罗非鱼低溶氧耐受性状的遗传网络解析

发布时间：2018-06-17 01:08

本文选题：罗非鱼 + 低氧性状　；参考：《中山大学》2017年硕士论文

【摘要】：罗非鱼(Oreochromis niloticus)原产于非洲,形似鲫鱼,俗称非洲鲫鱼,是世界水产业重点培养的淡水鱼类,被誉为未来动物性蛋白质的主要来源之一。罗非鱼有很强的繁殖能力和环境适应能力。低氧对水生生物乃至整个水生生态具有严重的危害,是水产养殖面临的最为严重的挑战之一。然而关于鱼类低氧适应的分子机制仍不是很清楚。罗非鱼对低溶氧环境具有较强的适应性,是研究低溶氧问题的理想材料。本论文以罗非鱼为研究材料从三个角度对低氧性状的遗传网络进行了解析。我们首先对保守的低氧相关候选基因,HIF1α及其抑制剂HIF1αn,进行了克隆、结构和功能分析;其次,对罗非鱼低氧相关转录组差异表达基因和差异表达外显子进行了鉴定和功能分析;最后,对一个尼罗罗非鱼家系中控制低氧性状的基因组QTL进行了鉴定与分析。本研究获得的相关结果如下:1.低氧相关候选基因的克隆、结构和功能分析。对HIF1α基因及其抑制基因HIF1αn的全CDS序列进行了克隆测序,并分析了其基因和蛋白序列的保守结构特征。利用SigniSite 2.1Server分别在HIF1α和HIF1αn蛋白序列上找到21个和2个与水生和陆生表型显著相关的氨基酸位点。利用PAML软件对HIF1α和HIF1αn序列进行分析发现两个基因都不存在强的正选择效应,但HIF1α序列上部分位点可能受到选择压力。时空表达结果显示,低氧条件下HIF1α基因在不同组织不同低氧处理时间点具有复杂的调节模式,而HIF1αn基因在低氧下的表达总体上调。2.转录组差异表达的基因和外显子鉴定以及功能分析。对罗非鱼低氧处理12h的心脏组织和鳃组织进行了转录组测序。利用cuffdiff软件鉴定了低氧处理组和正常组差异表达的基因和外显子。在心脏组织中鉴定了284个差异表达基因和103个具有差异表达外显子的选择性剪接基因;在鳃组织中鉴定了239个差异表达基因和34个具有差异表达外显子的选择性剪接基因。荧光定量PCR实验成功验证了几乎所有选取的基因及外显子的差异表达。GO注释和Pathway通路分析揭示这些差异表达基因和选择性剪接基因在与能量保存、蛋白合成和免疫反应等相关的过程中显著富集。同时,还发现这些差异表达基因和选择性剪接基因存在不同的富集分类。研究说明两者在低氧反应中具有不同的调节作用。3.罗非鱼低氧耐受性状基因组QTL分析。利用QTLseq和ddRADseq两种高通量测序方法对罗非鱼低氧性状的遗传调控网络进行了分析。两种方法均在LG3和LG14连锁群上各鉴定了一个与低氧性状显著关联的QTL。为了验证鉴定的QTLs的准确性,在两个QTL区间内开发了4个微卫星标记。研究证实这两个QTL区间标记基因型/耐低氧表型间存在显著相关。进一步分析在QTL区间内发现两个低氧候选基因,即LG3 QTL区间的GPR132基因以及LG14QTL区间的ABCG4基因。对这两个基因进行结构和功能分析显示两个基因的部分外显子上的SNPs与低氧性状存在显著相关性。荧光定量PCR实验显示这两个基因的表达量与罗非鱼低氧耐受性相关。结果表明这两个基因很有可能与罗非鱼低氧耐受性状相关。通过采用多种方法对罗非鱼低氧遗传网络进行综合分析,我们鉴定了罗非鱼基因组低氧性状相关的重要QTL区间及候选功能基因,获得了性状紧密连锁的分子标记。该研究为进一步通过应用分子标记(或基因)辅助育种技术开展罗非鱼耐低氧新品种选育奠定了基础。
[Abstract]:Oreochromis niloticus, native to Africa, resembles crucian carp and is commonly known as the African crucian carp, is a major freshwater fish cultivated in the world's water industry. It is known as one of the main sources of the future animal protein. The tilapia has strong breeding ability and environmental adaptability. Hypoxia has serious danger to aquatic organisms and even the whole aquatic ecology. Damage is one of the most serious challenges in aquaculture. However, the molecular mechanism of hypoxia adaptation is still not very clear. Tilapia has strong adaptability to low dissolved oxygen environment. It is an ideal material for the study of low dissolved oxygen. This paper uses tilapia as a research material for the genetic network of hypoxic characters from three angles. Analysis. We first clone, structure and functional analysis of the conservative hypoxic related candidate genes, HIF1 alpha and its inhibitor HIF1 alpha n. Secondly, the differential expression gene and the differential expression exons of the low oxygen related transcriptional group of tilapia were identified and functional analysis were carried out, and then the low oxygen trait was controlled in a Nile tilapia family. The genomic QTL was identified and analyzed. The results obtained in this study were as follows: 1. cloning, structure and function analysis of low oxygen related candidate genes. The whole CDS sequence of the HIF1 alpha gene and its suppressor HIF1 alpha N was cloned and sequenced, and the conservative structural characteristics of the gene and protein sequence were analyzed. The SigniSite 2.1Server score was used. 21 and 2 amino acid sites associated with aquatic and terrestrial phenotypes were not found on the sequence of HIF1 alpha and HIF1 alpha N protein. The analysis of HIF1 alpha and HIF1 alpha n sequences