仿刺参热胁迫响应的分子调控特征研究与SNP标记筛选

发布时间：2018-06-30 02:55

  本文选题：仿刺参 + 热胁迫　； 参考：《中国科学院研究生院(海洋研究所)》2016年博士论文

【摘要】：仿刺参(Apostichopus japonicus),又被称为刺参,是我国重要的经济物种,具有较高的营养价值和药用价值。水温是影响刺参生长和代谢的最重要的环境因素之一。近年来,由于极端温度的影响,在养殖池塘和浅海围堰更是出现了刺参的大面积死亡现象,造成惨重的资源和经济损失。因此,研究刺参对热胁迫的响应机制和刺参耐高温品种的选育工作具有非常重要的意义。本研究利用iTRAQ蛋白质组学技术、Real time PCR、Western blot等技术对刺参热胁迫的响应机制展开研究,并筛选与刺参耐热性状相互关联的SNP分子标记。探索了热胁迫下刺参肠道在组织、细胞和HSP70蛋白水平的变化;开展刺参热胁迫下iTRAQ蛋白质组学分析,揭示了热胁迫下刺参肠道组织蛋白水平的全局变化;获得关键响应基因hsf1、hsbp1、hsp60和hsp10的cDNA序列,探讨HSPs在热胁迫下表达特点及调控机制;筛选hsp90基因序列中与耐热性状关联的SNP分子标记,并应用于耐高温刺参品种的选育。主要研究结果如下:1、刺参肠道对热胁迫的组织学、细胞学和HSP70蛋白的响应热胁迫下,刺参肠道组织出现明显的组织退化,表现为肠道组织层厚度(粘膜层、粘膜下层和肌肉层)减小、环形皱襞的数目减少。刺参肠道细胞在热胁迫下出现明显的细胞凋亡现象,表现为线粒体和囊泡的数目减少、染色质聚集,甚至出现细胞质和细胞器流失的空细胞。HSP70免疫组化的结果显示,HSP70蛋白表达具有组织特异性,集中分布在粘膜层和浆膜层;常温下HSP70的染色为细胞质染色,热胁迫下细胞质和细胞核均有染色。HSP70的Western blot的结果显示,HSP70蛋白的表达水平随热胁迫持续时间一直增加,在热胁迫下对细胞起重要的保护作用。2、刺参热胁迫下iTRAQ蛋白质组学研究利用iTRAQ技术,分析了刺参肠道组织26°C热胁迫48 h后蛋白表达情况。结果显示,热胁迫下共有127种蛋白的表达量发生了显著变化,其中包括61个上调蛋白和66个下调蛋白。这些差异蛋白主要参与了组织保护和解毒、脂肪酸和氨基酸代谢、能量产生和利用、转录和翻译、细胞凋亡和细胞增殖等细胞活动。3、刺参热休克反应关键基因的克隆和表达(1)获得热休克反应的调控基因hsf1和hsbp1的cDNA全长。hsf1、hsbp1和hsp70的mRNA表达结果表明:热胁迫下三者表达量随热胁迫持续时间先增加后减小,均在热胁迫6 h达到峰值,表达量分别是对照组的11.67倍、4.71倍和955.30倍。刺参的hsf1在mRNA表达量方面会对热胁迫作出迅速的响应,有利于大量单体的结合,启动热休克反应。我们推测hsbp1和hsp70通过影响HSF1活性,对热休克反应起负面调节的作用。(2)获得热休克反应的重要基因hsp10和hsp60的cDNA全长。热胁迫下hsp10和hsp60的mRNA水平表达结果表明,热胁迫两者表达量随热胁迫持续时间先增加后减小,表达趋势相似:基因hsp10和hsp60的mRNA表达量均在热胁迫后2 h就显著上升,在8 h时达到峰值,最大表达量分别为193.3倍和60.0倍。HSP10作为HSP60的辅助伴侣蛋白,两者协同合作在热休克反应中起重要作用。4、刺参hsp90基因序列中多态性位点的筛选及其与耐热性状的关联分析获得hsp90基因序列6939 bp,含有外显子10个,内含子9个,多态性位点SNP共22个。通过32°C高温胁迫实验,获得对高温敏感和耐受的各50头刺参个体。对敏感组和耐受组个体进行基因分型,通过卡方检验得到5个在两组分布显著差异的SNP标记。进一步验证实验结果表明:SNP e10-1和SNP e11-6的基因型和等位基因分布在耐高温品系和普通群体中存在显著差异,其中SNP e10-6为三态SNP;SNP e2-3的等位基因分布在两个群体中也存在显著差异。这是在刺参中首次筛选到与耐温性状关联的SNP标记,并已应用于刺参耐高温选育中。
[Abstract]:Apostichopus japonicus, also known as the cucumbers, is an important economic species in China and has high nutritional and medicinal value. Water temperature is one of the most important environmental factors that affect the growth and metabolism of the cucumbers. In recent years, the large area of the japonicus japonicus appeared in the breeding ponds and the shallow sea cofferdams due to the extreme temperature. The phenomenon of death caused heavy resources and economic losses. Therefore, it is very important for the study of the response mechanism of the heat stress and the selection and breeding of the high temperature resistant varieties of the ginseng. This study uses iTRAQ proteomics technology, Real time PCR, Western blot and other techniques to study the response mechanism of the heat stress of the ginseng and screening and screening. SNP molecular markers linked to the heat resistance of the cucumbers were investigated. The changes in the tissue, cell and HSP70 protein levels of the intestine of the cucumbers under heat stress were explored, and the iTRAQ proteomic analysis under the heat stress of the cucumbers revealed the global changes in the protein level of the intestinal tissue under the heat stress, and the cD of the key response genes, HSF1, HSBP1, HSP60 and hsp10, was obtained. The NA sequence was used to investigate the expression and regulation mechanism of HSPs under heat stress; to screen the SNP molecular markers associated with the heat resistant character in the Hsp90 gene sequence and to apply it to the breeding of high temperature resistant varieties of the cucumbers. The main results are as follows: 1, the histology of heat stress in the intestine of the Cucumis japonicus and the response of the HSP70 protein to the intestinal tissue under the response to the heat stress of the HSP70 protein There were obvious tissue degeneration, which showed that the thickness of the intestinal tissue (the mucosa, the submucosa and the muscle layer) decreased and the number of ring folds decreased. The