麦长管蚜Cdc42基因的组织定位与定量分析以及保幼激素对翅型分化的影响
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摘要
麦长管蚜翅型具有二型现象,有翅型和无翅型之分。翅型分化是一个受多因子影响的调控体系决定的,二型的比例随内因和外界环境的变化而变化。因此利用分子生物学手段从基因水平上开展蚜虫翅型分化的机理研究,对丰富麦蚜基础生物学具有重要意义,并为今后基于蚜虫翅型分化调控研发新的蚜虫防控措施奠定理论基础。
本文以麦长管蚜翅型分化相关基因Cdc42为对象,采用原位杂交技术和实时荧光定量RT-PCR技术确定该基因在麦长管蚜中的组织定位及相对表达量。并通过体外涂抹保幼激素及其类似物测定其对麦长管蚜的翅型分化的调控作用。取得如下研究结果:
采用原位杂交技术,对Cdc42基因在二型麦长管蚜中的分布进行组织定位,结果表明在滴加杂交液的组织切片被特异性的染成棕褐色,而阴性对照的组织切片没有被染色。由此表明原位杂交技术可以灵敏检测到Cdc42基因在该蚜虫不同组织中的表达状况。原位杂交结果显示,Cdc42基因在有翅蚜和无翅蚜的头部和腹部均有表达,头部的表达集中在两复眼之间,腹部的表达几乎全集中在伪胚胎;有翅蚜的胸部几乎全部都有表达,根据着色点数量推断有翅蚜的胸部表达量最高;而无翅蚜的胸部几乎没有表达。
再运用实时荧光定量RT-PCR技术确定该基因的相对表达量,结果表明Cdc42基因在麦长管蚜不同组织及不同龄期均有表达,若蚜期高于成蚜期,有翅蚜高于无翅蚜,该基因在有翅蚜与无翅蚜胸部的表达成显著差异;从伪胚胎开始就有较高的转录水平。
对2龄若蚜经体外涂抹保幼激素JHⅢ及其类似物ZR-515的滴度效益结果表明,随着激素浓度的增加,无翅和有翅麦长管蚜成虫整虫Cdc42相对表达量逐渐减低,均低于正常有翅蚜的表达量,无翅蚜中的表达量只有正常表达量的千分之一,各浓度之间差异极显著;并且无翅型麦长管蚜成虫的个体数量不断增加,有翅蚜数量逐渐减少,各浓度之间差异极显著。
本研究为探讨建立微型昆虫基因的准确组织定位和定量分析方法奠定了基础,也为深入开展保幼激素、Cdc42基因与翅型分化的关系提供了理论依据。
The English grain aphid, Sitobion avenae, is of wing dimorphism, namely, winged and wingless aphid. The aphid’s wing differentiation is manipulated by a complicate factors system, and the ratio of the wing dimorphism depends on the intrinsic and external environment factors. So in order to uncover the mechanism of aphid’s wing differentiation, through the related genes expression level by molecular biology approach, which will be very important to enrich the basical biology of aphid, and also benefit to further develop new method to control the aphid based on the knowledge of the manipulation of aphid’s wing differentiation.
In this experiment thesis, Cdc42 gene tissue location and accurate relative expression levels of S. avenae were determined by using in situ hybridization (ISH) and real-time fluorescence quantitative PCR method to determine the Cdc42 gene in the location and relative expression levels of the organization in Sitobion avenae. Then the change dynamic of Cdc42 gene expression, and population and the ratio of alatae and apterae aphid were detected with dose variation of juvenile hormone (JH) and its analogues by smearing 2 instar nymphae aphid. The aim is to understand preliminarily for the interaction of JH in vitro, the Cdc42 gene expression, and the function of manipulation of wheat aphid’s wing dimorphism. The major results are as follows:
First, ISH results showed that drip liquid hybridization specificity of tissue biopsies were specific stained in brown with nucleic acid probe of Cdc42 gene, while without the same staining characteristic on the negative control, which indicated that ISH could be sensitive to identify the tissue locaton of the gene in the aphid. ISH results showed that Cdc42 expressed in the head and abdomen parts of the aphid, and focused between the two complicated eyes by aphid’s head.In the abdomen the gene expressions were almost concentrated the pseudo-embryos. According to the brown staining. The Cdc42 gene specific stainging spread all over on the alate thorax part, and it was deduced by the number staining dot, that the highest expression level occurred on the thorax part of alate aphids, but hardly not expressed on the thorax part of apterae aphid.
Tissue and stage specific transcriptive levels were determined by real-time fluorescence quantitative PCR and showed that Cdc42 was transcripted in multiples tissues and at different stages. There was higher transcriptive level in the nymphae stages than that in adult stage, and higher in alate aphid than that of apterous one, and significantly higher on the thorax part of alate than that in apterae aphid. The gene’s expression start up at the aphid’s pseudo-embryo stage, and even with very high expressed level in the stage.
Application juvenile hormone (JH) and its analogues ZR-515 on aphid’s thorax in vitro by three concentrations, to study the impact of JH dose on the expression of Cdc42 gene and wing differentiation, the results showed that with the dose increasing of JHⅢand its analogues ZR-515, the expression level of Cdc42 gene in the whole part of alate and apterae aphid decreasing gradually, which were significant lower in the three treated concentration than that in control of alate aphid, by real-time fluorescence quantitative PCR detection, and the expression level of Cdc42 in the treated apterae aphid was only one thousandth of the level compared with aphid in control.There were also significant difference among the different treatments. The number of apterae adults increasing, but the number of alate aphids decreasing under the JH and ZR-515 treatments.
