本文選題：過氧化氫酶基因 + 超氧化物歧化酶基因　； 參考：《廣東海洋大學(xué)》2017年碩士論文

【摘要】：凡納濱對蝦(Litopenaeus vannamei)也稱南美白對蝦,具有生長快、生長周期短、營養(yǎng)要求低、抗病能力強(qiáng)、適應(yīng)能力強(qiáng)、適合高密度養(yǎng)殖等優(yōu)點(diǎn),是我國養(yǎng)殖密度最大、產(chǎn)量最高、養(yǎng)殖范圍最廣的對蝦養(yǎng)殖品種。本研究以凡納濱對蝦的過氧化氫酶CAT基因和超氧化物歧化酶SOD基作為研究對象,利用直接測序法對兩個(gè)基因進(jìn)行多態(tài)性分析,同時(shí)將所得到SNP位點(diǎn)與凡納濱對蝦的低溶氧耐受性狀進(jìn)行相關(guān)性分析,以篩選出與凡納濱對蝦低溶氧耐受性狀具有關(guān)聯(lián)性的分子遺傳標(biāo)記。最后采用實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測在不同脅迫和恢復(fù)條件下,不同家系SOD基因和CAT基因表達(dá)量的差異,旨在為凡納濱對蝦的低溶氧耐受性和高氨氮耐受性研究提供分子方面的理論依據(jù)。具體研究結(jié)果如下:1.凡納濱對蝦CAT基因和SOD基因的SNPs及與低溶氧耐受性狀的關(guān)聯(lián)分析采用直接測序法研究凡納濱對蝦低溶氧敏感組和耐受組超氧化物歧化酶基因和過氧化氫酶基因的單核苷酸多態(tài)性,在凡納濱對蝦CAT基因的編碼區(qū)中發(fā)現(xiàn)1個(gè)SNP位點(diǎn),位于基因全長155bp處,發(fā)生A→G突變(CATg.155AG);在SOD基因的編碼區(qū)中發(fā)現(xiàn)2個(gè)SNP位點(diǎn),分別位于基因全長1575bp處,發(fā)生A→G突變(SODg.1575AG)和1602bp處,發(fā)生C→T突變(SODg.1602CT)。3個(gè)SNP位點(diǎn)的突變均為同義突變。對SNP位點(diǎn)進(jìn)行遺傳多樣性參數(shù)分析,結(jié)果表明:CAT g.155AG、SODg.1575AG和SODg.1602CT 3個(gè)SNP位點(diǎn)預(yù)期雜合度He的范圍為0.244~0.393,觀測雜合度Ho的范圍為0.281~0.344,均小于0.5,且3個(gè)SNP位點(diǎn)均符合哈迪-溫伯格平衡(HWE)(P0.05)。在多態(tài)信息含量PIC方面,CATg.155AG突變位點(diǎn)和SODg.1575AG突變位點(diǎn)的PIC值均小于0.25,處于低度多態(tài)(PIC0.25),而SODg.1602CT位點(diǎn)的PIC值為0.314,處于中度多態(tài)(0.25PIC0.5)。分別將3個(gè)SNP位點(diǎn)與凡納濱對蝦低溶氧耐受性狀進(jìn)行關(guān)聯(lián)性分析,結(jié)果表明:CAT g.155AG位點(diǎn)和SODg.1602CT位點(diǎn)與低溶氧耐受性存在著顯著相關(guān)性(P0.05),而SODg.1575AG位點(diǎn)與低溶氧耐受性不存在相關(guān)性(P0.05);在CATg.155AG位點(diǎn)中AG基因型的個(gè)體與GG基因型的個(gè)體相比具有較高的低溶氧耐受性,可推測CATg.155AG位點(diǎn)A基因?yàn)榉布{濱對蝦耐低溶氧性狀的增效基因;SODg.1602CT位點(diǎn)在應(yīng)激耐受組中基因型為CT和TT的個(gè)體數(shù)目比應(yīng)激敏感組的個(gè)體數(shù)要多,CT基因型和TT基因型的個(gè)體與CC基因型的個(gè)體相比具有較高的低溶氧耐受性,可推測SODg.1602CT位點(diǎn)T基因?yàn)榉布{濱對蝦耐低溶氧性狀的增效基因。2.低溶氧脅迫和恢復(fù)條件下凡納濱對蝦SOD基因和CAT基因表達(dá)量的變化利用實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測了在低溶氧脅迫48h和恢復(fù)24h條件下,凡納濱對蝦低溶氧耐受家系和低溶氧敏感家系SOD基因和CAT基因在肝胰臟組織中的表達(dá)情況。結(jié)果顯示,在低溶氧脅迫條件下,低溶氧敏感家系SOD基因和CAT基因的表達(dá)量均呈現(xiàn)出先降后升再降的波動(dòng)趨勢,而低溶氧耐受家系兩基因的表達(dá)量均表現(xiàn)出先升再降的趨勢。低氧脅迫6h時(shí),低溶氧敏感家系SOD基因和CAT基因的表達(dá)量顯著下降趨勢,在脅迫12h時(shí)上升至最高值,脅迫24h至脅迫48h時(shí)SOD基因和CAT基因的表達(dá)量又逐步下降;在低溶氧耐受家系中低氧脅迫6h時(shí)兩基因的表達(dá)量都表現(xiàn)出顯著上升趨勢,脅迫12h時(shí)后兩基因的表達(dá)量都明顯降低。在恢復(fù)條件下兩家系的SOD基因表達(dá)量都表現(xiàn)出先上升后下降的趨勢,而CAT基因的表達(dá)量則為先降再升趨勢。3.高氨氮脅迫和恢復(fù)條件下凡納濱對蝦SOD基因和CAT基因表達(dá)量的變化利用實(shí)時(shí)熒光定量RT-PCR技術(shù)檢測了在高氨氮脅迫48h和恢復(fù)24h條件下,凡納濱對蝦高氨氮耐受家系和高氨氮敏感家系SOD基因和CAT基因在肝胰臟組織中的表達(dá)情況。結(jié)果顯示,在高氨氮脅迫時(shí),高氨氮耐受家系和高氨氮敏感家系SOD基因的表達(dá)量均呈現(xiàn)先升后降的趨勢,在氨氮脅迫6h時(shí)SOD基因的表達(dá)量顯著上升,而脅迫48h時(shí)表達(dá)量降到最低值;兩家系CAT基因表達(dá)量表現(xiàn)出持續(xù)下降趨勢;在恢復(fù)24h內(nèi),SOD基因和CAT基因表達(dá)量均逐步上升的。
