本文選題：黃河鯉 + 基因克隆��； 參考：《西北農(nóng)林科技大學(xué)》2017年博士論文

【摘要】：豫選黃河鯉(Cyprinus carpio haematopterus)是由河南省水產(chǎn)科學(xué)研究院采用野生黃河鯉做親本,經(jīng)過(guò)近20年、連續(xù)8代精心繁育而成。迄今已得到廣泛推廣,有著顯著的經(jīng)濟(jì)和社會(huì)效益。然而,近幾年,黃河鯉各類疾病的頻發(fā),使黃河鯉養(yǎng)殖風(fēng)險(xiǎn)大大增大,造成嚴(yán)重的經(jīng)濟(jì)損失,明顯制約了該產(chǎn)業(yè)的可持續(xù)健康發(fā)展。感染錦鯉皰疹病毒(Koi herpesvirus,KHV)的黃河鯉具高度傳染性和極高的致死率。直到目前仍沒(méi)有好的防御措施和治愈手段。因而了解黃河鯉自身免疫系統(tǒng)對(duì)這種病毒的防御功能和培育抗病的優(yōu)良品種顯得尤為重要,也是維持健康水產(chǎn)養(yǎng)殖業(yè)的關(guān)鍵所在。先天免疫識(shí)別受體及信號(hào)通路相關(guān)基因在病毒檢測(cè)、干擾素誘導(dǎo)、保護(hù)機(jī)體免受侵害等方面具有關(guān)鍵作用。LGP2作為RIG-I樣模式識(shí)別受體的家族成員之一,首先識(shí)別存在于細(xì)胞質(zhì)中的病毒DNA或RNA,隨后下游系列信號(hào)級(jí)聯(lián)反應(yīng)被激活,誘導(dǎo)產(chǎn)生I型干擾素和促炎性因子。干擾素基因的刺激基因STING是宿主細(xì)胞中DNA病毒識(shí)別的感知器,通過(guò)RIG-TBK1-IRF3/IRF7信號(hào)級(jí)聯(lián)來(lái)誘導(dǎo)產(chǎn)生干擾素,對(duì)DNA病原體做出先天免疫反應(yīng)。為研究上述免疫相關(guān)基因的分子特性和抗病毒機(jī)制,本研究通過(guò)基因克隆及病毒感染,鑒定和分析了LGP2、STING基因和KHV病毒體內(nèi)外感染后基因表達(dá)譜以及poly(I:C)和poly(d A:d T)配體刺激CFC(尾鰭細(xì)胞)后的基因表達(dá)情況,探究LGP2和STING在病毒識(shí)別及抗病毒級(jí)聯(lián)反應(yīng)中的作用。為進(jìn)一步探究LGP2和STING基因與抗KHV的關(guān)聯(lián)性及遺傳特性,本研究提取感染KHV病毒的黃河鯉脾臟的DNA,利用特異PCR和染色體步移技術(shù)獲得LGP2和STING基因全長(zhǎng)及5’側(cè)翼序列,并應(yīng)用生物信息學(xué)和生物統(tǒng)計(jì)軟件SPSS等工具對(duì)克隆所得目標(biāo)基因的結(jié)構(gòu)特征及遺傳信息進(jìn)行系統(tǒng)的比較分析;通過(guò)構(gòu)建表達(dá)載體,驗(yàn)證啟動(dòng)子活性;以及利用基因池混合測(cè)序和PCR-RFLP方法,對(duì)目的基因的核苷酸多態(tài)位點(diǎn)與黃河鯉抗KHV感染進(jìn)行關(guān)聯(lián)分析,并對(duì)其表型關(guān)聯(lián)位點(diǎn)進(jìn)行驗(yàn)證。1.黃河鯉LGP2基因存在兩個(gè)選擇性剪接體,LGP2a c DNA全長(zhǎng)為3061bp,ORF開(kāi)放閱讀框位于第127位到第2066位核苷酸,編碼680個(gè)氨基酸的多肽,預(yù)測(cè)分子量為77,341.2Da,等電點(diǎn)為6.53。預(yù)測(cè)四個(gè)蛋白質(zhì)結(jié)構(gòu)域,包括細(xì)菌III型限制性內(nèi)切酶的保守結(jié)構(gòu)域,DEAD/DEAH盒解旋酶結(jié)構(gòu)域,C末端解旋酶超家族結(jié)構(gòu)域和調(diào)節(jié)結(jié)構(gòu)域。