【作者】 李芒；

【导师】 雷朝亮；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2011， 博士

【摘要】 肚倍蚜Kaburagia rhusicola Takagi隶属于半翅目Hemiptera蚜总科Aphidoidea瘿绵蚜科Pemphigidae铁倍蚜属Kaburagia,是一种异寄主全周期与同寄主不全周期并存的复迁式生活周期型资源昆虫。肚倍是肚倍蚜在夏寄主上产生的虫瘿即五倍子,其单宁含量约为70%左右(干重),为了解肚倍蚜对虫瘿内高单宁环境的适应机理,本研究以肚倍蚜为试验材料,研究了肚倍蚜对高单宁虫瘿环境的生理生化反应及相关机理。主要结果如下：1.肚倍组织切片观察及单宁含量测定通过肚倍的切片观察,分析了肚倍蚜瘿内生活所取食的部位。结果表明,肚倍的外壁、中层和内壁组织结构有较大差异,肚倍的外层布满非腺毛,外层的细胞较小；肚倍中层的细胞相对较大；而肚倍内壁的细胞较小且排列松散。除组织形态上不同外,肚倍的中层和内壁在单宁含量上也存在较大差异,肚倍的中层单宁含量为30.0%左右,内壁为13.2%,而汁液中的含量为7.2%。肚倍蚜以刺吸的方式吸取单宁含量较低的植物汁液,这是一种对高单宁环境的行为学适应。2.肚倍蚜体内单宁酶的研究研究表明,在pH值3.8-6.2范围的条件下,未发现肚倍蚜上清蛋白中具有水解单宁的活性成分。在进一步研究中,对肚倍蚜上清液进行了纯化和分离,并对分离的不同组分进行了单宁酶活性检测,结果表明,肚倍蚜蛋白经离子吸附所产生三个峰中的蛋白均无单宁酶活性。3.肚倍蚜抗氧化反应研究为验证单宁是否对肚倍蚜具有氧化胁迫作用,本研究以春迁蚜为对照,检测了瘿内不同时期总抗氧化能力以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和谷胱甘肽-S-转移酶(GST)的活性,分析了肚倍蚜体内抗氧化系统对高单宁环境胁迫所产生的相关反应。研究表明,与对照相比,瘿内时期肚倍蚜体内的总抗氧化活力、CAT和POD活性均显著升高；SOD和GST活性除在有翅蚜秋迁蚜阶段与对照无差异,在其它阶段的活性均显著高于对照。试验表明高单宁环境对肚倍蚜造成氧化胁迫作用。4.肚倍蚜Lac-1基因全长cDNA克隆及序列分析利用RT-PCR和RACE技术从肚倍蚜中得到了Lac-1基因2,327bp的全长cDNA序列,该序列推定的蛋白序列由591个氨基酸残基构成。利用ProtParam软件分析,推测蛋白的分子量为67.3583 kDa,等电点为6.31。对氨基酸序列进行疏水性分析,说明其氨基酸序列具有亲水性。基于氨基酸序列全长,对Lac-1蛋白进行了多重序列比对,并构建了系统发育树。结果表明肚倍蚜Lac-1基因具有很高的保守性,含有典型的铜结合蛋白结构,肚倍蚜Lac-1基因与豌豆蚜Lac-1基因的同源性最高。这也证实本实验所得基因为肚倍蚜相应基因序列,并为深入了解昆虫在抗胁迫环境下的分子适应机制奠定了基础。5.肚倍蚜抗氧化基因和Lac基因表达研究为了解肚倍蚜中肠相关基因在高单宁环境中的相应变化,克隆了肚倍蚜的β-Actin、Cu-Sod、Mn-Sod、Cat、Tpx和Gst基因片段,采用了荧光定量技术分析Cu-Sod、Mn-Sod、Cat、Tpx、Gst和Lac-1基因在肚倍蚜中肠组织的相对表达情况。结果表明,与对照春迁蚜相比,不同时期肚倍蚜中肠组织的Cu-Sod、Mn-Sod、Cat、Tpx、Gst和Lac-1基因表达差异显著。其中,Cu-Sod、Mn-Sod、Cat、Tpx和Gst所组成的抗氧化体系,在一代干雌、二代干雌和无翅秋迁蚜时期相对表达量较高,而Lac-1基因则在无翅秋迁蚜和有翅秋迁蚜时期相对表达量较高,这验证了肚倍蚜体内的相关基因在表达水平上对瘿内高单宁环境的应答表现,说明这些基因在肚倍蚜适应高单宁环境中起到了重要作用。更多还原

