|关键词||仿刺参 热胁迫 Itraq蛋白组 Hsf1 Hsbp1 Hsps Snp分子标记 耐热性状|
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：
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。
（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。
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。
|其他摘要||The sea cucumber Apostichopus japonicus has been exploited as a very important economic species in China for its nutritional value and medical properties. Water temperature is a key environmental factor for A. japonicus, and high temperature restricts their normal growth and metabolism. In recent years, rapid temperature increase or extreme high temperature in summer has led to widespread mortality of A. japonicus in the northern coasts and many aquacultural ponds of China, resulting in dramatic resource decline and enormous economic loss. Therefore, it has significant meaning to understand the heat shock response in A. japonicus and breed heat-resistance variety.|
In our study, we used iTRAQ sequencing, Western blot, Real-time PCR et al. to study the heat shock response in sea cucumbers, and screened SNP markers associated with heat-resistance trait from the sequences of hsp90 in A. japonicus. We investigated histological, ultrastructural and HSP70 responses to heat stress in the intestine of A. japonicus. We applied the iTRAQ technique to investigate the global protein expression profile under an acute short-term (48 h) heat stress. Furthermore, we cloned the cDNAs of key genes (hsf1, hsbp1, hsp10 and hsp60) involved in heat shock response, and analyzed their expression under heat stress. We also screened SNP markers associated with heat-resistance trait from the sequences of hsp90, which had been used in marker assisted selection of A. japonicus. Main results are as follows:
1. Histological, ultrastructural and heat shock protein 70 (HSP70) responses to heat stress
Histological, ultrastructural and HSP70 responses to heat stress were investigated in the intestine of A. japonicus. Tissue degradation was observed in muscular, submucosal and mucosal layers, with significant decrease in plicae circulares of the mucosal layer. Ultrastructural damage intensified with increasing stress time, and indicators of cell apoptosis were evident after 192 h heat stress. Immunostaining showed HSP70 mainly in mucosa and serosa, with faint staining in non-stressed individuals (the control group) and denser staining under stress (the 6, 48 and 192 h groups). Western blot detection confirmed ocurrence of HSP70 in all groups and significant up-regulation under stress. The rapid and persistent response of HSP70 implies its critical role in the heat shock response of A. japonicus.
2. Understanding the heat shock response using iTRAQ-based proteomics
We applied the iTRAQ technique to investigate the global protein expression profile under an acute short-term (48 h) heat stress. Totally 3,432 proteins were identified, and 127 proteins showed significant heat stress responses, with 61 upregulated proteins and 66 downregulated proteins. Our results suggest that heat stress influenced the expression of proteins involved in various biological processes, such as tissue protection and detoxification, lipid and amino acid metabolism, energy production and usage, transcription and translation, cell apoptosis, and cell proliferation. These findings provide a better understanding about the response and thermo-tolerance mechanisms of A. japonicus under heat stress.
3. Molecular cloning of key genes cDNAs and their expression analysis under heat stress
(1) We obtained full-length cDNAs of hsf1 and hsbp1 in A. japonicus for the first time. Analysis by real-time PCR showed the expression of hsf1, hsbp1 and hsp70 in the intestine responded significantly at 26 ºC. The mRNA expression of hsf1, hsbp1 and hsp70 increased at first, and all peaked at 6 h with a peak value of 11.67-fold, 4.71-fold and 955.30-fold repectively. Our results showed that the heat shock response (HSR) in A. japonicus is a rapid and transient gene-expression program. We assumed that hsf1 played an important role in the regulation of HSR, and hsbp1 and hsp70 repressed HSR by negatively affecting the activity of hsf1.
(2) We obtained full-length cDNAs of hsp10 and hsp60 in A. japonicus for the first time. Analysis by real-time PCR showed that their mRNA expression showed a time-dependent response with a similar tendency. Their mRNA expression both showed a significant increasement at 2 h, and reached the peak at 8 h. The peak value of hsp10 and hsp60 expression was 193.3-fold and 60.0-fold respectively, compared with that in control group. The results suggested hsp10 and hsp60 were involved in the heat-shock response in A. japonicus.
4. Polymorphisms of hsp90 and their association with heat-resistance
We identified 12 exons and 11 introns in the sequences of gene hsp90 in A. japonicus. Twenty-two single nucleotide polymorphisms (SNPs), including three non-synonymous mutations, were detected in the exons. Susceptible and resistant individuals were distinguished using a high-temperature (32 °C) challenge experiment. Five of the twenty-two SNPs were shown to be significantly associated with susceptibility/resistance to high temperature by correlation analysis (χ2 test). To confirm the importance of these five SNPs, a heat-resistance strain (HRS) was selected through three generations. It was shown that the distributions of genotypes and alleles of SNP e10-1 and e11-6 were significantly different between the two groups. SNP e10-1 was trimorphic, with three alleles (A, C and T) and five genotypes (AA, CC, AT, CT and AC). The allele frequency of SNP e2-3 was also significantly associated with this trait. This is the first demonstration of SNPs related to heat-resistance in A. japonicus and supports the use of SNP markers in the selective breeding of sea cucumbers.
|学科领域||海洋科学 ; 生态学|
|徐冬雪. 仿刺参热胁迫响应的分子调控特征研究与SNP标记筛选[D]. 北京. 中国科学院大学,2016.|