凡纳滨对虾CAT基因和SOD基因单核苷酸多态性及与抗逆性状的相关性分析

发布时间：2018-06-06 18:01

本文选题：过氧化氢酶基因 + 超氧化物歧化酶基因　；参考：《广东海洋大学》2017年硕士论文

【摘要】：凡纳滨对虾(Litopenaeus vannamei)也称南美白对虾,具有生长快、生长周期短、营养要求低、抗病能力强、适应能力强、适合高密度养殖等优点,是我国养殖密度最大、产量最高、养殖范围最广的对虾养殖品种。本研究以凡纳滨对虾的过氧化氢酶CAT基因和超氧化物歧化酶SOD基作为研究对象,利用直接测序法对两个基因进行多态性分析,同时将所得到SNP位点与凡纳滨对虾的低溶氧耐受性状进行相关性分析,以筛选出与凡纳滨对虾低溶氧耐受性状具有关联性的分子遗传标记。最后采用实时荧光定量RT-PCR技术检测在不同胁迫和恢复条件下,不同家系SOD基因和CAT基因表达量的差异,旨在为凡纳滨对虾的低溶氧耐受性和高氨氮耐受性研究提供分子方面的理论依据。具体研究结果如下:1.凡纳滨对虾CAT基因和SOD基因的SNPs及与低溶氧耐受性状的关联分析采用直接测序法研究凡纳滨对虾低溶氧敏感组和耐受组超氧化物歧化酶基因和过氧化氢酶基因的单核苷酸多态性,在凡纳滨对虾CAT基因的编码区中发现1个SNP位点,位于基因全长155bp处,发生A→G突变(CATg.155AG);在SOD基因的编码区中发现2个SNP位点,分别位于基因全长1575bp处,发生A→G突变(SODg.1575AG)和1602bp处,发生C→T突变(SODg.1602CT)。3个SNP位点的突变均为同义突变。对SNP位点进行遗传多样性参数分析,结果表明:CAT g.155AG、SODg.1575AG和SODg.1602CT 3个SNP位点预期杂合度He的范围为0.244~0.393,观测杂合度Ho的范围为0.281~0.344,均小于0.5,且3个SNP位点均符合哈迪-温伯格平衡(HWE)(P0.05)。在多态信息含量PIC方面,CATg.155AG突变位点和SODg.1575AG突变位点的PIC值均小于0.25,处于低度多态(PIC0.25),而SODg.1602CT位点的PIC值为0.314,处于中度多态(0.25PIC0.5)。分别将3个SNP位点与凡纳滨对虾低溶氧耐受性状进行关联性分析,结果表明:CAT g.155AG位点和SODg.1602CT位点与低溶氧耐受性存在着显著相关性(P0.05),而SODg.1575AG位点与低溶氧耐受性不存在相关性(P0.05);在CATg.155AG位点中AG基因型的个体与GG基因型的个体相比具有较高的低溶氧耐受性,可推测CATg.155AG位点A基因为凡纳滨对虾耐低溶氧性状的增效基因;SODg.1602CT位点在应激耐受组中基因型为CT和TT的个体数目比应激敏感组的个体数要多,CT基因型和TT基因型的个体与CC基因型的个体相比具有较高的低溶氧耐受性,可推测SODg.1602CT位点T基因为凡纳滨对虾耐低溶氧性状的增效基因。2.低溶氧胁迫和恢复条件下凡纳滨对虾SOD基因和CAT基因表达量的变化利用实时荧光定量RT-PCR技术检测了在低溶氧胁迫48h和恢复24h条件下,凡纳滨对虾低溶氧耐受家系和低溶氧敏感家系SOD基因和CAT基因在肝胰脏组织中的表达情况。结果显示,在低溶氧胁迫条件下,低溶氧敏感家系SOD基因和CAT基因的表达量均呈现出先降后升再降的波动趋势,而低溶氧耐受家系两基因的表达量均表现出先升再降的趋势。低氧胁迫6h时,低溶氧敏感家系SOD基因和CAT基因的表达量显著下降趋势,在胁迫12h时上升至最高值,胁迫24h至胁迫48h时SOD基因和CAT基因的表达量又逐步下降;在低溶氧耐受家系中低氧胁迫6h时两基因的表达量都表现出显著上升趋势,胁迫12h时后两基因的表达量都明显降低。在恢复条件下两家系的SOD基因表达量都表现出先上升后下降的趋势,而CAT基因的表达量则为先降再升趋势。3.高氨氮胁迫和恢复条件下凡纳滨对虾SOD基因和CAT基因表达量的变化利用实时荧光定量RT-PCR技术检测了在高氨氮胁迫48h和恢复24h条件下,凡纳滨对虾高氨氮耐受家系和高氨氮敏感家系SOD基因和CAT基因在肝胰脏组织中的表达情况。结果显示,在高氨氮胁迫时,高氨氮耐受家系和高氨氮敏感家系SOD基因的表达量均呈现先升后降的趋势,在氨氮胁迫6h时SOD基因的表达量显著上升,而胁迫48h时表达量降到最低值;两家系CAT基因表达量表现出持续下降趋势;在恢复24h内,SOD基因和CAT基因表达量均逐步上升的。
[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.
【学位授予单位】：广东海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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