桔小實(shí)蠅老熟幼蟲(chóng)濕度感受基因的功能研究
發(fā)布時(shí)間：2019-05-22 21:33
【摘要】：環(huán)境濕度對(duì)昆蟲(chóng)的發(fā)育、繁殖及生存有很嚴(yán)重的影響,能造成害蟲(chóng)不同的發(fā)生時(shí)期、發(fā)生數(shù)量和危害程度。昆蟲(chóng)感知環(huán)境濕度的能力是其在自然界生存的重要生物特征之一,昆蟲(chóng)體積較小且能敏感地感知環(huán)境中濕度的變化,是研究濕度感知能力的最好模型之一。桔小實(shí)蠅Bactrocera dorsalis(Hendel)是一種世界性檢疫害蟲(chóng),有很廣泛的寄主范圍,主要危害柑橘、香蕉等40多個(gè)科的250多種作物果實(shí)。目前桔小實(shí)蠅的防治策略僅局限于化學(xué)防治和雄蟲(chóng)誘殺技術(shù),而且這些防治方法很難取得較好的防治效果,嚴(yán)重影響了蔬菜水果產(chǎn)業(yè)的發(fā)展。事實(shí)上,桔小實(shí)蠅三齡老熟幼蟲(chóng)依賴濕度感知進(jìn)行化蛹,假設(shè)阻隔其選擇合適的濕度環(huán)境化蛹的過(guò)程,有望在此重要環(huán)節(jié)對(duì)其實(shí)施防治。本文從桔小實(shí)蠅老熟幼蟲(chóng)的彈跳行為出發(fā),以期在分子水平上研究其對(duì)沙土濕度的感知機(jī)制,濕度感受相關(guān)基因有望成為潛在的防治靶標(biāo)基因,從而為防治桔小實(shí)蠅提供新的防治策略。本研究以桔小實(shí)蠅老熟幼蟲(chóng)為試蟲(chóng),鑒定老熟幼蟲(chóng)可能與濕度感受相關(guān)TRP(Transient Receptor Potential)家族基因,并驗(yàn)證相關(guān)基因的功能。研究結(jié)果如下:1、桔小實(shí)蠅TRPPainless基因全長(zhǎng)的獲得及生物信息學(xué)分析本研究通過(guò)快速末端擴(kuò)增技術(shù)(RACE)和基因克隆技術(shù)獲得兩條濕度感知相關(guān)的TRPPainless基因全長(zhǎng)Bdor TRP1、Bdor TRP2。Bdor TRP1基因c DNA包括2793bp的開(kāi)放閱讀框,編碼930個(gè)氨基酸。Bdor TRP2基因c DNA包括2796bp的開(kāi)放閱讀框,編碼931個(gè)氨基酸。通過(guò)序列比對(duì)和同源性分析表明,全長(zhǎng)基因編碼的蛋白序列與已知黑腹果蠅(Drosophila melanogaster)、地中海實(shí)蠅(Ceratitis capitata)、家蠅(Musca domestica)的一致性分別為56%、88%、67%。與已知物種的TRPPainless基因進(jìn)行Clustal W對(duì)比分析得到五個(gè)保守結(jié)構(gòu)域。同時(shí)預(yù)測(cè)到TRPPainless基因的氨基酸序列含有六個(gè)跨膜結(jié)構(gòu)域。經(jīng)系統(tǒng)發(fā)育分析得到:TRP基因按同源性可分為七個(gè)分支,分別是TRPA、TRPN、TRPV、TRPM、TRPML、TRPP、TRPC。其中Bdor TRP1和Bdor TRP2高度相似,一致性為97.1%。同時(shí)與已報(bào)道的Deml Wtrw濕度感知基因都屬于TRPA家族,且與另一個(gè)屬TRP家族的濕度感知基因Deml Nan遺傳距離較近。因此Bdor TRP1和Bdor TRP2基因可能介導(dǎo)桔小實(shí)蠅老熟幼蟲(chóng)的濕度感知機(jī)制。2、桔小實(shí)蠅TRPPainless基因的功能研究本研究采用RNA干涉技術(shù)及熒光定量PCR技術(shù),比較干涉前后三天內(nèi)的濕度感知相關(guān)基因TRPPainless基因相對(duì)表達(dá)量的差異性。結(jié)果顯示:Bdor TRP1基因干涉后的平均相對(duì)表達(dá)量是干涉前的0.57倍,Bdor TRP2基因干涉后的平均相對(duì)表達(dá)量是干涉前的0.63倍,干涉效果明顯,差異性顯著。通過(guò)對(duì)桔小實(shí)蠅老熟幼蟲(chóng)對(duì)不同濕度沙土的選擇性、入土?xí)r間和彈跳高度等相關(guān)因素的研究,驗(yàn)證濕度感知基因干涉前后桔小實(shí)蠅老熟幼蟲(chóng)在行為學(xué)上的差異。結(jié)果表明,桔小實(shí)蠅老熟幼蟲(chóng)偏好濕度為40%和60%的沙土下鉆、化蛹。干涉前選擇40%和60%濕度沙土的蟲(chóng)數(shù)為70%,干涉后選擇40%和60%濕度沙土的蟲(chóng)數(shù)為53%,干涉后選擇適宜濕度沙土下鉆、化蛹的桔小實(shí)蠅老熟幼蟲(chóng)數(shù)下降,干涉前后差異性顯著;一定條件下,干涉前桔小實(shí)蠅老熟幼蟲(chóng)入土所需時(shí)間平均約為212s,干涉后桔小實(shí)蠅老熟幼蟲(chóng)入土所需時(shí)間平均約為197s,干涉后幼蟲(chóng)入土?xí)r間減少,干涉前后差異性顯著;干涉前后桔小實(shí)蠅老熟幼蟲(chóng)的彈跳高度均在5-6cm之間,無(wú)差異性。由以上結(jié)果推斷TRPPainless基因參與了桔小實(shí)蠅老熟幼蟲(chóng)感知不同濕度沙土的機(jī)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.
【學(xué)位授予單位】：仲愷農(nóng)業(yè)工程學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433
本文編號(hào)：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.
【學(xué)位授予單位】：仲愷農(nóng)業(yè)工程學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433
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