、禾谷缢管蚜hsp基因的克隆及序列分析本研究成功从禾谷缢管蚜中克隆到7个hsp基因(rphsp60、rphsc70、rphsp70-1、rphsp70-2、rphsc70-3、rphsc70-5和rphsp90),氨基酸序列分析显示,7个hsp基因分别具有hsp60家族,hsp70家族和hsp90家族的特征序列,其中rphsp60和rphsc70-5属于线粒体热激蛋白,rphsc70,rphsp70-1,rphsp70-2和rphsp90属于细胞质热激蛋白,而rphsc70-3为内质网hsp70基因。序列相似性分析表明,7个禾谷缢管蚜热激蛋白具有高度的保守性,并且与蚜科其他蚜虫的氨基酸序列在进化上关系较近。三、禾谷缢管蚜hsp基因在不同翅型、发育阶段和生殖方式的相对表达量分析禾谷缢管蚜5个hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)的相对表达量在不同翅型、生长发育阶段、生殖方式和蚜型中均表达,但差异显著。rphsp70-2表达量在不全周期有翅孤雌蚜中显著高于无翅孤雌蚜,而5个hsp基因表达量在全周期型禾谷缢管蚜两个翅型中无显著性差异,但rphsp70-1、rphsp70-2和rphsp90表达量在全周期型无翅和有翅孤雌蚜中显著高于不全周期型;rphsp60在若蚜期高表达;rphsc70在不同蚜型间表达量差异不显著,rphsp70-1和rphsp70-2在禾谷缢管蚜雄蚜中表达量最高显著高于其他蚜型,而rphsp90在禾谷缢管蚜卵的相对表达量最高。分析认为,5个hsp基因的表达与禾谷缢管蚜生长发育和生殖方式等密切相关,hsp基因在维持生命活动中发挥不同的作用。四、热胁迫下禾谷缢管蚜hsp基因的表达分析本研究表明,热胁迫后5个hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)均上调表达。热胁迫1h后,有翅孤雌蚜hsp基因在36℃处理后表达量最高,无翅孤雌蚜则在37℃或38℃处理后表达量达到最大值,而39℃和40℃胁迫下hsp基因表达量较37℃和38℃处理时下降,说明hsp基因对温度胁迫的响应有限度;36℃处理不同时间后,5个hsp基因的表达量虽有不同程度的变化,但表达水平基本稳定。此外,5个hsp基因对热胁迫的响应强度不同:rphsp70-1和rphsp70-1对热胁迫响应最强烈,rphsp90次之,rphsp60和rphsc70对热胁迫响应较温和。五、温度预处理对禾谷缢管蚜hsp基因表达量的影响热锻炼后再经历高温胁迫的存活率显著提高,并且显著上调hsp基因(rphsp60,rphsc70,rphsp70-1,rphsp70-2和rphsp90)的表达量,但长时冷驯化后hsp基因表达模式不同。热激处理后rphsp的表达量虽上调但与对照差异不显著,热锻炼和热锻炼后热激处理均显著诱导5个hsp基因的上调;冷驯化后禾谷缢管蚜两个翅型rphsp60和rphsc70表达量较对照无显著变化,对冷驯化不敏感,rphsp90表达量受冷驯化诱导表达,而冷驯化后RpHsp70-1和RpHsp70-2表达量显著下调,表明5个Hsp基因在低温适应中发挥不同作用。六、禾谷缢管蚜耐热能力和生殖力间的平衡本研究通过研究禾谷缢管蚜温度处理后的温度耐受性、生殖力和热激蛋白基因的表达,结果表明,Hsp基因在增强禾谷缢管蚜温度耐受性中具有重要作用。温度驯化显著提高禾谷缢管蚜无翅和有翅孤雌蚜的耐热能力,但对抗寒能力无明显提高;温度处理后禾谷缢管蚜无翅和有翅孤雌成蚜的前10天总产蚜量显著减少,说明温度驯化明显抑制了生殖能力;温和的温度驯化显著诱导RpHsp70-1、RpHsp70-2和RpHsp90基因的表达,一方面提高了禾谷缢管蚜的耐热能力,另一方面却抑制了成蚜的生殖能力。七、亚致死浓度高效氯氰菊酯对禾谷缢管蚜Hsp基因表达量的影响高效氯氰菊酯亚致死浓度LC10(0.3987 mg/L)和LC30(0.9280 mg/L)诱导处理12h、24 h和36 h后,禾谷缢管蚜5个Hsp基因(Rp Hsp60,Rp Hsc70,Rp Hsp70-1,RpHsp70-2和Rp Hsp90)具有不同的响应模式。其中,RpHsp60在处理后表达量显著下调且处理后不同时间表达量差异不显著;高效氯氰菊酯胁迫诱导其余4个Hsp基因的上调表达,在处理后24 h表达量最高,且对不同浓度的响应程度不同。除RpHsp60外,5个Hsp基因对高效氯氰菊酯胁迫的响应具有时间和浓度的依赖性。这4个Hsp基因对高效氯氰菊酯的响应强度明显低于高温胁迫,推测Hsp基因对高效氯氰菊酯和温度胁迫的响应机制不同。
[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.
【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】：2016
【分类号】：S435.122

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