Study The Gene Inversion And Deletion Mediated By Phage PhiC31/Att Site-specific Recombinant System

In the research of transgene,we can control a gene expression artificially by establish a "gene switch".The recombinant enzyme in a site-specific recombination system can recognizes the specific recombination sites to induce a recombination reaction. The recombination reactions can induce gene integration, deletion and inversion depended on the direction of recombination sites arrangemnt.These features of site-specific recombination system provides the possibility to design a "gene switch".The phiC31/att site-specific recombinant system in this study include a recombinase phiC31which is composed of613amino acids and two recognition sites attB and attP.The recognition sites attB and attP are non-homologous sequences and produce two new recombination sites attR and attL which can not be recognized by the recombinant enzyme phiC31after recombination.So the recombination induced by phiC31/att site-specific recombinant system is an irreversible reaction, which can be used to establish a locked " gene switch" in plant transgenic research.In present, there are lot of reports about the application of phiC31/att site-specific recombinant system in gene integration, and deletion,few reports in gene invertion. In this study, we constructed an prokaryotic expression vector PACYCNC31-1and two plant expression vector PCA23C31and PCA23NC31to express recombinant enzyme phiC31in Escherichia coli or plants. Meanwhile, three test vectors PE35SattB-H, PE35SattB-T and PEBar-D for prokaryotic as well as five plant expression vector PCABARattB-GUS, PCA13attB-BAR, PCA13attB-BT, PCA13BARattBoMYB2and PCA1335SBARattBoMYB2which contain recombinant enzyme binding sites attB and attP in different arrangement dirctions were constructed respectively in order to verify gene inversion function and gene deletion function induced by phiC31recombinant enzyme in prokaryotes and plants.This study will provide experimental evidence for using phiC31/att site-specific recombinant system to establish a stable "gene switch". which can achieve precise control of exogenous gene expression in plant.The main results are as following:(1) The prokaryotic expression vector PE35SattB-H,PE35SattB-T and PEBar-D were co-transformed into E. coli BL21(DE3) with PACYCNC31-1respectively. The dual-resistance clones on LB solid medium(contained Amp+Cam+IPTG) were identified by PCR using primers35S-F,Nos-R and C31-1,C31-4.The results showed that the recombinant enzyme gene phiNC31without intron can express the recombinant enzyme phiC31in E. coli, induce Bar-E9gene sequences deletion located between the recombination sites attB and attP arranged in the same direction as well as the inversion of35S promoter located between attB and attP arranged in the direction of "tail to tial".However, no inversion occurred when the35S promoter is located between attB and attP arranged in the direction of "head to head".(2) The phiC31(with an intron) and phiNC31(without intron) expression vectors and three plant expression vectors that use GUS, Bar and BT(MYB-TT8,a fusion gene control the anthocyanin biosynthesis) as reporter genes and contained binding sites of attB and attP arranged in "head to head" direction were intoduced into Brassica juncea and tobacco cells by Agrobacterium-mediated co-transformation or retransformation respectively.,The leaves of transgenic plants were perfomed for GUS staining, PPT resistance identification,PCR identification, cloning and sequencing.All the results indicated that the phiC31recombinase could not make the35S promoter located between the two recombination sites arranged in "head to head" direction to produce inversion,because no any report gene expression was onserved after co-transformation or retransformation with the recombinase gene phiC31(with an intron) and phiNC31(without intron).(3) PCA1335SBARattBoMY-B2vector (the binding sites attB and attP arranged in same direction) with a BoMYB reporter gene and PCA23C31or PCA23NC31were introduced into tobacco by,the Agrobacterium mediated co-transformation respectively..Red callus were observed on the selection medium after co-transformation,the result demonstrated the Bar-E9"Block sequence’located on the upstream of BoMYB gene had been deleted. A further PCR anlaysis was performed using the primers35S-1and BoMYB-R2, and an expected1.0kb fragment was amplified when the DNA of callus was taken as template.The1.0kb fragment was cloned and sequenced, from the sequencing results,we found a new recombination site attR (55bp) which contains section sequences of attB54and attP57produced..The results showed that the recombinase gene phiC31with a introninthe coding region can splicing properly and express functional recombinase in plant cell, the expression of the recombinant enzyme phiC31induced precise deletion of Bar-E9"Blocking gene",and made the BoMYB gene under the control of35S promoter, activated the expression of BoMYB gene..Due to the limitation of time,the experiments of gene inversion induced by recombinase phiC31between attB and attP arranged in "tail-to-tail" are in plant cells are in progress.