【Abstract】 The du-ensiform gall aphid, Kaburagia rhusicola Takagi (Hemiptera:Pemphigidae) is an important economic insect whose non-migratory and migratory life cycle are coexist in, the du-ensiform gallunts are rich in tannin concentration which is maintained at approximately 70% of dry weight and induced by K. rhusicola in the summer host. In this study, to clarify the mechanism for K. rhusicola to adapt to high tannin in gall environment, basic works about physiological and biochemical response and hydrolyzed protein were done. Main results were summarized as following:1. Observation on du-ensiform gallnuts slice and research on tanninconcentrationAfter making the slice of the gallnut and detecting the tannin concentration in different parts of the gallnut, we analyse the position which the galling aphid feeding in and the tannin concentration that aphid intake in. It showed that there were significent difference among the outer epideral, intraepidermal and middle layer of gallnut from microscope, the outer epideral covered with non-glandular hairs; the cells of middle layer were large than others and were closely linked; the cells in intraepidermal were loosely arranged. There also existed significent difference in tannin concentration among different parts of the gallnuts, the tannin concentration in middle layer of gallnut is about 30.0%, intraepidermal is 13.2% and the juice is 7.2%, the results indicated that the tannin concentrations from different parts of gallnuts were not at the same level, the aphids may intake lower concentration from the juice which is a adaptation to high tannin environment.2. Research on tannase in K. rhusicolaIn our study, we did not find any ingredient about tannase in the aphids’supernatant in the range of 3.8-6.2 pH condition. Afetr purifying and separating the aphids’ supernatant protein, we detected tannase in the three separation proteins and did not find any activity about the hydrolase yet.3. Response of the antioxidant system of K. rhusicola in gall environmentTo test the hypothesis that the effect of high tannin environment is to increase oxidative stress on the aphids. We treat spring migrant as contrast and measured the total antioxidant capacity and antioxidant enzymes activities of SOD, CAT, POD and GST in adult K rhusicola of different generation and analyse the response of the antioxidant system of K. rhusicola in the high tannin environment. We found that the activities of SOD and GST in aphid generations living within galls were enhanced except in AM generation when compared to control, T-AOC, CAT and POD activities increased over the course of the gall time. Our results show that the high tannin environment increase the level of oxidative stress in K. rhusicola.4. Cloning and sequence of cDNA of laccase-1 from K. rhusicolaThe cDNA of Lac-1 gene was cloned from K. rhusicola by means of reverse transcriptase and polymerase chain (RT-PCR) and 5’and 3’-rapid amplification of cDNA ends (RACE) analyses. The cDNA is 2,327 bp in length and encodes a putative protein of 591 amino acids (m.w.67.3583 kDa, pI 6.31). Multiple sequence alignments indicate that the deduced amino acid sequences of K. rhusicola show very high homology to their corresponding sequences in other species.The phylogenetic analysis of laccase sequences was consistent with well known classification of insects.5. Transcriptional analysis of antioxidant and detoxification genes in K. rhusicolaThe cDNA ofβ-Actin, Cu-Sod, Mn-Sod, Cat, Tpx and Gst genes were cloned from K. rhusicola by means of reverse transcriptase and polymerase chain (RT-PCR). In this study, we used quantitative real-time PCR method to describe the transcriptional and analysis of antioxidant genes in the aphids’midgut of different generations. The results showed that copper zinc superoxide dismutase (SOD-1), manganese superoxide dismutase (SOD-2), catalase (CAT), thioredoxin peroxidase (TPx), Glutathione-S-transferase (GST) and laccase-1 (LAC-1) transcripts were expressed differently in the midgut of different generations living in gall environment. Sod-1, Sod-2, Cat, Tpx and Gst established an antioxidant defense system and expressed at high levels in 1st and 2nd fundatrices, Lac-1 gene expressed at a high level in 3rd fundatrices when the antioxidant enzymes genes were low.Base upon these results, we postulate that K. rhusicola utilize antioxidant and phenol detoxification mechanisms together in the midgut to overcome the high phenolic environment.更多还原