桔小实蝇老熟幼虫湿度感受基因的功能研究
发布时间：2019-05-22 21:33
【摘要】：环境湿度对昆虫的发育、繁殖及生存有很严重的影响,能造成害虫不同的发生时期、发生数量和危害程度。昆虫感知环境湿度的能力是其在自然界生存的重要生物特征之一,昆虫体积较小且能敏感地感知环境中湿度的变化,是研究湿度感知能力的最好模型之一。桔小实蝇Bactrocera dorsalis(Hendel)是一种世界性检疫害虫,有很广泛的寄主范围,主要危害柑橘、香蕉等40多个科的250多种作物果实。目前桔小实蝇的防治策略仅局限于化学防治和雄虫诱杀技术,而且这些防治方法很难取得较好的防治效果,严重影响了蔬菜水果产业的发展。事实上,桔小实蝇三龄老熟幼虫依赖湿度感知进行化蛹,假设阻隔其选择合适的湿度环境化蛹的过程,有望在此重要环节对其实施防治。本文从桔小实蝇老熟幼虫的弹跳行为出发,以期在分子水平上研究其对沙土湿度的感知机制,湿度感受相关基因有望成为潜在的防治靶标基因,从而为防治桔小实蝇提供新的防治策略。本研究以桔小实蝇老熟幼虫为试虫,鉴定老熟幼虫可能与湿度感受相关TRP(Transient Receptor Potential)家族基因,并验证相关基因的功能。研究结果如下:1、桔小实蝇TRPPainless基因全长的获得及生物信息学分析本研究通过快速末端扩增技术(RACE)和基因克隆技术获得两条湿度感知相关的TRPPainless基因全长Bdor TRP1、Bdor TRP2。Bdor TRP1基因c DNA包括2793bp的开放阅读框,编码930个氨基酸。Bdor TRP2基因c DNA包括2796bp的开放阅读框,编码931个氨基酸。通过序列比对和同源性分析表明,全长基因编码的蛋白序列与已知黑腹果蝇(Drosophila melanogaster)、地中海实蝇(Ceratitis capitata)、家蝇(Musca domestica)的一致性分别为56%、88%、67%。与已知物种的TRPPainless基因进行Clustal W对比分析得到五个保守结构域。同时预测到TRPPainless基因的氨基酸序列含有六个跨膜结构域。经系统发育分析得到:TRP基因按同源性可分为七个分支,分别是TRPA、TRPN、TRPV、TRPM、TRPML、TRPP、TRPC。其中Bdor TRP1和Bdor TRP2高度相似,一致性为97.1%。同时与已报道的Deml Wtrw湿度感知基因都属于TRPA家族,且与另一个属TRP家族的湿度感知基因Deml Nan遗传距离较近。因此Bdor TRP1和Bdor TRP2基因可能介导桔小实蝇老熟幼虫的湿度感知机制。2、桔小实蝇TRPPainless基因的功能研究本研究采用RNA干涉技术及荧光定量PCR技术,比较干涉前后三天内的湿度感知相关基因TRPPainless基因相对表达量的差异性。结果显示:Bdor TRP1基因干涉后的平均相对表达量是干涉前的0.57倍,Bdor TRP2基因干涉后的平均相对表达量是干涉前的0.63倍,干涉效果明显,差异性显著。通过对桔小实蝇老熟幼虫对不同湿度沙土的选择性、入土时间和弹跳高度等相关因素的研究,验证湿度感知基因干涉前后桔小实蝇老熟幼虫在行为学上的差异。结果表明,桔小实蝇老熟幼虫偏好湿度为40%和60%的沙土下钻、化蛹。干涉前选择40%和60%湿度沙土的虫数为70%,干涉后选择40%和60%湿度沙土的虫数为53%,干涉后选择适宜湿度沙土下钻、化蛹的桔小实蝇老熟幼虫数下降,干涉前后差异性显著;一定条件下,干涉前桔小实蝇老熟幼虫入土所需时间平均约为212s,干涉后桔小实蝇老熟幼虫入土所需时间平均约为197s,干涉后幼虫入土时间减少,干涉前后差异性显著;干涉前后桔小实蝇老熟幼虫的弹跳高度均在5-6cm之间,无差异性。由以上结果推断TRPPainless基因参与了桔小实蝇老熟幼虫感知不同湿度沙土的机制。
[Abstract]:The environmental humidity has a serious effect on the development, reproduction and survival of the insects, and can cause different occurrence periods, quantity and degree of harm of the pests. The ability of the insect to sense the humidity of the environment is one of the important biological characteristics of the natural life of the insect, the volume of the insect is small, and the change of the humidity in the environment can be sensed in a sensitive manner, and is one of the best models for studying the humidity sensing ability. Bactrocera dorsalis (Hendel) is a world-wide quarantine pest. It has a wide range of host range, which is mainly harmful to more than 250 kinds of crop fruit of more than 40 families, such as citrus and banana. At present, the control strategy of the citrus fruit fly is limited to the chemical control and the male and insect trapping technology, and the prevention and control method is difficult to achieve better control effect, and the development of the vegetable and fruit industry is seriously affected. In fact, the three-instar old-mature larvae of the citrus fly-age are dependent on the humidity perception to make the pupae, and it is assumed that the process of selecting proper humidity and environment-friendly pupae is expected to be used for preventing and treating the pupae in this important link. In order to provide a new prevention and control strategy for the prevention and control of the citrus fruit fly, this paper starts with the bounce behavior of the old and mature larvae of the small fly-fly, in order to study its perception mechanism of the soil moisture level at the molecular level, and the relative gene of the humidity sensing is expected to be a potential target gene. The present study was to identify the family of TRP (TRP) family and to verify the function of the related genes. The results of the study are as follows:1. The full length of the TRPPainless gene of the fruit