兩種紫茉莉小熱休克蛋白基因的克隆及其融合基因在釀酒酵母中的遺傳轉(zhuǎn)化
發(fā)布時間：2019-02-21 09:56
【摘要】：伴隨著經(jīng)濟的快速的發(fā)展,我國的石油開采量日益增加,隨之而來的環(huán)境污染問題日漸顯著。近年來,植物修復技術被越來越多的應用在修復石油污染土壤等領域,但關于植物是如何耐受石油等多環(huán)芳烴類污染物的具體機理仍不明確,本研究以抗逆植物紫茉莉為供試材料,根據(jù)前期差異蛋白質(zhì)組實驗得到的的石油脅迫下差異蛋白的表達譜,發(fā)現(xiàn)石油污染組中兩種小熱休克蛋白(small heat shock protein, sHsp)Hsp18.2與Hsp17.7與清潔對照組相比表達量顯著上調(diào),并通過RACE(Rapid-amplification of cDNA ends)法獲得這兩種蛋白的編碼框序列,利用融合PCR及2A肽技術獲得共表達融合序列,并成功將上述3種ORF轉(zhuǎn)入釀酒酵母(Saccharomyces cerevisiae),以期研究其表達變化對釀酒酵母細胞抗石油脅迫能力的影響,并借此推測石油污染條件下紫茉莉較強耐受能力的機理,同時也為今后快速篩選石油污染修復植株提供分子理論依據(jù)。本研究結果如下所示：1、通過RACE技術獲得紫茉莉兩種小熱休克蛋白Hsp18.2與Hsp17.7的完整開放閱讀框,分別長為為483 bp和474 bp,編碼161和158個氨基酸,分子量分別約為18.2 kDa與17.7 kDa,均含有保守的ACD結構域(a-crystallin)。2、依據(jù)2A肽原理及融合PCR法獲得了HSP18.2與HSP17.7的胞內(nèi)共表達序列,片段長度1091 bp,融合片段可以通過一個完整的開放閱讀框同時表達HSP18.2-Flag與HSP17.7-HA。3、成功構建出三種含有BamHI和Xbal雙酶切位點的釀酒酵母胞內(nèi)表達載體pYES2-HSP18.2-Flag、pYES2-HSP17.7-HA及pYES2-HSP-fusion,且均經(jīng)過雙酶切鑒定及測序檢測。4、成功將上述三種釀酒酵母胞內(nèi)表達載體轉(zhuǎn)入酵母菌株Saccharomyces cerevisiae,并通過SC-U篩選培養(yǎng)基獲得陽性克隆,為進一步對紫茉莉小熱休克蛋白HSP18.2與HSP17.7的功能機制的研究提供了實驗材料。
[Abstract]:With the rapid development of economy, the oil exploitation in China is increasing day by day, and the environmental pollution problem is becoming more and more obvious. In recent years, phytoremediation technology has been used more and more in the field of remediation of petroleum contaminated soil. However, the mechanism of how plants tolerate polycyclic aromatic hydrocarbons (PAHs) such as petroleum is still unclear. In this study, the stress resistant plant Mirabilis was used as the experimental material. According to the differentially expressed protein profiles obtained from the preliminary differential proteome experiment, two small heat shock proteins (small heat shock protein,) were found in the oil-contaminated group. SHsp) Hsp18.2 was significantly up-regulated compared with Hsp17.7 compared with the clean control group. The coding frame sequences of the two proteins were obtained by RACE (Rapid-amplification of cDNA ends) method) and coexpressed fusion sequences were obtained by fusion PCR and 2A peptide techniques. The three kinds of ORF were successfully transferred into Saccharomyces cerevisiae (Saccharomyces cerevisiae), in order to study the effect of their expression changes on the resistance to oil stress of Saccharomyces cerevisiae cells. At the same time, it also provides molecular theory basis for rapid screening of oil contaminated remediation plants in the future. The results of this study are as follows: 1. The complete open reading frames of two small heat shock protein (Hsp18.2) and Hsp17.7 were obtained by RACE technique. The length of Hsp18.2 and Hsp17.7 were 483 bp and 474 bp, encoding 161 and 158 amino acids, respectively. The molecular weight of HSP18.2 and HSP17.7 were 18.2 kDa and 17.7 kDa, respectively, containing conserved ACD domain (a-crystallin). 2. According to 2A peptide principle and fusion PCR method, the intracellular coexpression sequences of HSP18.2 and HSP17.7 were obtained, and the length of the fragments was 1091 bp,. The fusion fragment can simultaneously express HSP18.2-Flag and HSP17.7-HA.3, through a complete open reading frame, and successfully construct three intracellular expression vectors pYES2-HSP18.2-Flag, containing double restriction sites of BamHI and Xbal in Saccharomyces cerevisiae. Both pYES2-HSP17.7-HA and pYES2-HSP-fusion, were identified by double enzyme digestion and sequenced. 4. The above three expression vectors were successfully transferred into yeast strain Saccharomyces cerevisiae, and positive clones were obtained by SC-U screening medium. The experimental materials were provided for further study on the functional mechanism of small heat shock protein (HSP18.2) and small heat shock protein (HSP17.7) in Mirabilis.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
