黄河鲤LGP2和STING基因的鉴定及其遗传多态性与鲤疱疹病毒感染的筛选

发布时间：2018-04-29 00:02

  本文选题：黄河鲤 + 基因克隆　； 参考：《西北农林科技大学》2017年博士论文

【摘要】：豫选黄河鲤(Cyprinus carpio haematopterus)是由河南省水产科学研究院采用野生黄河鲤做亲本,经过近20年、连续8代精心繁育而成。迄今已得到广泛推广,有着显著的经济和社会效益。然而,近几年,黄河鲤各类疾病的频发,使黄河鲤养殖风险大大增大,造成严重的经济损失,明显制约了该产业的可持续健康发展。感染锦鲤疱疹病毒(Koi herpesvirus,KHV)的黄河鲤具高度传染性和极高的致死率。直到目前仍没有好的防御措施和治愈手段。因而了解黄河鲤自身免疫系统对这种病毒的防御功能和培育抗病的优良品种显得尤为重要,也是维持健康水产养殖业的关键所在。先天免疫识别受体及信号通路相关基因在病毒检测、干扰素诱导、保护机体免受侵害等方面具有关键作用。LGP2作为RIG-I样模式识别受体的家族成员之一,首先识别存在于细胞质中的病毒DNA或RNA,随后下游系列信号级联反应被激活,诱导产生I型干扰素和促炎性因子。干扰素基因的刺激基因STING是宿主细胞中DNA病毒识别的感知器,通过RIG-TBK1-IRF3/IRF7信号级联来诱导产生干扰素,对DNA病原体做出先天免疫反应。为研究上述免疫相关基因的分子特性和抗病毒机制,本研究通过基因克隆及病毒感染,鉴定和分析了LGP2、STING基因和KHV病毒体内外感染后基因表达谱以及poly(I:C)和poly(d A:d T)配体刺激CFC(尾鳍细胞)后的基因表达情况,探究LGP2和STING在病毒识别及抗病毒级联反应中的作用。为进一步探究LGP2和STING基因与抗KHV的关联性及遗传特性,本研究提取感染KHV病毒的黄河鲤脾脏的DNA,利用特异PCR和染色体步移技术获得LGP2和STING基因全长及5’侧翼序列,并应用生物信息学和生物统计软件SPSS等工具对克隆所得目标基因的结构特征及遗传信息进行系统的比较分析;通过构建表达载体,验证启动子活性;以及利用基因池混合测序和PCR-RFLP方法,对目的基因的核苷酸多态位点与黄河鲤抗KHV感染进行关联分析,并对其表型关联位点进行验证。1.黄河鲤LGP2基因存在两个选择性剪接体,LGP2a c DNA全长为3061bp,ORF开放阅读框位于第127位到第2066位核苷酸,编码680个氨基酸的多肽,预测分子量为77,341.2Da,等电点为6.53。预测四个蛋白质结构域,包括细菌III型限制性内切酶的保守结构域,DEAD/DEAH盒解旋酶结构域,C末端解旋酶超家族结构域和调节结构域。LGP2b的全长为1928bp,编码346个氨基酸,5’-非翻译区(UTR)为114个核苷酸,3’UTR有603个核苷酸,其中包括AATAA终止信号。黄河鲤LGP2基因全长DNA为7655bp,有12个外显子,11个内含子,所有内含子均是由GT开头,AG结尾的,在LGP2 DNA全长与LGP2a和LGP2b m RNA比对结果显示,LGP2a和LGP2b来源于同一条DNA序列,在第七外显子处m RNA序列出现不同的选择性剪切,将第七内含子的部分序列转录为第七外显子,形成一条完整的m RNA序列。LGP2的5’侧翼序列预测有两个启动子,并且其活性均能被KHV诱导加强。病毒感染实验结果表明,LGP2有助于ds DNA病毒介导的细胞先天免疫应答的正调节。2.LGP2的16个有效的多态位点被探测到:其中包括12个SNP位点、4个有缺失核苷酸的位点;其中5’侧翼区的-294GCT三核苷酸缺失和414 GT二核苷酸缺失位点以及内含子中的3820 T/C和3836T/G位点的基因型和等位基因频率在抗性组和易感组间差异显著。经二次感染验证表明,-294 ins、414 ins、3820TT,3836TT基因型的黄河鲤分别比-294 del、414del,3820CC,3836GG型的黄河鲤死亡率低。因此该四个位点的突变可能是黄河鲤疱疹病毒病的遗传相关的危险因素。3.STING c DNA全长1528bp,编码402个氨基酸的多肽,分子量为46.184k Da,等电子点为6.08。推测的STING蛋白含有信号肽,N-末端区域中的三个跨膜基序和驻留的内质网蛋白中发现存在四个假定基序(RXR)。STING基因DNA全长为8068bp,由7个外显子和6个内含子组成,所有内含子均遵循GT开头,AG结尾的规律。5’-侧翼序列为901bp,序列预测有启动子活性,其活性可被KHV诱导加强。STING被鉴定为由DNA病原体介导的黄河鲤先天免疫应答的组成部分,并且在体外和体内都证实了STING对下游反应的正向调节中起关键作用。4.STING的14个有效的多态位点被探测到:13个SNP位点、1个有缺失核苷酸的位点;其中5’侧翼区的-873G/C和-687CCT三核苷酸缺失位点;外显子中的6767G/A和7034C/T位点的基因型和等位基因频率在抗性组和易感组间差异显著。二次病毒刺激实验表明,STING基因的-873GG、-687ins、6767GG和7034CC基因型的黄河鲤分别比-873CC、-687del、6767TT和7034TT型的黄河鲤死亡率低(P0.05),可以认为-873G/C、-687ins/del、6767G/A和7034C/T与黄河鲤抗KHV感染抗性-易感表型显著相关,可以作为抗病育种的分子标记。5.为了研究黄河鲤对KHV病毒的抗病防御机制和抗KHV的关联性,本研究将KHV病毒感染后黄河鲤抗性组和易感组的鳃和脾脏的RNA进行转录组测序,4个组织转录组测序共获得1.90亿个Raw reads,过滤后得到1.35亿Clean reads,经Trinity软件组装后,得到469,251个transcripts和366,783个unigene,unigene平均长度667,最长为18437bp,最短为201bp。其中长度在200-600 bp的序列有347659条,占74.08%。比对到Nr数据库的序列有31,837条,且与斑马鱼一致性最高,其次是墨西哥丽脂鲤和虹鳟,说明与鲤科鱼类的基因一致性最高。采用R with edge R package筛选差异表达基因,并对差异基因进行GO富集和KEGG通路富集分析,脾脏和鳃的抗性和易感组的差异基因数量相似,从天然免疫识别受体途径中选出78个基因在抗性组和易感组之间的表达差异表明,鱼类在脾中和鳃中抗KHV病毒的反应是不同的。本研究还对转录组文库中的基因进行SNP、SSR和选择性剪切进行概况分析。无论是鳃还是脾抗病组的SNP位点少于易感组;编码区SNP位点少于非编码区,但脾脏中编码区突变多于非编码区;每个组织转录组中,有意突变占总SNP的43.1%-50.58%,而无意突变仅占0.11%-0.13%。文库中由C转换为T的SNP最多,其次是由T转换为C,最少的是由C转换为G。本数据库中从检测序列168,510条中检测出31,084条存在SSR,而包含SSR的序列数为25,240,约占总检测序列数的15%,包含多于一个SSR的序列为4,549条,占所有SSR序列的18%;不同碱基SSR为1,401,含一个以上SSR的序列约占30.8%。重复的最大和最小长度分别为119和18,平均长度为24个核苷酸。SSR的模序类型主要以单核苷酸9-12重复最多,而4核苷酸及5核苷酸5-8重复最少。在本研究中,经transcripts与unigenes比对,共有16,491个unigenes含有多个转录本(29.88%),可能受选择性剪切调节。这些unigenes中,某些不同表达转录本的基因DET共4,183占总Unigene的7.58%。本实验结果提供易感组鳃和脾脏以及抗病组鳃和脾脏的转录组概况,为黄河鲤抗疱疹病毒病的免疫生物学的第一次评估、也为后续的抗病分子育种奠定了理论基础。
