【作者】 张学军；

【导师】 艾秀莲； 赵震宇；

【作者基本信息】 新疆农业大学 ， 植物病理， 2007， 硕士

【摘要】 雪莲PEBP基因是本实验室分离出的新抗寒相关基因，共有510个核苷酸，网上进行BLAST对比，与拟南芥(Arabidopsis thaliana)中的磷脂酰乙醇胺结合蛋白基因类似，相似性81％。本实验以实验室构建的Teasy-PEBP质粒为模板，扩增PEBP基因，克隆到表达载体pET30(+)和pPIC9k并导入大肠杆菌BL21(DE3)和毕赤酵母(Pichia pastoris)GS115细胞内表达，纯化表达产物，优化表达条件，主要结果如下：根据pET30(+)多克隆位点设计PEBP基因特异性引物，引入EcoRⅠ和NcoⅠ酶切位点，以Teasy-PEBP质粒为模板，扩增PEBP基因，连接到原核表达载体pET30(+)上，转化感受态表达菌株BL21(DE3)，在低温下，低浓度IPTG诱导融合蛋白表达，纯化表达产物，Western blot鉴定目的蛋白，SDS-PAGE分析：其相对分子量约为28KD，与预期相符，表达量约占菌体蛋白的26．8％，并且通过亲和层析纯化了重组融合蛋白，Western blot鉴定为阳性。根据pPIC9k多克隆位点设计PEBP基因引物，分别加入EcoRⅠ和NotⅠ酶切位点，以Teasy-PEBP质粒为模板，扩增PEBP基因，克隆到表达载体pPIC9k，得到重组质粒并命名为pPIC9k-PEBP，将pPIC9k-PEBP用SalⅠ进行酶切，线性化后的pPIC9k-PEBP以电转化法导入到酵母GS115细胞中，利用MM和MD平板筛选Mut~s(methanol utilization slow)表型，PCR鉴定重组酵母菌株，乙醇诱导表达。SDS-PAGE分析：其相对分子量约为20KD，蛋白表达量可占总蛋白的90％，上清液抗冻检测表明，PEBP能降低液体的冰点，具有一定的抗冻作用。优化重组质粒pPIC9k-PEBP在毕赤酵母(Pichia pastoris)GS115中的表达条件，初步得到最佳的重组蛋白表达条件为：诱导剂甲醇浓度为0．5％，诱导培养基的PH为6．0，诱导温度为32℃，诱导时间为216h。实验成功构建了表达载体pET-PEBP和pPIC9k-PEBP，获得了高效表达产物，并为进一步研究雪莲PEBP的抗冻功能奠定基础。更多还原

【Abstract】 PEBP gene is new Antifreeze gene that was separated by our laboratory in Saussurea involucrate Kar.er kir. The length of PEBP is 510bp,Homology search with PEBP were performed against GenBank using BLAST, the identity with PEBP of Arabidopsis thaliana were 81%.ln this research, Using Teasy-PEBP plasmid as a template ,the PEBP gene was amplified by PCR .PEBP gene was inserted into pET30a（+） andpPIC9K, then expressed in E. coli BL21 and pichia pastoris GS115, respectively. The expression products were purified by affinity chromatography, and the optimal expression condition was obtained by preliminary research. The main results were as follows:A pair of specific primers PEBP sense and PEBP anti-sense were designed based on the multi clone site （MCS） of pET30a（+）, introduced restriction site EcoRⅠand NcoⅠrespectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the pET30a（+） vector. The recombinant vector was transferred into Ecoli BL21（DE3）and induced the expression of protein by low concentration of IPTG and low temperature overnight. Fusion protein was purified by Ni-NTA affinity chromatography. SDS-PAGE and Western blot analysis showed that the fusion protein relative molecular weight was 28KD that was consistent with the theoretical value, and its product was 26.8% in total protein.Expression primers PEBP sense and PEBP anti-sense were designed based on the MCS)of pPIC9K, introduced restriction site EcoRⅠand NotⅠrespectively. The PEBP gene was amplified from Teasy-PEBP plasmid by PCR and inserted into the Pichia pastoris expression vector. The recombinant plasmid named as pPIC9K-PEBP was linearized by SalⅠ,and men transformed into Pichia pastoris GS115. Mut^s （Methanol utilization slow） transformants were screened by MD and MM plates, confirmed by PCR. Supernatant of the culture was analyzed by SDS-PAGE, a size of specific strip was found, indicating that the PEBP gene was expressed and its relative molecular weight is 20KD, and target protein was 90% in total protein. The PEBP has been definiteanti-freeze character by the biological activity detection of the supernatant.The optimal expression parameters of GS115 were 0.5% （ V/V ） CH₃OH concentration, PH 6.0 culture medium. 30℃culture temperature, 216 hours inducing time.The prokaryotic and Eukaryotic expression system of PEBP is successfully constructed. It lays the foundation for the further study on the Antifreeze characters of the PEBP更多还原