In this thesis, a micro-insect gene tissue localization and quantitative analysis, method has been established, and the results provide a primary data for the interaction of JH, Cdc42 gene and wing differentiation in aphid.
本文以麦长管蚜翅型分化相关基因Cdc42为对象,采用原位杂交技术和实时荧光定量RT-PCR技术确定该基因在麦长管蚜中的组织定位及相对表达量。并通过体外涂抹保幼激素及其类似物测定其对麦长管蚜的翅型分化的调控作用。取得如下研究结果:
采用原位杂交技术,对Cdc42基因在二型麦长管蚜中的分布进行组织定位,结果表明在滴加杂交液的组织切片被特异性的染成棕褐色,而阴性对照的组织切片没有被染色。由此表明原位杂交技术可以灵敏检测到Cdc42基因在该蚜虫不同组织中的表达状况。原位杂交结果显示,Cdc42基因在有翅蚜和无翅蚜的头部和腹部均有表达,头部的表达集中在两复眼之间,腹部的表达几乎全集中在伪胚胎;有翅蚜的胸部几乎全部都有表达,根据着色点数量推断有翅蚜的胸部表达量最高;而无翅蚜的胸部几乎没有表达。
再运用实时荧光定量RT-PCR技术确定该基因的相对表达量,结果表明Cdc42基因在麦长管蚜不同组织及不同龄期均有表达,若蚜期高于成蚜期,有翅蚜高于无翅蚜,该基因在有翅蚜与无翅蚜胸部的表达成显著差异;从伪胚胎开始就有较高的转录水平。
对2龄若蚜经体外涂抹保幼激素JHⅢ及其类似物ZR-515的滴度效益结果表明,随着激素浓度的增加,无翅和有翅麦长管蚜成虫整虫Cdc42相对表达量逐渐减低,均低于正常有翅蚜的表达量,无翅蚜中的表达量只有正常表达量的千分之一,各浓度之间差异极显著;并且无翅型麦长管蚜成虫的个体数量不断增加,有翅蚜数量逐渐减少,各浓度之间差异极显著。
本研究为探讨建立微型昆虫基因的准确组织定位和定量分析方法奠定了基础,也为深入开展保幼激素、Cdc42基因与翅型分化的关系提供了理论依据。
The English grain aphid, Sitobion avenae, is of wing dimorphism, namely, winged and wingless aphid. The aphid’s wing differentiation is manipulated by a complicate factors system, and the ratio of the wing dimorphism depends on the intrinsic and external environment factors. So in order to uncover the mechanism of aphid’s wing differentiation, through the related genes expression level by molecular biology approach, which will be very important to enrich the basical biology of aphid, and also benefit to further develop new method to control the aphid based on the knowledge of the manipulation of aphid’s wing differentiation.
In this experiment thesis, Cdc42 gene tissue location and accurate relative expression levels of S. avenae were determined by using in situ hybridization (ISH) and real-time fluorescence quantitative PCR method to determine the Cdc42 gene in the location and relative expression levels of the organization in Sitobion avenae. Then the change dynamic of Cdc42 gene expression, and population and the ratio of alatae and apterae aphid were detected with dose variation of juvenile hormone (JH) and its analogues by smearing 2 instar nymphae aphid. The aim is to understand preliminarily for the interaction of JH in vitro, the Cdc42 gene expression, and the function of manipulation of wheat aphid’s wing dimorphism. The major results are as follows:
First, ISH results showed that drip liquid hybridization specificity of tissue biopsies were specific stained in brown with nucleic acid probe of Cdc42 gene, while without the same staining characteristic on the negative control, which indicated that ISH could be sensitive to identify the tissue locaton of the gene in the aphid. ISH results showed that Cdc42 expressed in the head and abdomen parts of the aphid, and focused between the two complicated eyes by aphid’s head.In the abdomen the gene expressions were almost concentrated the pseudo-embryos. According to the brown staining. The Cdc42 gene specific stainging spread all over on the alate thorax part, and it was deduced by the number staining dot, that the highest expression level occurred on the thorax part of alate aphids, but hardly not expressed on the thorax part of apterae aphid.
Tissue and stage specific transcriptive levels were determined by real-time fluorescence quantitative PCR and showed that Cdc42 was transcripted in multiples tissues and at different stages. There was higher transcriptive level in the nymphae stages than that in adult stage, and higher in alate aphid than that of apterous one, and significantly higher on the thorax part of alate than that in apterae aphid. The gene’s expression start up at the aphid’s pseudo-embryo stage, and even with very high expressed level in the stage.
Application juvenile hormone (JH) and its analogues ZR-515 on aphid’s thorax in vitro by three concentrations, to study the impact of JH dose on the expression of Cdc42 gene and wing differentiation, the results showed that with the dose increasing of JHⅢand its analogues ZR-515, the expression level of Cdc42 gene in the whole part of alate and apterae aphid decreasing gradually, which were significant lower in the three treated concentration than that in control of alate aphid, by real-time fluorescence quantitative PCR detection, and the expression level of Cdc42 in the treated apterae aphid was only one thousandth of the level compared with aphid in control.There were also significant difference among the different treatments. The number of apterae adults increasing, but the number of alate aphids decreasing under the JH and ZR-515 treatments.
In this thesis, a micro-insect gene tissue localization and quantitative analysis, method has been established, and the results provide a primary data for the interaction of JH, Cdc42 gene and wing differentiation in aphid.
引文
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