Cloning and Sequence Analysis of ras Promoter from Auricuralia auricular Strain YBS-3

[Objective] The ras promoters were cloned from genomic DNA of Auricuralia auricular so as to provide promoters for breeding better species by genetic technology. [Method] PCR was used to clone promoters with the genomic DNA of A. auricular strain YBS-3 as template,and then the sequences of four ras promoters were obtained. The analysis of promoter sequences was made by three promoter analysis software：Promoter prediction,Place and TFSEARCH ver.1.3. [Result] Four fragments contained core elements of promoter including TATA-box and CAAT-box,and many other important cis-elements such as GATA BOX,GCGC BOX,CCAAT BOX1,etc. At the same time,there was at least one transcriptional start site in these sequences. And several transcription factor binding sites were detected in these sequences. [Conclusion] Four promoter fragments all had promoter function theoretically.

黑木耳核糖体失活蛋白的质谱分析

目的:对悬浮培养黑木耳菌丝体中分离纯化得到的核糖体失活蛋白进行质谱分析,测定其肽的指纹图谱。为用5′-RACE与3′-RACE方法,克隆和表达核糖体失活蛋白的基因,设计5′和3′末端的PCR引物。方法:使用HPLC和MALDI-TOF-MS(基质辅助激光解吸附电离飞行时间质谱)分析方法。结果:测定出肽的指纹图谱及三个随机肽段的氨基酸序列。结论:可根据所测肽段氨基酸序列设计5’-和3’-RACE的引物。

黑木耳YBS-3菌株ras启动子的克隆与分析

[目的]从黑木耳(Auricuralia auricular)基因组中克隆内源ras启动子,为黑木耳利用基因工程培育优良品种提供启动子元件材料。[方法]利用PCR技术,以黑木耳菌株YBS-3基因组DNA为模版,克隆得到4条ras启动子片段。采用启动子序列分析软件Place、Promoter prediction和TFSEARCHver.1.3对其进行序列结构分析。[结果]4条启动子片段均含有核心启动子序列,除典型的基本作用元件TATAbox和CAATbox外,还有许多其他重要作用元件如GATABOX、GCGCBOX和CCAATBOX1等,同时每条片段中均至少具有1个转录起始位点,且检测到HSF、AP-1、GCN4等多个转录因子结合位点。[结论]4条目的序列在理论上具有较强的启动子活性功能。

黑木耳核糖体失活蛋白cDNA 3′-端的克隆与分析

为了克隆黑木耳核糖体失活蛋白cDNA 3′-端,根据随机测得的中间一段蛋白质序列设计简并引物,应用RT-PCR和3′-RACE反应方法,将得到的基因片段与质粒连接,并转化至大肠杆菌中,进行蓝白筛选,再通过琼脂糖凝胶电泳法和PCR法验证白斑,从而得到阳性重组质粒,最后克隆出该目的蛋白基因片段。结果表明,克隆出的cDNA 3′-端为330bp的开放阅读框(ORF),编码107个氨基酸和2个终止密码子。经试验证明和文献检索,克隆得到的cDNA 3′-端为一种新基因。