[Abstract]:Yu selected the Yellow River carp (Cyprinus carpio haematopterus) is a parent of the wild the Yellow River carp by the Henan Academy of Fisheries Sciences. After nearly 20 years, it has been developed carefully for 8 generations. So far, it has been widely popularized and has remarkable economic and social benefits. However, in recent years, the frequent occurrence of various diseases of the carp in the Yellow River has made the the Yellow River carp aquiculture a great risk. Large increase, causing serious economic loss, obviously restricting the sustainable and healthy development of the industry. The the Yellow River carp virus (Koi herpesvirus, KHV) has highly infectious and high mortality rate. Until now there are still no good defensive measures and cure means. Therefore, understand the prevention of this virus by the autoimmune system of the Yellow River carp. .LGP2 is one of the family members of the RIG-I like pattern recognition receptor, the key role of the innate immune recognition receptor and signal pathway related genes in virus detection, interferon induction, and protection from the organism. First identify the virus DNA or RNA that exist in the cytoplasm, then the cascade reaction of the downstream signal is activated to induce the production of I interferon and proinflammatory factors. The stimulating gene STING of the interferon gene is the perceptron of the DNA virus identification in the host cell, which is induced by the cascade of RIG-TBK1-IRF3/ IRF7 signals to induce the production of interferon, and the pathogen of DNA. In order to study the molecular and antiviral mechanisms of the immune related genes mentioned above, the gene expression profiles of LGP2, STING and KHV viruses in vivo and in vivo, as well as the gene expression of poly (I:C) and poly (D A:d T) ligand stimulation CFC (tail fin cells) were identified and analyzed by gene cloning and virus infection. To explore the role of LGP2 and STING in virus identification and antiviral cascade. In order to further explore the association and genetic characteristics of LGP2 and STING genes and anti KHV, this study extracts DNA of the spleen of the Yellow River carp infected with KHV virus, and uses specific PCR and chromosome step technique to obtain the total length of LGP2 and STING genes and the 5 'flanking sequence, and should be used. The structural features and genetic information of the target genes were compared and analyzed by bioinformatics and biostatistics software SPSS, and the promoter activity was verified by constructing the expression vector, and the gene pool sequencing and PCR-RFLP method were used to resist KHV infection of the target nucleotide polymorphisms and the the Yellow River carp. .1. the Yellow River carp LGP2 gene has two selective splice bodies, the total length of LGP2a C DNA is 3061bp, the ORF open reading frame is located in 127th to 2066th bits, the peptide of 680 amino acids is encoded, the predicted molecular weight is 77341.2Da, and the isoelectric point is 6.53. prediction of four protein structures. Domain, including the conservative domain of bacterial III type restriction endonuclease, DEAD/DEAH