酿酒酵母论文：酿酒酵母Sfil功能性多肽的体外表达与分析

酿酒酵母论文：酿酒酵母Sfil功能性多肽的体外表达与分析
【中文摘要】Sfi1蛋白是与酿酒酵母细胞周期密切相关的一种蛋白,其主要功能是维持细胞分裂过程中纺锤体的正确装配,在细胞分裂、细胞形态维持等方面具有重要的作用,但是迄今鲜有文献从分子水平对Sfi1蛋白结构与功能进行系统研究。

Sfilp具有21个LLX3F/LX2W模式的内部重复序列,以及20个间隔序列,位于该蛋白的中央区域。

Sfilp中央区域的功能主要在于,通过内部重复序列结合酵母中心体蛋白Cdc31p,从而作用于SPB的复制过程。

然而,Sfilp的氨基端与羧基端区域虽不含有内部重复序列,但是生物信息学分析发现,Sfilp的羧基端存在七个,氨基端存在两个可能的细胞周期蛋白依赖激酶Cdc28的保守磷酸化位点；同时酵母细胞水平发现,将其缺失会导致细胞分裂出现异常,在调控SPB的复制功能上起关键性作用。

因此对Sfi1蛋白的深入研究,本课题重点是从其功能性多肽的研究角度来切入,其中包括对内部保守多肽和两端的磷酸化多肽的分析。

本论文从两个方面来阐述对酿酒酵母Sfilp功能性多肽的研究,一方面通过对其内部21个保守多肽的同源分析,发现第20个保守多肽具有研究代表性,我们针对此段多肽构建肽库,其中包括野生型,定点突变型以及随机突变型Sfi1多肽,来研究突变对Sfi1多肽与中心体蛋白Cdc31p相互作用的影响,以及对Sfilp生物学功能的影响；一方面
针对Sfilp氨基端和羧基端的磷酸化多肽进行体外表达,为后期的体外磷酸化研究奠定基础。

本实验通过计算机同源模建来比对位于第20个保守多肽上的定点突变型Sfi1多肽,即Sfi1p-763的三维构象,将此位置的氨基酸进行随机突变,发现L763H的末端构象稍有差
异,a-螺旋较为舒展,然后利用常规PCR技术及融合PCR技术,分别扩增得到SFIl野生型及定点突变型的基因序列,再利用酵母表面展示技术,在酵母细胞中诱导表达Sfi1多肽,发现其诱导表达的最佳条件为诱导温度20℃,乳糖浓度0.02g/ml,诱导时间24h。

通过SDS-PAGE 检测,发现Sfi1p定点突变L763H并没有影响Sfi1多肽与Cdc31p的相互作用。

而通过流式细胞检测DNA含量发现,L763H使细胞周期停滞在G2/M期,并且影响酵母细胞的生长状态。

本实验还采用错误倾向PCR技术,扩增得到Sfi1多肽的编码基因库；将编码基因克隆至表面展示载体,获得Sfi1多肽表达载体库；将表达载体库转化酵母细胞,诱导表达多肽,获得Sfi1多肽肽库,并通过SDS-PAGE检测Sfi1随机突变多肽与Cdc31p的相互作用。

此外,通过常规PCR技术扩增得到sFIl-N端和C端基因序列,构建至蛋白表达载体pET上,在大肠杆菌BL21细胞中,使用诱导剂IPTG对Sfilp-N端和C端多肽进行体外诱导表达,其中Sfilp-N端多肽诱导表达的最佳条件是：诱导温度为25℃,IPTG终浓度为1.0mmol/L,诱导时间为4h:Sfilp-C端多肽诱导表达的最佳条件是诱导温度为30℃,IPTG终浓度为1.0mmol/L,诱导时间为8h.Sfilp-N端和C端磷酸化多肽成功地体外表达,为研究
Sfilp-N端和C端的磷酸化机制奠定了基础。

