取食香根草對(duì)水稻二化螟中腸轉(zhuǎn)錄組影響及相關(guān)基因鑒定分析
發(fā)布時(shí)間：2018-08-05 10:30
【摘要】：水稻二化螟(Chilo suppressalis)屬鱗翅目螟蛾科,是為害水稻等禾本科植物最重要的常發(fā)性害蟲之一。近年來,由于我國(guó)耕作制度、水稻品種的改變、以及全球氣候變暖,二化螟的生存環(huán)境得到極大的改善,種群數(shù)量顯著上升。長(zhǎng)久以來,水稻螟蟲的防治主要依靠化學(xué)防治為主,由于長(zhǎng)期依賴化學(xué)藥劑進(jìn)行防治,導(dǎo)致了害蟲抗藥性、農(nóng)藥殘留和環(huán)境污染等一系列問題。因此,尋求更加安全的生物防治措施迫在眉睫,已有研究表明在田埂種植香根草(Vetiveria zizanioideis)可以有效的誘集二化螟雌成蟲在其上產(chǎn)卵,并且二化螟在香根草上不能完成生活史。雖然香根草作為二化螟的致死型誘集植物已開展田間試驗(yàn),但是其對(duì)二化螟的致死作用機(jī)制還未見報(bào)道。中腸是昆蟲體內(nèi)的第二大器官,作為分泌消化酶、解毒酶和其它酶類的主要存在部位,是消化食物、吸收營(yíng)養(yǎng)和解毒代謝的主要場(chǎng)所。本研究利用第二代高通量測(cè)序技術(shù)對(duì)香根草對(duì)二化螟的致死作用機(jī)制進(jìn)行初步探究。主要研究結(jié)果總結(jié)如下:1.二化螟幼蟲中腸轉(zhuǎn)錄組測(cè)序結(jié)果二化螟幼蟲分別取食水稻和香根草2d后,測(cè)定中腸轉(zhuǎn)錄組。測(cè)序獲得數(shù)據(jù)產(chǎn)量堿基數(shù)為48,666,252nt,產(chǎn)生總的unigene數(shù)量為48,944,最長(zhǎng)的unigene為35,885nt,最短的為205nt,平均組裝長(zhǎng)度為994.325nt,GC含量為39.6%,N50和N90分別為1874nt和375nt。根據(jù)blast比對(duì)結(jié)果可以看出53.69%序列與數(shù)據(jù)庫中匹配有顯著同源性(比1.0E10-50小),而46.31%的序列介于1.0E10-5和1.0E10-50之間。GO功能分類注釋結(jié)果顯示6738條Unigene歸入細(xì)胞構(gòu)成組分,9993條歸入生物學(xué)過程,9052條歸入分子功能。共有716條序列比對(duì)到KEGG數(shù)據(jù)庫,此數(shù)據(jù)庫含14條Pathway,其中比對(duì)到序列最多的Pathway是:代謝途徑(Metabolic pathways)和核糖體(Ribosome,accounting),分別占 9.02%和 3.67%。2.轉(zhuǎn)錄組測(cè)定結(jié)果驗(yàn)證以分別取食水稻和香根草2天的二化螟幼蟲中腸為樣本,從上述測(cè)定的轉(zhuǎn)錄組數(shù)據(jù)中選取部分與消化、免疫、解毒代謝及保護(hù)作用相關(guān)的重要基因進(jìn)行熒光定量PCR(Real-Time PCR)驗(yàn)證。結(jié)果表明:上調(diào)的基因有19個(gè)分別是:糜蛋白酶(Chymotrypsin)、胰蛋白酶(Trypsin)、氨肽酶 N2(Aminopeptidase N2)、羧肽酶A1(Carboxypeptidase A1)、羧膚酶(Carboxypeptidase)、二肽基肽(Dipeptidyl-peptidase)、絲氨酸蛋白酶20(Serine protease 20)、胰脂肪酶(Pancreatic lipase)、中性脂肪酶(Neutral lipase)、葡萄糖苷酶(Glucosidase)、α-淀粉酶 2(alpha-amylase 2)、α-淀粉酶 1(alpha-amylase 1)、肽聚糖識(shí)別蛋白(Peptidoglycan recognition protein)、p-葡聚糖識(shí)別蛋白(Beta-glucan recognition protein)、Hddl、Hdd13、細(xì)胞色素 306A1(CYP306A1)、類谷胱甘肽轉(zhuǎn)移酶蛋白(GSTs-like protein)、過氧化氫酶(Catalase),下調(diào)的基因有4個(gè)分別是:組織蛋白酶L2(Cathepsin L2)、羧肽酶抑制劑(Carboxypeptidase inhibitor)、糖轉(zhuǎn)運(yùn)蛋白 4(Sugar transporter 4)、細(xì)胞色素 6A2(CYP6A2)。2/3的基因表達(dá)量驗(yàn)證結(jié)果和轉(zhuǎn)錄組測(cè)定結(jié)果一致,說明轉(zhuǎn)錄組測(cè)定結(jié)果可信。3.取食香根草不同時(shí)間后二化螟中腸基因變化利用Real-Time PCR測(cè)定上述基因在二化螟取食水稻、香根草2,4,6,8,10d后的表達(dá)量。結(jié)果表明:消化酶相關(guān)基因:相對(duì)于水稻對(duì)照組來說,Pancreatic lipase和Serine protease 20在取食香根草的二化螟中腸中表達(dá)水平較高,相對(duì)表達(dá)水平維持在80%左右,絲氨酸蛋白酶抑制劑(Serineprotease inhibitor)在二化螟取食香根草8d之后,其相對(duì)表達(dá)水平也達(dá)到了 80%,其他基因的表達(dá)水平基本都是在水稻對(duì)照組中表達(dá)量高或者取食一定天數(shù)之后在水稻對(duì)照組中的表達(dá)量高。免疫相關(guān)基因:Hddl在取食香根草的二化螟中腸中表達(dá)水平高,在香根草處理組中的相對(duì)表達(dá)水平在60%-70%之間,而Hdd 13的表達(dá)則恰恰相反,其在水稻對(duì)照組中相對(duì)表達(dá)水平在85%左右,Peptidoglycan recognition protein 和 Beta-glucan recognition protein 表達(dá)量在兩組中始終差異不大。解毒代謝相關(guān)基因:取食香根草一定天數(shù)之后,5個(gè)解毒代謝相關(guān)基因均在取食水稻的二化螟中腸中表達(dá)水平較高,尤其是CYP306A1和羧酸酯酶(carboxylesterase)在食香根草處理組中表達(dá)水平始終都顯著低于水稻對(duì)照組,兩者在水稻對(duì)照組中的相對(duì)表達(dá)水平在75%-100%之間。保護(hù)作用相關(guān)基因:硫氧還蛋白過氧化物酶(Thioredoxin peroxidase)表達(dá)水平在兩組中隨時(shí)間變化差異不大,其它基因在香根草處理組中的表達(dá)水平較低,相對(duì)表達(dá)水平都在50%以下�？偟膩碚f,以上這些基因可能在香根草對(duì)二化螟幼蟲具致死過程中發(fā)揮不同程度的作用。4.重要相關(guān)功能基因在二化螟幼蟲體內(nèi)不同組織中的表達(dá)利用Real-Time PCR測(cè)定了 35個(gè)基因在二化螟頭、中腸、脂肪體和表皮4個(gè)組織中的表達(dá)。結(jié)果表明:與消化相關(guān)的基因Cathepsin L2在二化螟幼蟲頭部特異表達(dá),在其他三個(gè)組織中的相對(duì)表達(dá)水平在15%以下,Cathepsin B和Carboxypeptidase inhibitor在表皮組織中特異表達(dá)相對(duì)表達(dá)水平在60%左右,在其他三個(gè)組織中的相對(duì)表達(dá)水平在20%以下。其余基因都在脂肪體和中腸這兩個(gè)組織中特異表達(dá)的。大部分與消化相關(guān)的基因以及免疫(Hdd1除外)、解毒代謝、防御保護(hù)酶類相關(guān)基因都在脂肪體中具有較高的表達(dá)水平。從上述分析結(jié)果看來,大多數(shù)消化、解讀代謝以及保護(hù)作用相關(guān)的基因都是在取食香根草的二化螟中腸中具有較低的表達(dá)水平,二化螟幼蟲體內(nèi)消化酶合成減少,進(jìn)而影響了蛋白質(zhì)、脂肪及糖等營(yíng)養(yǎng)物質(zhì)的吸收代謝,可能造成幼蟲營(yíng)養(yǎng)不良而死亡;解毒代謝酶和保護(hù)酶類減少影響了二化螟體內(nèi)有毒物質(zhì)的代謝和防御機(jī)制發(fā)揮作用,這些都和香根草對(duì)二化螟具致死作用機(jī)制有關(guān)。