apoptosis of the intestinal cells in the intestinal tract was obvious under heat stress, which showed the decrease in the number of mitochondria and vesicles, the accumulation of chromatin, and even the loss of cytoplasm and organelles. The results of.HSP70 immunohistochemical staining showed that the expression of HSP70 protein was tissue specific and concentrated in the mucosa and serous layer. The staining of HSP70 at normal temperature was cytoplasmic staining. The result of Western blot staining of.HSP70 in cytoplasm and nucleus under heat stress showed that the expression level of HSP70 protein was consistent with the duration of heat stress. In addition,.2 plays an important role in the protection of cells under heat stress. ITRAQ proteomics under the heat stress of the Cucumis japonicus was used to analyze the expression of protein expression after 26 degrees of heat stress of 48 h in the intestinal tissue of the cucumbers. The results showed that there were significant changes in the expression of 127 proteins under heat stress, including 61 up-regulated proteins and 66 H. These differential proteins are mainly involved in tissue protection and detoxification, fatty acid and amino acid metabolism, energy production and utilization, transcription and translation, cell apoptosis and cell proliferation, such as cell activity.3, the cloning and expression of key genes of the heat shock response (1) obtained the control gene HSF1 and cDNA full length.H of the heat shock response. The mRNA expression of SF1, HSBP1 and HSP70 showed that the expression of three in heat stress increased first and then decreased with the duration of heat stress, and reached the peak value at 6 h in the heat stress, and the expression amount was 11.67 times, 4.71 times and 955.30 times of the control group respectively. The HSF1 of the cucumbers would respond quickly to the heat stress in the mRNA expression, which was beneficial to a large number of monomers. Combine, start the heat shock response. We speculate that HSBP1 and HSP70 play a negative role in the heat shock response by affecting the HSF1 activity. (2) obtain the important gene hsp10 and HSP60 cDNA full length of the heat shock response. The mRNA level expression of hsp10 and Hsp60 under heat stress shows that the expression of heat stress increases with the duration of heat stress first. The expression trend was similar: the mRNA expression of gene hsp10 and Hsp60 increased significantly at 2 h after heat stress, and reached the peak at 8 h. The maximum expression was 193.3 times and 60 times.HSP10 as the auxiliary companion protein of HSP60, which played an important role in the heat shock reaction,.4, and polymorphisms in the sequence of the Hsp90 gene. Hsp90 gene sequence 6939 BP, including exon 10, intron 9, and 22 polymorphic loci SNP, was obtained by screening of the loci and the correlation analysis with the heat resistant character. Through the high temperature stress experiment of 32 degree C, the individuals with sensitive and tolerable high temperature were obtained. The genotyping of sensitive and tolerant individuals was carried out by chi square test To 5 SNP markers with significant differences in the two groups, the experimental results showed that the genotype and allele distribution of SNP e10-1 and SNP e11-6 were significantly different in high temperature resistant strains and common populations, of which SNP e10-6 was three SNP, and the distribution of allele in SNP e2-3 was also significantly different in two groups. For the first time, SNP markers linked to temperature tolerance traits were screened and used in high temperature breeding of sea cucumber.
【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4
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本文编号：2084543