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The Investigation of Interactions between Proteins Encoded by Banana Streak Virus

Author: LinZhanSong
Tutor: LiuZhiZuo
School: Hainan University
Course: Microbiology
Keywords: Banana streak virus protein interaction bacterial two-hybrid system yeast two-hybrid system
CLC: S436.68
Type: Master's thesis
Year: 2011
Downloads: 72
Quote: 0
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Abstract


Banana streak virus (BSV) which is a kind of plant virus detected and identified in the recent several decades belongs to Caulimoviridae, Badnavirus, there is no effective detecting methods till now. The genome of BSV is a circle double-strained DNA, which is 7500bp long and could be divided into three open reading frames (ORFs) among which the function of the translation products of both ORFI and ORFII still remain unknown.In the present study, both of bacterial two-hybrid system and yeast two-hybrid system were utilized to analyze the interaction between PI, PII and CP encoded by BSV1 Constructions of bait and prey plasmids with the three target segments in bacterial two-hybrid system, tests of self-activation and interaction between different pairs of proteins.This set of experiments were carried out aimed to construct both bait and prey plasmids of ORFI, ORFII and CP, then to test the interaction between each two of them. The three segments were firstly generated by PCR amplification and then inserted into both pBT and pTRG plasmids. The results of sequencing suggested that both the sequence and the reading frame of the 6 plasmids were accurately right. Then the plasmids were transformed into competent cells of E. coli strain XL 1-Blue MRF’and the transformants were then poured on medium plates with or without 3-AT and incubated on 37℃, in the meantime, the transformants with only empty pTRG and pBT plasmids were utilized as negative controls. The results suggested that all of the 6 plasmids can not independently activate the report genes and thus could be further utilized in the following interaction tests. Each of the prey plasmids and bait plasmids were thereafter co-transformed into competent cells of E. coli strain XL 1-Blue MRF’in order to test the existence of interactions, the results suggested that PⅡ/CP, PⅡ/PⅡ, CP/CP had strong interactions, with the rest of the pairs only had extremely weak interactions or even no interaction. 2 Constructions of bait and prey plasmids with the three target segments in yeast two-hybrid system, testing self-activation and interaction between different pairs of proteins.The three segments were firstly generated by PCR amplification and then inserted into both pDESTTM32 and pDESTTM22 plasmids by BP and LR recombination of GatewayTM technology. The results of sequencing suggested that both the sequence and the reading frame of the 6 plasmids were accurately right. Then the plasmids are transformed into competent cells of yeast strain MaV203, the transformants were proved to have weak self-activations by the comparisons with a set of controls however could be utterly suppressed by 3-AT with the concentration of less than 50 mM and thus could be further utilized in the following interaction tests. Each of the prey plasmids and bait plasmids were thereafter co-transformed into competent cells of yeast strain MaV203 in order to test the existence of interaction, and the results coincided with the results in bacterial two-hybrid system.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Horticultural Crops Pest and Disease Control > Fruit tree pests and diseases > Perennial herbaceous fruit pests and diseases
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