【作者】 郝炜；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2005， 硕士

【摘要】 芪合成酶(Stilbene Synthase STS)是催化植物体内芪类化合物合成的关键酶,自从STS 基因被分离以来,有关STS 基因工程的研究成为植物次生代谢基因工程成功地典范。乙醛脱氢酶(Aldehyde dehydrogenase ALDH)是广泛存在于动物、植物和微生物体内的一类酶,参与体内多种代谢途径,目前它在植物抗病反应中的作用还有待进一步了解。本实验室研究人员通过mRNA差异显示技术(mRNA　differential　display　reverse　transcription-PCR　,　DDRT-PCR)及cDNA 末端快速扩增技术(Rapid　amplification　of　cDNA　ends,　RACE),从高抗白粉病的华东葡萄白河-35-1(Vitis　pseudoreticulata)中克隆分离得到中国野生葡萄STS 基因、ALDH 基因。本研究利用所获基因通过构建该基因原核表达载体,试图从体外表达途径对该基因的功能进行验证,为该基因在基因工程上的进一步应用和研究提供依据,主要取得以下结果:　1.　　根据已获STS 基因、ALDH 基因cDNA　全长序列,分别设计一对特异性PCR 引物,以华东葡萄白河-35-1的5’RACE　cDNA第一链为模板,经PCR扩增,分别获得大小约1200bp和1600bp 的DNA 片段,经过克隆测序,结果显示:这两个片段各自分别含有实际大小约1179bp 和1611bp 的阅读读码框架,序列分别与已知中国野生葡萄STS 基因、ALDH基因完全一致,从而体外扩增获得两端带有相应酶切位点的STS 基因和ALDH 基因全长。　2.　　将ALDH基因定向克隆到原核表达载体上pGEX-4T-1上,重组表达载体pGEX-ALDH　同样经BamHI 和SmaI 双酶切处理,切下片段与已知ALDH 基因片段大小一致,证明ALDH基因正确连接到表达载体上,重组表达载体构建成功。将重组表达载体pGEX-ALDH 转化表达菌E.coli　BL21 中,经诱导培养后,未见有明显特异性表达。将该重组表达载体重新转入表达菌株E.coli　BL21(coden　plus)中,经IPTG 诱导培养,聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示重组菌在85kD 大小的位置有特异表达条带,与所推算的GST-ALDH 融合蛋白大小相符合,证明ALDA 基因在大肠杆菌中获得了表达。　3. 将STS 基因从定向克隆到原核表达载体pGEX-4T-1 上,重组表达载体pGEX-STS同样经BamHI 和XhoI 双酶切处理,切下片段与已知STS 基因片段大小一致,证明STS基因正确连接到表达载体上,重组表达载体构建成功。将pGEX-STS 表达载体转化大肠杆菌BL21,经IPTG 诱导培养,聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示重组菌在66kD 大小的位置有特异表达条带,与根据已知STS 大小所推算的GST-STS 融合蛋白大小相符合,证明STS 基因在大肠杆菌中获得表达。通过诱导剂IPTG不同浓度诱导对照和不同诱导培养时间对照,确定了最佳表达条件。4. GST-STS 融合蛋白主要以包涵体形式表达,通过高浓度变性剂溶解包涵体和低浓度变性剂梯度透析对该包涵体进行了复性。复性的蛋白可通过GST亲和介质进行富集、更多还原

【Abstract】 This paper presents that Stilbene Synthase (STS) is a key enzyme of the synthsis of resveratrol in plants. Since the gene was cloned , the related studies of STS gene engineering becomes a successful example of the gene engineering of secondary metabolism .The Aldehyde dehydrogenase (ALDH) is an enzyme widely existed in animals, plants and microorganisms ,it engage in many important metabolism actions ,but its function in disease resistance of plants still needs more study . In our lab , By mRNA DDRT-PCR (mRNA differential display reverse transcription-PCR ) and RACE (Rapid amplification of cDNA ends ) , the STS gene and ALDH gene of Chinese wild grape was cloned and separated from Vitis pseudoreticulata Baihe-35-1 , which has a high resistance to Uncinula necator schw.Burr.On this basis , we try to test and verify the gene’s function from the way of gene’s expression in E.coli , so that this could supply a basis for the STS gene’s further study and use in gene engineering . Some results of this study are as follows : 1. Based on the cDNA full sequence of STS gene and ALDH gene of Vitis pseudoreticulata , we respectively designed a pair of specific primers , and obtained two DNA bands of about 1200 bp and 1700bp length respectively by PCR from the first strain of 5’RACE cDNA of Vitis pseudoreticulata Baihe-35-1 . The sequencing result shows that the factual length of the open reading frames are1179 bp and 1611bp and respectively their sequence is identical to the known STS gene and ALDH gene of Vitis pseudoreticulata , so we obtained the full sequence of STS gene and ALDH gene in vitro. 2. The ALDH gene was subcloned into the expression vector pGEX-4T-1. The recombinant expression vector was digested by BamHI and SmaI and the result shows that the ALDH gene was correctly linked to the pGEX-4T-1.The recombinant expression vector pGEX-ALDH was transformed into E.coli BL21 and induced by IPTG ,but cells had not any special expression . So we retransformed the pGEX-ALDH into E.coli BL21(coden plus) and induced by IPTG ,the cellsspecially expressed a protein of about Mr. 85 kD displayed in SDS-PAGE gel , it is identical to the GST-ALDH fusion protein we had calculated , so the ALDH gene was expressed in E.coli BL21 (coden plus). 3. The STS gene was subcloned into the expression vector pGEX-4T-1 . The recombinant expression vector was digested by BamHI and XhoI and the result shows that the STS gene was correctly linked to the pGEX-4T-1 . The recombinant expression vector pGEX-STS was transformed into E.coli BL21 cells and was induced by IPTG , the cells specifically expressed a protein of about Mr.66kD displayed in SDS-PAGE gel , it is identical to the known GST-STS fusion protein , so the STS gene was expressed in E.coli BL21 . We also fixed the best expression condition by compare the expression result of different IPTG density and cell induced culture times . 4. The GST-RS fusion protein was expressed in inclusion body , we dissolved the inclusion body by high density denaturant and renatured the protein by gradual dialysis low density denaturant . The renatured protein could be purified by Glutathione Sepharose 4B Fast flow . It supply a basis for further study of the gene’s function . 5. We also used the washed inclusion body to immunolize rabbit to prepare polyclonal serum of GST-STS . Western blotting shows that the serum (diluted to 1:3000) had specific antigen-antibody recognition to the renatured GST-STS fusion protein . It supply a basis for the antiserum’s more use in the localization of STS in plants and the testing of trans-STS gene plants .更多还原