[Abstract]:Penaeus vannamei (Litopenaeus vannamei), also known as Penaeus vannamei, has the advantages of fast growth, short growth cycle, low nutrition requirements, strong resistance to disease, strong adaptability, and suitable for high density breeding. It is the largest aquaculture breed in China with the largest density, the highest yield and the wide range of aquaculture. This study is based on the catalase CAT of Penaeus vannamei. The gene and superoxide dismutase (SOD) SOD base was used as the research object, and the two genes were analyzed by direct sequencing. At the same time, the correlation between the SNP site and the low dissolved oxygen tolerance of Penaeus vannamei was analyzed in order to screen the molecular genetic markers associated with the low dissolved oxygen tolerance of Penaeus vannamei. Real time fluorescence quantitative RT-PCR was used to detect the difference of SOD gene and CAT gene expression in different families under different stress and recovery conditions. The aim is to provide the theoretical basis for the low oxygen tolerance and high ammonia tolerance study of Penaeus vannamei. The results are as follows: 1. the CAT gene and SOD gene of Penaeus vannamei. SNPs and the correlation analysis with low dissolved oxygen tolerance characteristics, direct sequencing was used to study the single nucleotide polymorphism of superoxide dismutase (SOD) gene and catalase gene in the low dissolved oxygen sensitive group and tolerance group of Penaeus vannamei, and 1 SNP loci were found in the coding region of the CAT gene of Penaeus vannamei, located at the full length of 155bp and A to G. Mutation (CATg.155AG); 2 SNP loci were found in the coding region of the SOD gene, located at the total length of 1575bp, A to G mutation (SODg.1575AG) and 1602bp. The mutation of C to T mutation (SODg.1602CT) was all synonymous mutation. The range of expected heterozygosity He of 3 SNP loci of SODg.1602CT was 0.244~0.393, and the range of heterozygosity Ho was 0.281~0.344, less than 0.5, and 3 SNP loci were consistent with Hardy Weinberg equilibrium (HWE) (P0.05). In PIC of polymorphic information content, CATg.155AG and SODg.1575AG mutation sites were lower than 0.25, at a low degree. Polymorphism (PIC0.25), while the PIC value of SODg.1602CT site was 0.314, and was in moderate polymorphism (0.25PIC0.5). The correlation analysis between 3 SNP loci and low dissolved oxygen tolerance of Penaeus vannamei was analyzed. The results showed that there was a significant correlation between CAT g.155AG site and SODg.1602CT site and low dissolved oxygen tolerance (P0.05), while SODg.1575AG loci and low levels were low. There is no correlation between dissolved oxygen tolerance (P0.05); in the CATg.155AG locus, the AG genotype individuals have higher low dissolved oxygen tolerance compared with those of the GG genotype, and the CATg.155AG loci A gene is an synergistic gene for the low dissolved oxygen tolerance of Penaeus vannamei, and the SODg.1602CT site in the stress tolerance group