LGP2b的全長(zhǎng)為1928bp,編碼346個(gè)氨基酸,5’-非翻譯區(qū)(UTR)為114個(gè)核苷酸,3’UTR有603個(gè)核苷酸,其中包括AATAA終止信號(hào)。黃河鯉LGP2基因全長(zhǎng)DNA為7655bp,有12個(gè)外顯子,11個(gè)內(nèi)含子,所有內(nèi)含子均是由GT開(kāi)頭,AG結(jié)尾的,在LGP2 DNA全長(zhǎng)與LGP2a和LGP2b m RNA比對(duì)結(jié)果顯示,LGP2a和LGP2b來(lái)源于同一條DNA序列,在第七外顯子處m RNA序列出現(xiàn)不同的選擇性剪切,將第七內(nèi)含子的部分序列轉(zhuǎn)錄為第七外顯子,形成一條完整的m RNA序列。LGP2的5’側(cè)翼序列預(yù)測(cè)有兩個(gè)啟動(dòng)子,并且其活性均能被KHV誘導(dǎo)加強(qiáng)。病毒感染實(shí)驗(yàn)結(jié)果表明,LGP2有助于ds DNA病毒介導(dǎo)的細(xì)胞先天免疫應(yīng)答的正調(diào)節(jié)。2.LGP2的16個(gè)有效的多態(tài)位點(diǎn)被探測(cè)到:其中包括12個(gè)SNP位點(diǎn)、4個(gè)有缺失核苷酸的位點(diǎn);其中5’側(cè)翼區(qū)的-294GCT三核苷酸缺失和414 GT二核苷酸缺失位點(diǎn)以及內(nèi)含子中的3820 T/C和3836T/G位點(diǎn)的基因型和等位基因頻率在抗性組和易感組間差異顯著。經(jīng)二次感染驗(yàn)證表明,-294 ins、414 ins、3820TT,3836TT基因型的黃河鯉分別比-294 del、414del,3820CC,3836GG型的黃河鯉死亡率低。因此該四個(gè)位點(diǎn)的突變可能是黃河鯉皰疹病毒病的遺傳相關(guān)的危險(xiǎn)因素。3.STING c DNA全長(zhǎng)1528bp,編碼402個(gè)氨基酸的多肽,分子量為46.184k Da,等電子點(diǎn)為6.08。推測(cè)的STING蛋白含有信號(hào)肽,N-末端區(qū)域中的三個(gè)跨膜基序和駐留的內(nèi)質(zhì)網(wǎng)蛋白中發(fā)現(xiàn)存在四個(gè)假定基序(RXR)。STING基因DNA全長(zhǎng)為8068bp,由7個(gè)外顯子和6個(gè)內(nèi)含子組成,所有內(nèi)含子均遵循GT開(kāi)頭,AG結(jié)尾的規(guī)律。5’-側(cè)翼序列為901bp,序列預(yù)測(cè)有啟動(dòng)子活性,其活性可被KHV誘導(dǎo)加強(qiáng)。STING被鑒定為由DNA病原體介導(dǎo)的黃河鯉先天免疫應(yīng)答的組成部分,并且在體外和體內(nèi)都證實(shí)了STING對(duì)下游反應(yīng)的正向調(diào)節(jié)中起關(guān)鍵作用。4.STING的14個(gè)有效的多態(tài)位點(diǎn)被探測(cè)到:13個(gè)SNP位點(diǎn)、1個(gè)有缺失核苷酸的位點(diǎn);其中5’側(cè)翼區(qū)的-873G/C和-687CCT三核苷酸缺失位點(diǎn);外顯子中的6767G/A和7034C/T位點(diǎn)的基因型和等位基因頻率在抗性組和易感組間差異顯著。