box helicase domain, C terminal helicase superfamily and regulatory domain.LGP2b full length 1928bp, encoding 346 amino acids, 5 '- non translation region (UTR) 114 nucleotides, 3' UTR having 603 nucleotides, including AATAA termination signal. The Yellow River common carp LGP2 The total length DNA of the gene is 7655bp, with 12 exons and 11 introns, all the introns are beginning with GT and end of AG. In the LGP2 DNA full length and LGP2a and LGP2b m RNA comparison results, LGP2a and LGP2b are derived from the same sequence, and there are different selective clipping at the seventh exons and the partial sequences of the seventh introns The seventh exons were transcribed into seventh exons, and the 5 'flanking sequence of a complete M RNA sequence predicted that there were two promoters, and their activity could be induced by KHV. The results of virus infection experiment showed that LGP2 was helpful to the detection of 16 effective polymorphic loci of the positive regulatory.2.LGP2 of the cell congenital immune response mediated by DS DNA virus. There were 12 SNP loci and 4 loci with missing nucleotides; the frequencies of the -294GCT trinucleotide deletion and 414 GT dinucleotide deletion sites in the 5 'flanking region and the genotype and allele frequencies of the 3820 T/C and 3836T/G loci in the introns were significantly different between the resistant and susceptible groups. Two infection tests showed that -294 ins, 414 INS, 3820TT, 3836TT genotypes of the Yellow River carp were lower than -294 del, 414del, 3820CC, and 3836GG type the Yellow River carp, so the mutation of the four loci may be the genetic related risk factor of the Yellow River Cyprinus herpes virus disease.3.STING C DNA full length 1528bp, encoding the 402 amino acid polypeptide, the molecular weight is 46.184k, and the electron point is pushed. The measured STING protein contains a signal peptide. In the three transmembrane sequence and residing endoplasmic reticulum in the N- terminal region, there are four assumed base sequence (RXR).STING gene DNA full length 8068bp, which are composed of 7 exons and 6 introns. All the introns follow GT and the regular.5 '- flanking sequence is 901bp. The sequence is predicted. Promoter activity, its activity can be induced by KHV to strengthen.STING to be identified as a component of the congenital immune response of the Yellow River carp mediated by the DNA pathogen, and in vitro and in vivo, 14 effective polymorphic loci of STING, which play a key role in the forward regulation of downstream responses, are detected: 13 SNP loci and 1 deletion. Nucleotide sites; the -873G/C and -687CCT trinucleotide deletion sites in the 5 'flanking region; the genotype and allele frequencies of the 6767G/A and 7034C/T loci in exons were significantly different between the resistant and susceptible groups. The two viral stimulation experiments showed that the STING gene of the -873GG, -687ins, 6767GG and 7034CC genotypes of the Yellow River carp were respectively compared. The mortality rate of -873CC, -687del, 6767TT and 7034TT type the Yellow River carp is low (P0.05). It is considered that -873G/C, -687ins/del, 6767G/A and 7034C/T are closely related to the resistance phenotype of the Yellow River