is the individual number of CT and TT. The number of individuals in the stress sensitive group was more than that of the stress sensitive group. The CT genotype and the TT genotype had a higher low solubility in oxygen tolerance compared with those of the CC genotype, and the SODg.1602CT locus T based on the low dissolved oxygen stress of the low dissolved oxygen trait of Penaeus vannamei,.2. and the SOD gene and CAT gene expression in the recovery strip of Penaeus vannamei. The expression of SOD and CAT genes in low dissolved oxygen tolerant pedigrees and low dissolved oxygen sensitive pedigrees of Penaeus vannamei and low dissolved oxygen sensitive families in hepatopancreas under low dissolved oxygen stress 48h and 24h were detected by real time fluorescence quantitative RT-PCR. The results showed that the SOD gene and CAT base of low dissolved oxygen sensitive family under low dissolved oxygen stress. The expression of the two genes in the low dissolved oxygen tolerant family two showed a tendency to rise first and then descend. When low oxygen stress 6h, the expression of SOD and CAT genes in the low dissolved oxygen sensitive family decreased significantly, and increased to the highest value at the time of stress 12h, and stressed the SOD gene from 24h to the stress 48h. And the expression of CAT gene decreased gradually; the expression of the two gene in the low oxygen tolerant family 6h showed a significant increase in the expression of the two gene, and the expression of the two gene was obviously reduced when the stress was stressed. The expression of the SOD gene expression in the two lines showed a trend of rising and descending, and the expression of the CAT gene. The changes of SOD gene and CAT gene expression of Penaeus vannamei in.3. high ammonia nitrogen stress and recovery conditions were measured by real time fluorescence quantitative RT-PCR technique. The SOD gene and CAT gene of high ammonia nitrogen tolerant family and high ammonia nitrogen sensitive family of Penaeus vannamei were detected under the condition of high ammonia nitrogen stress and 24h, and the SOD gene and CAT gene in the hepatopancreas were in the liver and pancreas. The expression of SOD gene in high ammonia nitrogen tolerant family and high ammonia nitrogen sensitive family showed a tendency to rise first and then descend when high ammonia nitrogen stress was stressed. The expression of SOD gene increased significantly when ammonia nitrogen stress was 6h, while the expression of 48h was reduced to the lowest value, and the expression of CAT gene expression in two lines showed a continuous decline. The expression of SOD and CAT genes gradually increased in 24h.
【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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本文編號：1987622