[Abstract]:The rice borer (Chilo suppressalis) is a Lepidoptera borer, which is one of the most important frequent pests of rice and other gramineous plants. In recent years, the living environment of the rice borer has been greatly improved and the population number has risen significantly. The control of insects mainly depends on chemical control. Due to the long-term dependence of chemical agents, the pest resistance, pesticide residues and environmental pollution are a series of problems. Therefore, it is imminent to seek more safe biological control measures. The research has shown that the planting of Vetiveria zizanioideis in the field bank can be effective. The female adult worm of the suppressalis suppressalis is spawned on it, and the borer can not complete the life history on the vetiver. Although Vetiver has been tested as a lethal type of luring plant of the borer, the mechanism of its lethal effect has not yet been reported. The midgut is the second organ in the insect, as the secretory digestive enzyme and detoxification enzyme. The main location of the enzymes and other enzymes is the main place to digest food, absorb nutrition and detoxify the metabolism. The second generation high-throughput sequencing technology was used to investigate the lethal mechanism of vetiver on borer. The main results are summarized as follows: 1. the result of the sequence of the midgut transcriptome of the larvae of borer borer Do not eat rice and vetiver 2D, determine the midgut transcriptional unit. The base number of the output base is 48666252nt, the total number of UniGene is 48944, the longest UniGene is 35885nt, the shortest is 205nt, the average assembly length is 994.325nt, the GC content is 39.6%, N50 and N90 are divided into 1874nt and 375nt. according to the result comparison results. The 53.69% sequence has a significant homology with the database (smaller than 1.0E10-50), and the 46.31% sequence between 1.0E10-5 and 1.0E10-50 shows that the.GO functional classification annotation results show that 6738 Unigene is classified into the constituent components of the cell, 9993 into the biological process and 9052 into the molecular power. A total of 716 sequences are compared to the KEGG database. The library contains 14 Pathway, of which the Pathway of the highest alignment is the metabolic pathway (Metabolic pathways) and ribosome (Ribosome, accounting), accounting for 9.02% and 3.67%.2. transcripts, respectively, to verify the sample of the midgut of the larvae of the rice and vetiver for 2 days respectively. The important genes related to digestion, immunity, detoxification, and protection were verified by fluorescence quantitative PCR (Real-Time PCR). The results showed that the up regulated genes were 19: chymotrypsin (Chymotrypsin), trypsin (Trypsin), aminopeptidase N2 (Aminopeptidase N2), A1 (Carboxypeptidase A1), carboxypeptidase (Carboxypeptidase A1), carboxypeptidase (Carboxypeptidase), and two Peptidepeptide (Dipeptidyl-peptidase), serine protease 20 (Serine protease 20), pancreatic lipase (Pancreatic lipase), neutral lipase (Neutral lipase), glucosidase (Glucosidase), alpha amylase 2 (alpha-amylase 2), alpha amylase 1 (alpha-amylase 1), peptidoglycan identification protein (Peptidoglycan recognition), dextran Beta-glucan recognition protein, Hddl, Hdd13, cytochrome 306A1 (CYP306A1), glutathione transferase protein (GSTs-like protein), catalase (Catalase), and down regulated genes are 4: cathepsin L2 (Cathepsin), carboxypeptidase inhibitor, and sugar transporter 4 Ansporter 4), the results of the gene expression of cytochrome 6A2 (CYP6A2).2/3 