by PAML software found that there was no strong positive selection effect in two genes, but some of the loci on HIF1 a sequence may be subjected to selective pressure. Under oxygen conditions, the HIF1 alpha gene has a complex regulation mode in different tissues and different hypoxia treatment time points, while the expression of HIF1 alpha N gene under hypoxia generally up-regulated the differentially expressed genes and exons of the.2. transcriptional group and the functional analysis. The transcriptional sequence of the cardiac tissue and gill tissue of 12h in the hypoxic treatment of tilapia was sequenced. C The uffdiff software identified the differentially expressed genes and exons in the hypoxia treatment group and the normal group. In the cardiac tissue, 284 differentially expressed genes and 103 selective splicing genes with differentially expressed exons were identified, and 239 differentially expressed genes and 34 selective splicing groups with differentially expressed exons were identified in the gill tissue. The difference expression of almost all the selected genes and exons was verified by the fluorescence quantitative PCR experiment. The.GO annotation and Pathway pathway analysis revealed that these differentially expressed genes and selective splicing genes were significantly enriched in the processes related to energy preservation, protein synthesis and immune response. There are different enrichment and classification of selective splicing genes. The study shows that both of them have different regulatory roles in the hypoxia response, and the QTL analysis of genomic.3. for low oxygen tolerance of tilapia. Two high throughput sequencing methods of QTLseq and ddRADseq were used to analyze the genetic regulation network of hypoxic traits of tilapia. The two methods were all in LG3 and in LG3. The LG14 chain identified a QTL. with a significant association with hypoxic traits to verify the accuracy of the identified QTLs. 4 microsatellite markers were developed within two QTL intervals. The study confirmed that there was a significant correlation between the two QTL interval markers / hypoxia tolerance phenotypes. Further segregation of two hypoxia candidate genes was found in the QTL interval. The GPR132 gene in the LG3 QTL interval and the ABCG4 gene in the LG14QTL interval. The structural and functional analysis of the two genes showed that there was a significant correlation between the SNPs and the hypoxia in the partial exons of the two genes. The fluorescence quantitative PCR test showed that the expression of the two genes was related to the hypoxia tolerance of the tilapia. The results showed that the expression of the two genes was related to the low oxygen tolerance of the tilapia. The two genes are likely to be related to the low oxygen tolerance of tilapia. By using a variety of methods to analyze the low oxygen genetic network of tilapia, we identified the important QTL interval and candidate functional genes related to the hypoxic traits of the tilapia, and obtained the molecular markers for the tight linkage of the traits. Molecular marker (or gene) assisted breeding technology was used to breed new varieties of tilapia resistant to hypoxia.
【学位授予单位】：中山大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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