Construction of a Three-frame Yeast Two-hybrid cDNA Library of Fusarium oxysporum

A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.

Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 （SOD1） using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins （protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain）, and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.

鸡胚成纤维细胞cDNA酵母表达文库的构建与鉴定

试验提取CEF细胞总RNA,运用SMART和LD-PCR技术合成cDNA,通过同源重组克隆入真核表达质粒pGADT7,转化酵母菌Y187,构建cDNA表达文库。文库滴度高达6.94×107cfu/mL,重组率达100%。随机挑选10个克隆进行测序,全部为原鸡序列。本研究为进一步研究重要禽源病毒与宿主细胞的相互作用奠定了基础。

黄瓜子叶细菌性果斑病菌侵染初期cDNA文库的构建及质量评价

【目的】构建黄瓜子叶细菌性果斑病菌侵染初期的cDNA文库,研究细菌性果斑病菌与黄瓜相互作用的分子机制奠定基础。【方法】以接种细菌性果斑病菌4d的Chineselong品种自交系9930黄瓜子叶为材料,分离纯化mRNA,反转录合成双链cDNA,利用同源重组的方法将双链cDNA转移到酵母双杂交载体pGADT7中,获得cDNA文库,对该文库质量进行分析。【结果】提取的黄瓜子叶总RNA电泳检测出3条特异性条带,分离纯化的mRNA电泳检测呈弥散条带;反转录合成的双链cDNA电泳条带呈弥散状均匀分布,大小分布在850~4000bp;黄瓜子叶cDNA文库的库容量达2.2×10 6CFU/mL,文库重组率为95.8%,文库插入片段的平均长度在1.5kb左右,达到了标准cDNA文库的要求。【结论】所获得的文库质量较高,可以用于进行与功能蛋白相互作用的筛选及研究细菌性果斑病菌与黄瓜相互作用的分子机制。

利用SMART技术构建鸡肺酵母双杂交cDNA文库

以1月龄SPF鸡肺为材料,用Trizol方法提取总RNA,并用mRNA纯化试剂盒纯化PolyA+mRNA,利用SMART技术合成双链cDNA(ds cDNA)。合成的ds cDNA通过CHROMA SPINTM TE-400 Column进行纯化,纯化后的ds cDNA与线性pGADT7-Rec共转化酵母感受态细胞AH109中,以同源重组的方式,在酵母细胞内构建成鸡肺的cDNA文库。获得的文库容量为3.9×106cfu,随机选取20个克隆进行PCR检测,插入片段大小集中在0.3～3.0kb之间,平均插入片段约为1.4kb左右,文库重组率为100%。结果表明该文库达到了高质量文库所应具备的条件,为酵母双杂交技术筛选与IBV-N相互作用的宿主细胞蛋白奠定基础。

Vero细胞酵母双杂交cDNA文库的构建与鉴定

为了研究新孢子虫与Vero细胞的相互作用蛋白,试验以体外培养的Vero细胞为材料,构建了Vero细胞酵母双杂交c DNA文库。首先,提取Vero细胞的总RNA,经反转录合成第一链c DNA,以SMART方法合成双链c DNA,通过柱层析去除200 bp以下片段;然后,将纯化的双链c DNA与p GADT7-Rec一起转到酵母Y187感受态细胞中,并发生同源重组;最后,计算文库效价、文库容量及文库的重组率。结果表明：文库效价为7.1×108cfu/m L,文库容量为1.8×1010cfu,重组率为100%,插入片段长度在500~2 000 bp之间,达到了建库要求,可用于Vero细胞和新孢子虫蛋白质相互作用的筛选。

酵母双杂交系统筛选与水稻条纹病毒Pc2互作的寄主蛋白基因片段

水稻条纹病毒编码的Pc2蛋白在病毒侵染及宿主症状发生中起作用。研究其与寄主间的分子互作有助于揭示病毒侵染与致病的分子机制。分别将编码Pc2N端（Pc2-N）与Pc2C端（Pc2-C）的基因片段克隆到酵母双杂交诱饵载体pGBKT7上．并构建水稻叶片cDNA文库．然后分别用Pc2-N和Pc2-C为诱饵筛选文库寻找与其互作的寄主因子。结果表明．诱饵载体中插入的Pc2-N和Pc2-C编码框和氨基酸序列均正确。Pc2-N对酵母菌株AHl09无毒性和自主激活能力，但Pc2-C对酵母具有细胞毒性。文库滴度为4x107CFU／mL．且大多数插入片段在500～2000bp之间。利用Pc2-N为诱饵筛选水稻cDNA文库．获得有8个候选阳性克隆。经序列测定和BLAST分析结果表明．这些克隆共编码5种蛋白．主要为胁迫相关蛋白与光系统相关蛋白。