【英文摘要】Sfil protein is closely related to the cell cycle of Saccharomyces cerevisiae,and its main function is to maintain spindle assembled correctly in cell division process.It has an important role in cell division process and cell shape maintenance,but so far there are little literature to study systematacially that the Sfil protein structure and function at the molecular level.Sfilp has 21 internal repeats of LLX3F/LX2W mode, and 20 interval sequences,which they are located in the central region of the protein. The main function of the central region is to combine with yeast centrosome protein Cdc31p through the internal repeat,by which to act on the replication process of SPB.Meanwhile,the N-terminal and C-terminal regions of Sfilp do not contain the internal repeats,but bioinformatics analysis revealed that there are several possible conserved phosphoric sites of cyclin dependent kinase Cdc28.Among the sites C-terminal region has seven and N-terminal region has two.It also found that the site deletion would lead to the cell divided abnormally at the level of yeast cells.In all these ways,it plays a key role in the regulation of SPB replication function.So the project
emphasized on the functional peptides to study Sfilp deeply,including conservative peptides and phosphorylation peptides.This paper discussed the functional peptides of Sfilp in the Saccharomyces cerevisiae mainly from two aspects.One side is to analyse the 21 reapeats at the central region of Sfilp,and found the 20th conservative peptide is typical to research.Then we focus on this peptide to build peptide library which consists of the wild type,the site-directed mutate,and the random mutate.We study the influence to interact with Cdc31p,and discuss the biological effect which the mutate brings to.On the other side we express the phosphorylation peptides in vitro which peptides are in the N-terminal and C-terminal of Sfilp,and it lays a foundation for the phosphorylation research.We compared the three-dimensional conformations of Sfilp-763 amino acids random mutation by the computer homology modeling,and found that the end of the L763H conformation is slightly different that the a-helix is more stretch.We cloned the wild sequence and site-directed mutational sequence,respectively using the conventional PCR and fusion PCR technology.Then We expressed the Sfi 1 peptide in the yeast cell utilizing the yeast phage display
technology,and found that the best expresstion condition is temperature 20℃,IPTG concentration 0.02g/ml,induction time 24h.By the SDS-PAGE analysis,we found that Sfilp-L763H did not affect the interaction with Cdc31p.We also found that
Sfilp-L763H can arrest the cell cycle in G2/M phase by the flow cytometry detecting the DNA content.We also used error-prone PCR technology to clone the coding gene bank of Sfil peptide,then cloned to pYD1 to obtain the expression vector library,finally transformed to the yeast cell to induce the peptide expresstion to obtain Sfil peptide library. We detected the effect of the interaction to Cdc31p by SDS-PAGE。

