T1083 substitution mutant in tail sequence of tobacco NtKRP was amplified by overlapping PCR from pBI121KE plasmid template.The fragment was then cloned into pGBKT7 vector to construct BD fusion expression plasmid pGBKT7-Ts,which laid an experimental foundation for further understanding of the effect of T1083 substitution mutation on the interaction of NtKRP and its target partner.
[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.