fly and the bioinformatics analysis. The full length of the TRPPainless gene is obtained by the rapid terminal amplification technology (RACE) and the gene cloning technique. The full length of the TRPPainless gene is Bdor TRP1, Bor TRP2. Bdor TRP1 gene c DNA includes an open reading frame of 279bp. 930 amino acids are encoded. The Bor TRP2 gene c DNA includes an open reading frame of 2796 bp, encoding 931 amino acids. The homology of the whole-length gene with the known Drosophila melanogaster, Certitis capitata and Musca domestica was 56%,88% and 67%, respectively. The five conserved domains were obtained by the analysis of Clustal W with the TRPPainless gene of the known species. At the same time, the amino acid sequence of the TRPPainless gene is predicted to contain six transmembrane domains. The results of the phylogenetic analysis showed that the homology of the TRP gene can be divided into seven branches, namely, TRPA, TRPN, TRPV, TRPM, TRPML, TRPP and TRPC. The height of Bor TRP1 and Bor TRP2 was similar and the consistency was 97.1%. At the same time, both the reported Dl Wtrw humidity sensing genes belong to the TRPA family and are closer to the humidity-aware gene Deml Nan of the other TRP family. Therefore, the Bdor TRP1 and Bor TRP2 genes may mediate the humidity sensing mechanism of the mature and mature larvae of the Bactrocera dorsalis, and the function of the TRPPainless gene of the fruit fly is studied by using the RNA interference technology and the fluorescence quantitative PCR technology. The difference of the relative expression of the related gene, TRPPainless gene, was measured in three days before and after the intervention. The results showed that the average relative expression of the Bdor TRP1 gene was 0.57 times before the intervention, and the average relative expression of the Bdor TRP2 gene was 0.63 times before the intervention, and the interference effect was significant and the difference was significant. Based on the study of the related factors, such as the selectivity, the time of soil and the height of the spring, the difference of the behavior of the old and mature larvae of the citrus and fly larvae before and after the intervention of the humidity-sensing gene was verified by the study of the related factors such as the selectivity, the soil-in time and the jumping height of the old and mature larvae of the Bactrocera citri. The results showed that the old and mature larvae of the fruit fly had a preference of 40% and 60% of the sand to drill and pupae. The number of insects with 40% and 60% humidity in sand before intervention is 70%, and the number of insects with 40% and 60% humidity in the soil after interference is 53%. After the interference, the number of the old and mature larvae of the small and small fly larvae of the pupae is decreased, and the difference between the front and the back of the interference is significant; under certain conditions, The time required to interfere with the entry of the old-mature larvae of the small fly-fly larvae was about 211s, and the time required for the late-stage larvae to reach the soil was about 197s, the time of the larvae entering the soil after the intervention was reduced, and the difference of the difference before and after the intervention was significant. The bounce height of the old and mature larvae of the orange-and-fly larvae before and after the intervention was between 5 and 6 cm, and there was no difference. It is concluded from the above results that the TRPPainless gene is involved in the mechanism of sensing the sandy soil with different humidity.