In addition,We cloned the SFI1-N terminal and C-terminal sequences by the conventional PCR technology, and builded to the protein expression vector pET.Then we expressed the peptides of Sfilp-N terminal and C-terminal using the inducer IPTG in the E. coli cells of BL21.Sfilp N-terminal peptide inducing expression condition is that the induction temperature is 20℃, IPTG final concentration is 1.0mmol/L,and the timeis 4h;the Sfi 1 p-C-terminal peptide inducing expression condition is that the induction temperature is 30℃,IPTG final concentration is 1.0mmol/L,and the induction
time is 8h.Sfilp N-terminal peptide and C-terminal peptide expressed in vitro successfully,and it lays the foundation for studing the Sfilp-N and C-terminal peptide phosphorylation mechanism.
【关键词】酿酒酵母 Sfi1p 保守多肽磷酸化多肽体外表达蛋白质相互作用
【英文关键词】saccharomyces cerevisia Sfi1p conservative peptide phosphorylated peptide expression protein interaction
【目录】酿酒酵母Sfil功能性多肽的体外表达与分析摘要
4-6Abstract6-7表格目录12-13图片目录13-14第一章前言14-28 1 酿酒酵母的研究优势14-16 1.1 酿酒酵母是一种重要的真核模式生物14-15 1.2 以酿酒酵母作为模式生物的研究意义15-16 2 酿酒酵母的纺锤体极体16-21 2.1 SPB的结构16-17 2.2 SPB的复制与细胞周期17-19 2.3 SPB的关键蛋白组分19-20 2.4 影响SPB功能的重要调控蛋白20-21 3 酿酒酵母纺锤体极体蛋白组分Sfi1p21-23 4 酿酒酵母Sfi1p的研究进展23-25 5 本课题的研究思路25-28第二章材料与方法
28-48 1 实验材料28-36 1.1 质粒,菌株与引物28-30 1.2 主要实验仪器30-31 1.3 药品与试剂31-34 1.3.1 主要药品
31-32 1.3.2 主要试剂32-34 1.4 培养基34-36 2 实验方
法36-48 2.1 菌种的培养与保存36-38 2.2 大肠杆菌感受态细胞Top10的制备与转化36-38 2.3 CTAB法提取质粒
DNA38-39 2.4 DNA的限制性内切酶消化39 2.5 DNA的纯化
39-40 2.6 酚抽提纯化DNA40-41 2.7 线性DNA的去磷酸化
41 2.8 DNA的连接反应41-42 2.9 琼脂糖凝胶电泳验证方法
42 2.10 常规PCR扩增技术42-43 2.11 融合PCR技术
43-44 2.12 错误倾向PCR技术44 2.13 酵母细胞的快速转化法44-45 2.14 PCR法验证酵母交配型45 2.15 HO质粒法构建二倍体细胞45 2.16 酵母高效产孢法45-46 2.17 聚丙烯酰胺凝胶电泳(SDS-PAGE)验证方法46-48第三章酿酒酵母Sfi1保守多肽肽库的构建与分析48-73 1 实验材料与方法50-59 1.1 Sfi1保守多肽的同源分析50-51 1.1.1 Sfi1保守多肽中关键氨基酸的特点分析50 1.1.2 Sfi1保守多肽的二级结构分析50-51 1.2 Sfi1p 20~(th)保守多肽中特定氨基酸突变的结构与功能分析
51-56 1.2.1 Sfi1p-L763随机突变的同源模拟51-52 1.2.2 SFI1-W763和SFI1-M763基因序列的扩增52-53 1.2.3 重组质粒pYD1-W763和pYD1-M763的构建及其鉴定53-54 1.2.4 Sfi1多肽在酵母中的诱导表达与检测54-55 1.2.5 Sfi1p与Cde31p的相互作用与检测55 1.2.6 Sfi1p的流式细胞周期及DNA含量的检测55 1.2.7 Sfi1p突变对酵母细胞生长的影响55-56 1.3 酿酒酵母Sfi120~(th)保守多肽中随机氨基酸突变的肤库的建立
56-59 1.3.1 多肽编码基因库的建立56-57 1.3.2 多肽表达载体库pYD1-S20的构建57-58 1.3.3 多肽表达菌株库的构建
58 1.3.4 Sifl多肽肽库的构建58 1.3.5 Sfil随机突变多肽与Cdc31p的相互作用与检测58-59 2 实验结果59-70 2.1 Sfi1保守多肽的同源分析59-60 2.1.1 Sfi1保守多肽中关键氨基酸的特点分析59 2.1.2 Sfi1保守多肽的二级结构分析59 2.1.3 Sfi1第20个保守多肽的特点59-60 2.2 Sfi1p 20~(th)保守多肽中特定氨基酸突变的结构与功能分析60-67 2.2.1 Sfi1p-L763氨基酸随机突变的三维结构的拟合60 2.2.2 SFI1-W763和
SFI1-M763基因序列的扩增结果验证60 2.2.3 重组载体
pYD1-W763和pYD1-M763的酶切验证60-61 2.2.4 SFI1-W763和SFI1-M763基因序列和相应的氨基酸序列比对61-62 2.2.5 中心体蛋白Cdc31p诱导表达的SDS-PAGE验证62-63 2.2.6 野生型Sfi1多肽体外表达的SDS-PAGE检测63-64 2.2.7 突变型Sfi1多肽体外表达的SDS-PAGE检测64-65 2.2.8 Sfi1多肽与Cdc31p相互作用结果检测65-66 2.2.9 野生型Sfi1多肽和突变型Sfi1多肽的流式细胞周期检测66-67 2.2.10 Sfi1p突变对酵母细胞有丝分裂的影响67 2.3 酿酒酵母Sfi120~(th)保守多肽中随机氨基酸突变的肽库的建立67-70 2.3.1 错误倾向PCR产物的琼脂糖凝胶电泳验证67-68 2.3.2 重组载体pYD1-S20的酶切验证
68 2.3.3 Sfi1随机突变多肽与Cdc31p相互作用结果检测
68-70 3 讨论70-73第四章酿酒酵母Sfi1p磷酸化多肽的体外表达与分析73-84 1 实验材料与方法73-75 1.1 SFI1氨基端和羧基端基因序列的扩增73-74 1.2 重组质粒pET-SN和pET-SC 的构建与鉴定74 1.3 Sfi1p磷酸化多肽在大肠杆菌中的诱导表达74-75 2 实验结果75-82 2.1 重组载体pET-SN和pET-SC的构建示意图75 2.2 SFI1氨基端和羧基端基因序列的扩增验证
75-76 2.3 重组载体pET-SN和pET-SC的酶切验证76-77 2.4 SFI1-N端和SFI1-C端基因的测序比对77-79 2.5 Sfi1p-N端和Sfi1p-C端编码氨基酸的序列比对79-80 2.6 Sfi1磷酸化多肽体外诱导表达条件的优化80-82 3 讨论82-84结论与展望
84-85参考文献85-90个人简历90获得学士、硕士学位的学校、时间90在学期间发表的学术论文与研究成果90-91致谢91。