利用修饰的随机引物构建非洲爪蟾酵母双杂交定向cDNA文库

描述了一种新的构建cDNA文库的方法,其中用来合成cDNA第一链的随机引物5′-端被加上碱基d(AC),在cDNA双链合成后所添加的连接头的末端含有碱基d(GTCG)。在cDNA双链3′-端,连接头连接上后会形成一个完整的SalI酶切位点d(GTCGAC),而在5′-端几乎不会形成SalI位点。在SalI酶切后利用形成的3′-粘性末端与5′-EcoRI粘性末端一起将cDNA双链定向导入线性化的质粒载体,再通过转化细菌获得cDNA文库。利用此方法构建了一个不同发育时期非洲爪蟾胚胎酵母双杂交cDNA文库。检测了文库中空载体的比例,插入片段大小和不同基因的表达水平几个指标,都符合预期,但是同时发现定位于mRNA的3′-端的插入片段比例比较低。这种倾向性符合文献报道,应该是实验系统的倾向性,不影响进一步的酵母双杂交实验。这些数据证明成功地构建了定向非洲爪蟾酵母双杂交cDNA文库。

Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库的构建

为了筛选柔嫩艾美耳球虫子孢子入侵相关的蛋白,利用酵母双杂交体系构建了柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库。提取纯化的子孢子总RNA,经MMLV-RT反转录合成cDNA第一链后,利用LD-PCR扩增合成双链cDNA（ds cDNA）,dscDNA经纯化柱CHROMA SPINT＋TE-400 Columns纯化剔除小于200 bp的片段。将纯化后dscDNA、pGADT7-Rec及Carrier DNA共转化到已经制备好的酵母感受态细胞Y187中,dscDNA与pGADT7-Rec以同源重组的方式在细胞内进行连接,经过缺陷性培养基（SD/-Leu）的筛选得到酵母双杂交cDNA文库。结果显示,构建的柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库的转化率为4.33×10^5/μg pGADT7-Rec,文库滴度为3.62×10^7 cfu/m L。随机挑取32个单克隆进行PCR检测,插入片段大小为200~2000bp,重组率为93.75%,并以该文库作为模板,用柔嫩艾美耳球虫5对特异性引物进行PCR扩增,获得了这5个基因片段。结果表明成功构建了柔嫩艾美耳球虫子孢子酵母双杂交cDNA文库,为进一步筛选和研究球虫入侵互作蛋白奠定了基础。

An improved yeast two-hybrid approach for detection of interacting proteins

Yeast two-hybrid approach is popularly used nowadays as an important technical method in the field of studying protein-protein interactions.Although yeast two-hybrid system is obviously advantageous in searching interacting proteins and setting up the network of proteins interaction,not all of proteins can use routine yeast two-hybrid method to search interacting proteins.Many important proteins,such as some nucleoprotein transcriptional factor,carry out the regular method and construct the bait-BD vector to screen the library containing AD vector.However,it usually results in failures because it contains the activate domain and can self-activate the reporter gene.In this study,we changed the research strategy,fused the bait gene(FOXA3)with the AD vector to screen the library containing BD vector,so that we constructed a two-hybrid library containing BD vector and can bypass the interference of self-activation.And we used this two-hybrid library to screen FOXA3,a hepatocyte nuclear factor,and found out an interacting protein:complement component C3.

大豆花叶病毒诱导的应用于膜蛋白酵母双杂交的大豆cDNA文库构建

前言大豆花叶病毒（Soybean mosaic virus，SMV）是大豆上最普遍的病害之一，严重影响大豆的产量和品质，通过筛选与病毒互作的寄主蛋白，根据互作蛋白的功能和两者的作用机制可揭示病毒侵染的分子机制。对于病毒编码的膜蛋白或者膜相关特性的蛋白，由于具有膜结合特性，在生物体内的互作多发生在膜上。