二次病毒刺激實(shí)驗(yàn)表明,STING基因的-873GG、-687ins、6767GG和7034CC基因型的黃河鯉分別比-873CC、-687del、6767TT和7034TT型的黃河鯉死亡率低(P0.05),可以認(rèn)為-873G/C、-687ins/del、6767G/A和7034C/T與黃河鯉抗KHV感染抗性-易感表型顯著相關(guān),可以作為抗病育種的分子標(biāo)記。5.為了研究黃河鯉對(duì)KHV病毒的抗病防御機(jī)制和抗KHV的關(guān)聯(lián)性,本研究將KHV病毒感染后黃河鯉抗性組和易感組的鰓和脾臟的RNA進(jìn)行轉(zhuǎn)錄組測(cè)序,4個(gè)組織轉(zhuǎn)錄組測(cè)序共獲得1.90億個(gè)Raw reads,過(guò)濾后得到1.35億Clean reads,經(jīng)Trinity軟件組裝后,得到469,251個(gè)transcripts和366,783個(gè)unigene,unigene平均長(zhǎng)度667,最長(zhǎng)為18437bp,最短為201bp。其中長(zhǎng)度在200-600 bp的序列有347659條,占74.08%。比對(duì)到Nr數(shù)據(jù)庫(kù)的序列有31,837條,且與斑馬魚(yú)一致性最高,其次是墨西哥麗脂鯉和虹鱒,說(shuō)明與鯉科魚(yú)類的基因一致性最高。采用R with edge R package篩選差異表達(dá)基因,并對(duì)差異基因進(jìn)行GO富集和KEGG通路富集分析,脾臟和鰓的抗性和易感組的差異基因數(shù)量相似,從天然免疫識(shí)別受體途徑中選出78個(gè)基因在抗性組和易感組之間的表達(dá)差異表明,魚(yú)類在脾中和鰓中抗KHV病毒的反應(yīng)是不同的。本研究還對(duì)轉(zhuǎn)錄組文庫(kù)中的基因進(jìn)行SNP、SSR和選擇性剪切進(jìn)行概況分析。無(wú)論是鰓還是脾抗病組的SNP位點(diǎn)少于易感組;編碼區(qū)SNP位點(diǎn)少于非編碼區(qū),但脾臟中編碼區(qū)突變多于非編碼區(qū);每個(gè)組織轉(zhuǎn)錄組中,有意突變占總SNP的43.1%-50.58%,而無(wú)意突變僅占0.11%-0.13%。文庫(kù)中由C轉(zhuǎn)換為T的SNP最多,其次是由T轉(zhuǎn)換為C,最少的是由C轉(zhuǎn)換為G。本數(shù)據(jù)庫(kù)中從檢測(cè)序列168,510條中檢測(cè)出31,084條存在SSR,而包含SSR的序列數(shù)為25,240,約占總檢測(cè)序列數(shù)的15%,包含多于一個(gè)SSR的序列為4,549條,占所有SSR序列的18%;不同堿基SSR為1,401,含一個(gè)以上SSR的序列約占30.8%。重復(fù)的最大和最小長(zhǎng)度分別為119和18,平均長(zhǎng)度為24個(gè)核苷酸。SSR的模序類型主要以單核苷酸9-12重復(fù)最多,而4核苷酸及5核苷酸5-8重復(fù)最少。在本研究中,經(jīng)transcripts與unigenes比對(duì),共有16,491個(gè)unigenes含有多個(gè)轉(zhuǎn)錄本(29.88%),可能受選擇性剪切調(diào)節(jié)。這些unigenes中,某些不同表達(dá)轉(zhuǎn)錄本的基因DET共4,183占總Unigene的7.58%。本實(shí)驗(yàn)結(jié)果提供易感組鰓和脾臟以及抗病組鰓和脾臟的轉(zhuǎn)錄組概況,為黃河鯉抗皰疹病毒病的免疫生物學(xué)的第一次評(píng)估、也為后續(xù)的抗病分子育種奠定了理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S943
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本文編號(hào)：1817469