本文選題：仿刺參 + 熱脅迫　； 參考：《中國(guó)科學(xué)院研究生院(海洋研究所)》2016年博士論文

【摘要】：仿刺參(Apostichopus japonicus),又被稱為刺參,是我國(guó)重要的經(jīng)濟(jì)物種,具有較高的營(yíng)養(yǎng)價(jià)值和藥用價(jià)值。水溫是影響刺參生長(zhǎng)和代謝的最重要的環(huán)境因素之一。近年來(lái),由于極端溫度的影響,在養(yǎng)殖池塘和淺海圍堰更是出現(xiàn)了刺參的大面積死亡現(xiàn)象,造成慘重的資源和經(jīng)濟(jì)損失。因此,研究刺參對(duì)熱脅迫的響應(yīng)機(jī)制和刺參耐高溫品種的選育工作具有非常重要的意義。本研究利用iTRAQ蛋白質(zhì)組學(xué)技術(shù)、Real time PCR、Western blot等技術(shù)對(duì)刺參熱脅迫的響應(yīng)機(jī)制展開(kāi)研究,并篩選與刺參耐熱性狀相互關(guān)聯(lián)的SNP分子標(biāo)記。探索了熱脅迫下刺參腸道在組織、細(xì)胞和HSP70蛋白水平的變化;開(kāi)展刺參熱脅迫下iTRAQ蛋白質(zhì)組學(xué)分析,揭示了熱脅迫下刺參腸道組織蛋白水平的全局變化;獲得關(guān)鍵響應(yīng)基因hsf1、hsbp1、hsp60和hsp10的cDNA序列,探討HSPs在熱脅迫下表達(dá)特點(diǎn)及調(diào)控機(jī)制;篩選hsp90基因序列中與耐熱性狀關(guān)聯(lián)的SNP分子標(biāo)記,并應(yīng)用于耐高溫刺參品種的選育。主要研究結(jié)果如下:1、刺參腸道對(duì)熱脅迫的組織學(xué)、細(xì)胞學(xué)和HSP70蛋白的響應(yīng)熱脅迫下,刺參腸道組織出現(xiàn)明顯的組織退化,表現(xiàn)為腸道組織層厚度(粘膜層、粘膜下層和肌肉層)減小、環(huán)形皺襞的數(shù)目減少。刺參腸道細(xì)胞在熱脅迫下出現(xiàn)明顯的細(xì)胞凋亡現(xiàn)象,表現(xiàn)為線粒體和囊泡的數(shù)目減少、染色質(zhì)聚集,甚至出現(xiàn)細(xì)胞質(zhì)和細(xì)胞器流失的空細(xì)胞。HSP70免疫組化的結(jié)果顯示,HSP70蛋白表達(dá)具有組織特異性,集中分布在粘膜層和漿膜層;常溫下HSP70的染色為細(xì)胞質(zhì)染色,熱脅迫下細(xì)胞質(zhì)和細(xì)胞核均有染色。HSP70的Western blot的結(jié)果顯示,HSP70蛋白的表達(dá)水平隨熱脅迫持續(xù)時(shí)間一直增加,在熱脅迫下對(duì)細(xì)胞起重要的保護(hù)作用。2、刺參熱脅迫下iTRAQ蛋白質(zhì)組學(xué)研究利用iTRAQ技術(shù),分析了刺參腸道組織26°C熱脅迫48 h后蛋白表達(dá)情況。結(jié)果顯示,熱脅迫下共有127種蛋白的表達(dá)量發(fā)生了顯著變化,其中包括61個(gè)上調(diào)蛋白和66個(gè)下調(diào)蛋白。這些差異蛋白主要參與了組織保護(hù)和解毒、脂肪酸和氨基酸代謝、能量產(chǎn)生和利用、轉(zhuǎn)錄和翻譯、細(xì)胞凋亡和細(xì)胞增殖等細(xì)胞活動(dòng)。3、刺參熱休克反應(yīng)關(guān)鍵基因的克隆和表達(dá)(1)獲得熱休克反應(yīng)的調(diào)控基因hsf1和hsbp1的cDNA全長(zhǎng)。hsf1、hsbp1和hsp70的mRNA表達(dá)結(jié)果表明:熱脅迫下三者表達(dá)量隨熱脅迫持續(xù)時(shí)間先增加后減小,均在熱脅迫6 h達(dá)到峰值,表達(dá)量分別是對(duì)照組的11.67倍、4.71倍和955.30倍。刺參的hsf1在mRNA表達(dá)量方面會(huì)對(duì)熱脅迫作出迅速的響應(yīng),有利于大量單體的結(jié)合,啟動(dòng)熱休克反應(yīng)。我們推測(cè)hsbp1和hsp70通過(guò)影響HSF1活性,對(duì)熱休克反應(yīng)起負(fù)面調(diào)節(jié)的作用。(2)獲得熱休克反應(yīng)的重要基因hsp10和hsp60的cDNA全長(zhǎng)。熱脅迫下hsp10和hsp60的mRNA水平表達(dá)結(jié)果表明,熱脅迫兩者表達(dá)量隨熱脅迫持續(xù)時(shí)間先增加后減小,表達(dá)趨勢(shì)相似:基因hsp10和hsp60的mRNA表達(dá)量均在熱脅迫后2 h就顯著上升,在8 h時(shí)達(dá)到峰值,最大表達(dá)量分別為193.3倍和60.0倍。HSP10作為HSP60的輔助伴侶蛋白,兩者協(xié)同合作在熱休克反應(yīng)中起重要作用。4、刺參hsp90基因序列中多態(tài)性位點(diǎn)的篩選及其與耐熱性狀的關(guān)聯(lián)分析獲得hsp90基因序列6939 bp,含有外顯子10個(gè),內(nèi)含子9個(gè),多態(tài)性位點(diǎn)SNP共22個(gè)。通過(guò)32°C高溫脅迫實(shí)驗(yàn),獲得對(duì)高溫敏感和耐受的各50頭刺參個(gè)體。對(duì)敏感組和耐受組個(gè)體進(jìn)行基因分型,通過(guò)卡方檢驗(yàn)得到5個(gè)在兩組分布顯著差異的SNP標(biāo)記。進(jìn)一步驗(yàn)證實(shí)驗(yàn)結(jié)果表明:SNP e10-1和SNP e11-6的基因型和等位基因分布在耐高溫品系和普通群體中存在顯著差異,其中SNP e10-6為三態(tài)SNP;SNP e2-3的等位基因分布在兩個(gè)群體中也存在顯著差異。這是在刺參中首次篩選到與耐溫性狀關(guān)聯(lián)的SNP標(biāo)記,并已應(yīng)用于刺參耐高溫選育中。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院研究生院(海洋研究所)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S917.4
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本文編號(hào)：2084543