取食香根草对水稻二化螟中肠转录组影响及相关基因鉴定分析

发布时间：2018-08-05 10:30

【摘要】：水稻二化螟(Chilo suppressalis)属鳞翅目螟蛾科,是为害水稻等禾本科植物最重要的常发性害虫之一。近年来,由于我国耕作制度、水稻品种的改变、以及全球气候变暖,二化螟的生存环境得到极大的改善,种群数量显著上升。长久以来,水稻螟虫的防治主要依靠化学防治为主,由于长期依赖化学药剂进行防治,导致了害虫抗药性、农药残留和环境污染等一系列问题。因此,寻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种植香根草(Vetiveria zizanioideis)可以有效的诱集二化螟雌成虫在其上产卵,并且二化螟在香根草上不能完成生活史。虽然香根草作为二化螟的致死型诱集植物已开展田间试验,但是其对二化螟的致死作用机制还未见报道。中肠是昆虫体内的第二大器官,作为分泌消化酶、解毒酶和其它酶类的主要存在部位,是消化食物、吸收营养和解毒代谢的主要场所。本研究利用第二代高通量测序技术对香根草对二化螟的致死作用机制进行初步探究。主要研究结果总结如下:1.二化螟幼虫中肠转录组测序结果二化螟幼虫分别取食水稻和香根草2d后,测定中肠转录组。测序获得数据产量碱基数为48,666,252nt,产生总的unigene数量为48,944,最长的unigene为35,885nt,最短的为205nt,平均组装长度为994.325nt,GC含量为39.6%,N50和N90分别为1874nt和375nt。根据blast比对结果可以看出53.69%序列与数据库中匹配有显著同源性(比1.0E10-50小),而46.31%的序列介于1.0E10-5和1.0E10-50之间。GO功能分类注释结果显示6738条Unigene归入细胞构成组分,9993条归入生物学过程,9052条归入分子功能。共有716条序列比对到KEGG数据库,此数据库含14条Pathway,其中比对到序列最多的Pathway是:代谢途径(Metabolic pathways)和核糖体(Ribosome,accounting),分别占 9.02%和 3.67%。2.转录组测定结果验证以分别取食水稻和香根草2天的二化螟幼虫中肠为样本,从上述测定的转录组数据中选取部分与消化、免疫、解毒代谢及保护作用相关的重要基因进行荧光定量PCR(Real-Time PCR)验证。结果表明:上调的基因有19个分别是:糜蛋白酶(Chymotrypsin)、胰蛋白酶(Trypsin)、氨肽酶 N2(Aminopeptidase N2)、羧肽酶A1(Carboxypeptidase A1)、羧肤酶(Carboxypeptidase)、二肽基肽(Dipeptidyl-peptidase)、丝氨酸蛋白酶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)、肽聚糖识别蛋白(Peptidoglycan recognition protein)、p-葡聚糖识别蛋白(Beta-glucan recognition protein)、Hddl、Hdd13、细胞色素 306A1(CYP306A1)、类谷胱甘肽转移酶蛋白(GSTs-like protein)、过氧化氢酶(Catalase),下调的基因有4个分别是:组织蛋白酶L2(Cathepsin L2)、羧肽酶抑制剂(Carboxypeptidase inhibitor)、糖转运蛋白 4(Sugar transporter 4)、细胞色素 6A2(CYP6A2)。2/3的基因表达量验证结果和转录组测定结果一致,说明转录组测定结果可信。3.取食香根草不同时间后二化螟中肠基因变化利用Real-Time PCR测定上述基因在二化螟取食水稻、香根草2,4,6,8,10d后的表达量。结果表明:消化酶相关基因:相对于水稻对照组来说,Pancreatic lipase和Serine protease 20在取食香根草的二化螟中肠中表达水平较高,相对表达水平维持在80%左右,丝氨酸蛋白酶抑制剂(Serineprotease inhibitor)在二化螟取食香根草8d之后,其相对表达水平也达到了 80%,其他基因的表达水平基本都是在水稻对照组中表达量高或者取食一定天数之后在水稻对照组中的表达量高。免疫相关基因:Hddl在取食香根草的二化螟中肠中表达水平高,在香根草处理组中的相对表达水平在60%-70%之间,而Hdd 13的表达则恰恰相反,其在水稻对照组中相对表达水平在85%左右,Peptidoglycan recognition protein 和 Beta-glucan recognition protein 表达量在两组中始终差异不大。解毒代谢相关基因:取食香根草一定天数之后,5个解毒代谢相关基因均在取食水稻的二化螟中肠中表达水平较高,尤其是CYP306A1和羧酸酯酶(carboxylesterase)在食香根草处理组中表达水平始终都显著低于水稻对照组,两者在水稻对照组中的相对表达水平在75%-100%之间。保护作用相关基因:硫氧还蛋白过氧化物酶(Thioredoxin peroxidase)表达水平在两组中随时间变化差异不大,其它基因在香根草处理组中的表达水平较低,相对表达水平都在50%以下。总的来说,以上这些基因可能在香根草对二化螟幼虫具致死过程中发挥不同程度的作用。4.重要相关功能基因在二化螟幼虫体内不同组织中的表达利用Real-Time PCR测定了 35个基因在二化螟头、中肠、脂肪体和表皮4个组织中的表达。结果表明:与消化相关的基因Cathepsin L2在二化螟幼虫头部特异表达,在其他三个组织中的相对表达水平在15%以下,Cathepsin B和Carboxypeptidase inhibitor在表皮组织中特异表达相对表达水平在60%左右,在其他三个组织中的相对表达水平在20%以下。其余基因都在脂肪体和中肠这两个组织中特异表达的。大部分与消化相关的基因以及免疫(Hdd1除外)、解毒代谢、防御保护酶类相关基因都在脂肪体中具有较高的表达水平。从上述分析结果看来,大多数消化、解读代谢以及保护作用相关的基因都是在取食香根草的二化螟中肠中具有较低的表达水平,二化螟幼虫体内消化酶合成减少,进而影响了蛋白质、脂肪及糖等营养物质的吸收代谢,可能造成幼虫营养不良而死亡;解毒代谢酶和保护酶类减少影响了二化螟体内有毒物质的代谢和防御机制发挥作用,这些都和香根草对二化螟具致死作用机制有关。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.112.1

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