carp resistance to KHV infection resistance phenotype, and can be used as a molecular marker for disease resistance breeding. The KHV virus infection group of the Yellow River carp resistance group and the susceptible group's gills and spleen RNA were sequenced. The 4 tissue transcripts were sequenced and 190 million Raw reads were sequenced. After filtration, 135 million Clean reads were obtained. After assembly of Trinity software, 469251 transcripts and 366783 UniGene were obtained. The average length of UniGene was 667, the longest was 18437bp, the longest. There were 347659 sequences in the short 201bp. length of 200-600 BP, which accounted for 31837 of the 74.08%. alignment to the Nr database, and the highest consistency with zebrafish, followed by Mexico carp and rainbow trout, indicating the highest gene consistency with the cyprinid fishes. R with edge R package was used to screen the differentially expressed genes and the differential genes were entered. GO enrichment and KEGG pathway enrichment analysis, the resistance of spleen and gill and the number of different genes in the susceptible group are similar. The differences in the expression of 78 genes between the resistance group and the susceptible group from the natural immune recognition receptor pathway indicate that the response of the fish to the anti KHV virus in the spleen and the gills is different. A general survey of SNP, SSR and selective shear. The SNP loci in the gill and spleen resistance groups were less than those of the susceptible group; the SNP locus in the coded region was less than that in the non coding region, but the mutation of the coded region in the spleen was more than that in the non coding region; in each tissue transcript, the intentional mutation accounted for 43.1%-50.58% of the total SNP, while the unintentional mutation only accounted for the 0.11%-0.13%. library. The SNP converted from C to T is the most, followed by T conversion to C, and the least is that C is converted to G. to detect 31084 SSR in 168510 detection sequences, and the number of SSR is 25240, accounting for 15% of the total number of detection sequences, containing more than one SSR sequence of 4549, 18% of all SSR sequences, and different base SSR. For 1401, the sequences containing more than one SSR accounted for approximately 119 and 18 of the maximum and minimum length of 30.8%. repetition, respectively, and the average length of 24 nucleotides.SSR was mainly repeated in single nucleotide 9-12, while 4 nucleotides and 5 nucleotides were repeated at least 5-8. In this study, there were 16491 unigenes containing unigenes in comparison with unigenes. There are multiple transcripts (29.88%), which may be regulated by selective shear. In these unigenes, some different transcriptional transcripts, DET, 4183 of the total Unigene, provide an overview of the gills and spleens, the gills and spleen of the disease resistant group, and the first assessment of the immunology of the anti herpes virus disease of the Yellow River carp. It also laid a theoretical foundation for further molecular breeding of disease resistance.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S943
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本文编号：1817469