were consistent with the results of the transcriptional test, indicating that the transcriptional group determination results trusted.3. to feed the midgut gene of the borer in different time. Real-Time PCR was used to determine the expression of the above gene in rice and the expression of Vetiver 2,4,6,8,10d. The expression of digestive enzyme related genes: the expression level of Pancreatic lipase and Serine protease 20 in the midgut of the suppressalis suppressalis is higher than that of the rice control group, and the relative expression level is about 80%. The relative expression of the serine protease inhibitor (Serineprotease inhibitor) after the feeding of vetiver 8D by the serine suppressalis (Serineprotease inhibitor) The expression level of other genes is 80%, and the expression level of other genes is high in rice control group or after feeding a certain number of days in rice control group. Immuno related genes: Hddl is high in the midgut of the midgut of the suppressalis suppressalis, and the relative expression level in 60%-70%. The expression of Hdd 13 was the opposite, and the relative expression level in the rice control group was about 85%. The expression of Peptidoglycan recognition protein and Beta-glucan recognition protein was not very different in the two groups. The expression level in the midgut of rice borer was higher, especially the expression level of CYP306A1 and carboxylesterase (carboxylesterase) in the treatment group of vetiver was significantly lower than that of the control group. The relative expression level of the two in the rice control group was between 75%-100% and the related genes of the protective effect: Th The expression level of ioredoxin peroxidase is not very different in the two groups, the expression level of other genes in the vetiver treatment group is lower and the relative expression level is below 50%. In general, these genes may play an important role in different degrees of function of.4. in the death process of vetiver borer larvae. The expression of the gene in different tissues of the larvae of the suppressalis suppressalis using Real-Time PCR was used to determine the expression of 35 genes in the head, midgut, fat body and epidermis of 4 tissues. The results showed that the gene Cathepsin L2 associated with digestion was specifically expressed in the head of the larvae of the suppressalis suppressalis, and the relative expression level in the other three tissues was 15%. The specific expression level of Cathepsin B and Carboxypeptidase inhibitor in the epidermal tissue is about 60%, and the relative expression level in the other three tissues is below 20%. The other genes are expressed specifically in the fat bodies and the two tissues of the midgut. Most of the genes associated with digestion and immunity (except Hdd1) are detoxified. Metabolism, defense and protective enzymes related genes all have high levels of expression in the fat body. From the above analysis, most of the digestibility, interpretation of metabolism and protection related genes are low expression levels in the midgut of the suppressalis suppressalis, and the digestion enzyme synthesis in the body of the suppressalis suppressalis decreases. The absorption and metabolism of nutrients such as protein, fat and sugar may cause the death of the malnutrition of the larvae; detoxification metabolic enzymes and protective enzymes affect the metabolism and defense mechanism of toxic substances in the body of the suppressalis suppressalis, all of which are related to the mechanism of killing action of vetiver to the borer.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.112.1