本文編號：2427442
[Abstract]:With the rapid development of economy, the oil exploitation in China is increasing day by day, and the environmental pollution problem is becoming more and more obvious. In recent years, phytoremediation technology has been used more and more in the field of remediation of petroleum contaminated soil. However, the mechanism of how plants tolerate polycyclic aromatic hydrocarbons (PAHs) such as petroleum is still unclear. In this study, the stress resistant plant Mirabilis was used as the experimental material. According to the differentially expressed protein profiles obtained from the preliminary differential proteome experiment, two small heat shock proteins (small heat shock protein,) were found in the oil-contaminated group. SHsp) Hsp18.2 was significantly up-regulated compared with Hsp17.7 compared with the clean control group. The coding frame sequences of the two proteins were obtained by RACE (Rapid-amplification of cDNA ends) method) and coexpressed fusion sequences were obtained by fusion PCR and 2A peptide techniques. The three kinds of ORF were successfully transferred into Saccharomyces cerevisiae (Saccharomyces cerevisiae), in order to study the effect of their expression changes on the resistance to oil stress of Saccharomyces cerevisiae cells. At the same time, it also provides molecular theory basis for rapid screening of oil contaminated remediation plants in the future. The results of this study are as follows: 1. The complete open reading frames of two small heat shock protein (Hsp18.2) and Hsp17.7 were obtained by RACE technique. The length of Hsp18.2 and Hsp17.7 were 483 bp and 474 bp, encoding 161 and 158 amino acids, respectively. The molecular weight of HSP18.2 and HSP17.7 were 18.2 kDa and 17.7 kDa, respectively, containing conserved ACD domain (a-crystallin). 2. According to 2A peptide principle and fusion PCR method, the intracellular coexpression sequences of HSP18.2 and HSP17.7 were obtained, and the length of the fragments was 1091 bp,. The fusion fragment can simultaneously express HSP18.2-Flag and HSP17.7-HA.3, through a complete open reading frame, and successfully construct three intracellular expression vectors pYES2-HSP18.2-Flag, containing double restriction sites of BamHI and Xbal in Saccharomyces cerevisiae. Both pYES2-HSP17.7-HA and pYES2-HSP-fusion, were identified by double enzyme digestion and sequenced. 4. The above three expression vectors were successfully transferred into yeast strain Saccharomyces cerevisiae, and positive clones were obtained by SC-U screening medium. The experimental materials were provided for further study on the functional mechanism of small heat shock protein (HSP18.2) and small heat shock protein (HSP17.7) in Mirabilis.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q943.2
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