Screening Of Interactor Protein With Soybean Transcription Factor, GmDREB5, Using Yeast Two-Hybrid System And Construction Of A Nuclear Transcription Trap (NTT) System

Transcription factors play important role in the regulation of plant growth and development, as well as its response to environment. A typical higher plant transcription factor usually contains a DNA-binding domain, a transcription regulation domain, a oligomerization site and a nuclear localization domain. Transcription factors regulate the expression of downstream genes by the binding of these domains and cis-acting elements. The DREB (dehydration responsive element binding protein) is the stress-related transcription factor family. It were reported that DREB transcription factor can regulate expression of several downstream genes, which will enhance plants tolerance to stresses. The activity of DREB is regulated by other interactor proteins. Therefore, screening of novel DREB interactor proteins is the foundation to clarify the moleculer mechanism of DREB transcription factors.The provious work in our lab indicated that the soybean GmDREB5 transcription factor had essential characters like the other DREB proteins. Furthermore, Construction the bait vector: the function analysis shown that overexpression of GmDREB5 could enhance the drought- and salt-tolerance of the transgenic plant. In this work, yeast two-hybrid system was utilized to screen interactor proteins with GmDREB5 from soybean cDNA library treated under drought condition for 5h. In order to avoid the auto-activation of the GmDREB5, using website database(http://myhits.isb-sib.ch/cgi-bin/motif_scan)to predict the modification site of GmDREB5. And then we divided the GmDREB5 gene into three fragments, and inserted these fragment into bait-vector (pGBKT7)of the yeast two-hybrid system for identifying antonomous activation of three fragments;Construction cDNA library and selection: After analysis, we choosed the region from 73 to 226 amino acid of the GmDREB5 as bait fragment. We extracted the total RNA from soybean Tiefeng8, and used SMART method(Switching Mechanism At 5' end of RNA Transcript) to synthesize the first strand of cDNA. Then obtain the double-strand cDNA using LD-PCR(long distance PCR). The cDNA library was synthesized and inserted into the screen vector(pGADT7)through site-sepecific recombinant enzyme, and transformed into the yeast AH109 altogether; Finally,Identified the clones: we obtained more than 500 yeast clone. Sequencing of these inserting fragment indicated that one of these yaest clone contains TRP(Tetratricopeptide repeat containing protein) conserve domain , named as GmTPR1. Aligning it with other six protein containing TPR showed GmTPR1 shares only 14% homology with them. The expression analysis of GmTPR1 indicated that GmTPR1 induced by drought, low temperature, high salt and ABA.The plant nuclear proteins play vital roles in the growth and development of plants. The research on the component and the dynamic change of plant nuclear protein is essential to study on the regulation network of resistance-related genes.To study on nuclear proteins and its dynamic composing under various of environmental condition, a high-efficiency nuclear transportation trap (NTT) system was constructed in this research, which aimed at cloning of genes encoding nuclear proteins that contain all kind of nuclear localization signals (NLS) sequence. To identify the nuclear transportation trap system, NLS sequence of the large T antigen of SV40 protein, was synthesized and inserted into multi-cloning site of selective vector of NTT system, which were transferred into yeast strain, EGY48. The results shown that yeast transferred with selecitve vector with NLS sequence could grow on SD/His-/Leu- selective medium, whereas yeast without NLS sequence couldn't grow, which indicated that NTT system could work. Moreover, comparing analysis shown that selective efficiency of vector constructed in this study is higher than that of vector reported in other article. The NTT system will be helpful in the screening of genes encoding nuclear proteins from cDNA library, which is very important to research dynamic composing of nuclear proteins and modulating network of all genes in nuclear. To data, we had screened 500 yeast clone from wheat Xiobaimai cDNA library treated under drought condition for 5h using NTT system. The identification and sequencing were in progress.the two of clones are putative enoyl CoA hydratase and Maxi-KH type RNA binding protein.