【学位授予单位】：仲恺农业工程学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
本文编号：2483280
[Abstract]:The environmental humidity has a serious effect on the development, reproduction and survival of the insects, and can cause different occurrence periods, quantity and degree of harm of the pests. The ability of the insect to sense the humidity of the environment is one of the important biological characteristics of the natural life of the insect, the volume of the insect is small, and the change of the humidity in the environment can be sensed in a sensitive manner, and is one of the best models for studying the humidity sensing ability. Bactrocera dorsalis (Hendel) is a world-wide quarantine pest. It has a wide range of host range, which is mainly harmful to more than 250 kinds of crop fruit of more than 40 families, such as citrus and banana. At present, the control strategy of the citrus fruit fly is limited to the chemical control and the male and insect trapping technology, and the prevention and control method is difficult to achieve better control effect, and the development of the vegetable and fruit industry is seriously affected. In fact, the three-instar old-mature larvae of the citrus fly-age are dependent on the humidity perception to make the pupae, and it is assumed that the process of selecting proper humidity and environment-friendly pupae is expected to be used for preventing and treating the pupae in this important link. In order to provide a new prevention and control strategy for the prevention and control of the citrus fruit fly, this paper starts with the bounce behavior of the old and mature larvae of the small fly-fly, in order to study its perception mechanism of the soil moisture level at the molecular level, and the relative gene of the humidity sensing is expected to be a potential target gene. The present study was to identify the family of TRP (TRP) family and to verify the function of the related genes. The results of the study are as follows:1. The full length of the TRPPainless gene of the fruit fly and the bioinformatics analysis. The full length of the TRPPainless gene is obtained by the rapid terminal amplification technology (RACE) and the gene cloning technique. The full length of the TRPPainless gene is Bdor TRP1, Bor TRP2. Bdor TRP1 gene c DNA includes an open reading frame of 279bp. 930 amino acids are encoded. The Bor TRP2 gene c DNA includes an open reading frame of 2796 bp, encoding 931 amino acids. The homology of the whole-length gene with the known Drosophila melanogaster, Certitis capitata and Musca domestica was 56%,88% and 67%, respectively. The five conserved domains were obtained by the analysis of Clustal W with the TRPPainless gene of the known species. At the same time, the amino acid sequence of the TRPPainless gene is predicted to contain six transmembrane domains. The results of the phylogenetic analysis showed that the homology of the TRP gene can be divided into seven branches, namely, TRPA, TRPN, TRPV, TRPM, TRPML, TRPP and TRPC. The height of Bor TRP1 and Bor TRP2 was similar and the consistency was 97.1%. At the same time, both the reported Dl Wtrw humidity sensing genes belong to the TRPA family and are closer to the humidity-aware gene Deml Nan of the other TRP family. Therefore, the Bdor TRP1 and Bor TRP2 genes may mediate the humidity sensing mechanism of the mature and mature larvae of the Bactrocera dorsalis, and the function of the TRPPainless gene of the fruit fly is studied by using the RNA interference technology and the fluorescence quantitative PCR technology. The difference of the relative expression of the related gene, TRPPainless gene, was measured in three days before and after the intervention. The results showed that the average relative expression of the Bdor TRP1 gene was 0.57 times before the intervention, and the average relative expression of the Bdor TRP2 gene was 0.63 times before the intervention, and the interference effect was significant and the difference was significant. Based on the study of the related factors, such as the selectivity, the time of soil and the height of the spring, the difference of the behavior of the old and mature larvae of the citrus and fly larvae before and after the intervention of the humidity-sensing gene was verified by the study of the related factors such as the selectivity, the soil-in time and the jumping height of the old and mature larvae of the Bactrocera citri. The results showed that the old and mature larvae of the fruit fly had a preference of 40% and 60% of the sand to drill and pupae. The number of insects with 40% and 60% humidity in sand before intervention is 70%, and the number of insects with 40% and 60% humidity in the soil after interference is 53%. After the interference, the number of the old and mature larvae of the small and small fly larvae of the pupae is decreased, and the difference between the front and the back of the interference is significant; under certain conditions, The time required to interfere with the entry of the old-mature larvae of the small fly-fly larvae was about 211s, and the time required for the late-stage larvae to reach the soil was about 197s, the time of the larvae entering the soil after the intervention was reduced, and the difference of the difference before and after the intervention was significant. The bounce height of the old and mature larvae of the orange-and-fly larvae before and after the intervention was between 5 and 6 cm, and there was no difference. It is concluded from the above results that the TRPPainless gene is involved in the mechanism of sensing the sandy soil with different humidity.
【学位授予单位】：仲恺农业工程学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
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