本文編號(hào)：2165523
[Abstract]:The rice borer (Chilo suppressalis) is a Lepidoptera borer, which is one of the most important frequent pests of rice and other gramineous plants. In recent years, the living environment of the rice borer has been greatly improved and the population number has risen significantly. The control of insects mainly depends on chemical control. Due to the long-term dependence of chemical agents, the pest resistance, pesticide residues and environmental pollution are a series of problems. Therefore, it is imminent to seek more safe biological control measures. The research has shown that the planting of Vetiveria zizanioideis in the field bank can be effective. The female adult worm of the suppressalis suppressalis is spawned on it, and the borer can not complete the life history on the vetiver. Although Vetiver has been tested as a lethal type of luring plant of the borer, the mechanism of its lethal effect has not yet been reported. The midgut is the second organ in the insect, as the secretory digestive enzyme and detoxification enzyme. The main location of the enzymes and other enzymes is the main place to digest food, absorb nutrition and detoxify the metabolism. The second generation high-throughput sequencing technology was used to investigate the lethal mechanism of vetiver on borer. The main results are summarized as follows: 1. the result of the sequence of the midgut transcriptome of the larvae of borer borer Do not eat rice and vetiver 2D, determine the midgut transcriptional unit. The base number of the output base is 48666252nt, the total number of UniGene is 48944, the longest UniGene is 35885nt, the shortest is 205nt, the average assembly length is 994.325nt, the GC content is 39.6%, N50 and N90 are divided into 1874nt and 375nt. according to the result comparison results. The 53.69% sequence has a significant homology with the database (smaller than 1.0E10-50), and the 46.31% sequence between 1.0E10-5 and 1.0E10-50 shows that the.GO functional classification annotation results show that 6738 Unigene is classified into the constituent components of the cell, 9993 into the biological process and 9052 into the molecular power. A total of 716 sequences are compared to the KEGG database. The library contains 14 Pathway, of which the Pathway of the highest alignment is the metabolic pathway (Metabolic pathways) and ribosome (Ribosome, accounting), accounting for 9.02% and 3.67%.2. transcripts, respectively, to verify the sample of the midgut of the larvae of the rice and vetiver for 2 days respectively. The important genes related to digestion, immunity, detoxification, and protection were verified by fluorescence quantitative PCR (Real-Time PCR). The results showed that the up regulated genes were 19: chymotrypsin (Chymotrypsin), trypsin (Trypsin), aminopeptidase N2 (Aminopeptidase N2), A1 (Carboxypeptidase A1), carboxypeptidase (Carboxypeptidase A1), carboxypeptidase (Carboxypeptidase), and two Peptidepeptide (Dipeptidyl-peptidase), serine protease 20 (Serine protease 20), pancreatic lipase (Pancreatic lipase), neutral lipase (Neutral lipase), glucosidase (Glucosidase), alpha amylase 2 (alpha-amylase 2), alpha amylase 1 (alpha-amylase 1), peptidoglycan identification protein (Peptidoglycan recognition), dextran Beta-glucan recognition protein, Hddl, Hdd13, cytochrome 306A1 (CYP306A1), glutathione transferase protein (GSTs-like protein), catalase (Catalase), and down regulated genes are 4: cathepsin L2 (Cathepsin), carboxypeptidase inhibitor, and sugar transporter 4 Ansporter 4), the results of the gene expression of cytochrome 6A2 (CYP6A2).2/3 were consistent with the results of the transcriptional test, indicating that the transcriptional group determination results trusted.3. to feed the midgut gene of the borer in different time. Real-Time PCR was used to determine the expression of the above gene in rice and the expression of Vetiver 2,4,6,8,10d. The expression of digestive enzyme related genes: the expression level of Pancreatic lipase and Serine protease 20 in the midgut of the suppressalis suppressalis is higher than that of the rice control group, and the relative expression level is about 80%. The relative expression of the serine protease inhibitor (Serineprotease inhibitor) after the feeding of vetiver 8D by the serine suppressalis (Serineprotease inhibitor) The expression level of other genes is 80%, and the expression level of other genes is high in rice control group or after feeding a certain number of days in rice control group. Immuno related genes: Hddl is high in the midgut of the midgut of the suppressalis suppressalis, and the relative expression level in 60%-70%. The expression of Hdd 13 was the opposite, and the relative expression level in the rice control group was about 85%. The expression of Peptidoglycan recognition protein and Beta-glucan recognition protein was not very different in the two groups. The expression level in the midgut of rice borer was higher, especially the expression level of CYP306A1 and carboxylesterase (carboxylesterase) in the treatment group of vetiver was significantly lower than that of the control group. The relative expression level of the two in the rice control group was between 75%-100% and the related genes of the protective effect: Th The expression level of ioredoxin peroxidase is not very different in the two groups, the expression level of other genes in the vetiver treatment group is lower and the relative expression level is below 50%. In general, these genes may play an important role in different degrees of function of.4. in the death process of vetiver borer larvae. The expression of the gene in different tissues of the larvae of the suppressalis suppressalis using Real-Time PCR was used to determine the expression of 35 genes in the head, midgut, fat body and epidermis of 4 tissues. The results showed that the gene Cathepsin L2 associated with digestion was specifically expressed in the head of the larvae of the suppressalis suppressalis, and the relative expression level in the other three tissues was 15%. The specific expression level of Cathepsin B and Carboxypeptidase inhibitor in the epidermal tissue is about 60%, and the relative expression level in the other three tissues is below 20%. The other genes are expressed specifically in the fat bodies and the two tissues of the midgut. Most of the genes associated with digestion and immunity (except Hdd1) are detoxified. Metabolism, defense and protective enzymes related genes all have high levels of expression in the fat body. From the above analysis, most of the digestibility, interpretation of metabolism and protection related genes are low expression levels in the midgut of the suppressalis suppressalis, and the digestion enzyme synthesis in the body of the suppressalis suppressalis decreases. The absorption and metabolism of nutrients such as protein, fat and sugar may cause the death of the malnutrition of the larvae; detoxification metabolic enzymes and protective enzymes affect the metabolism and defense mechanism of toxic substances in the body of the suppressalis suppressalis, all of which are related to the mechanism of killing action of vetiver to the borer.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.112.1
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