Effects Of Deletion Of VKOR On Expression Of AnnAT1, A Stress-resistant Gene From Arabidopsis

Vitamin K epoxide reductase (VKOR) act as membrane protein in the cells ofanimals, plants, and microorganisms, and recent results suggest that they located inthe chloroplast and had the oxidoreductase activity. Annexins are a multigene,multifunctional family of Ca2+-dependent membrane-binding proteins playing a keyrole in stress. Some study have indicated that expression of Annexin1had closerelationship with plant stress. Annexin1was up-regulated in stress and had partlyperoxidase activity. Arabidopsis leaves of wide type(WT), knockout (lto1-2), andcompensation (lto1-2C) plants were used to study effects of Arabidopsis VKOR onthe expression of Annexin1on mRNA and protein level in stress. This research mayprovide certain theory basis for improving survival ability of crops and clarifying themolecular mechanism of plant stress response. The main results are as follows:(1) Cloning and expression of AnnAT1and antibody preparation. We designprimer of AnnAT1gene (AnnAT1) and construct recombinant plasmid. The BL21cellswere transformed with recombinant plasmid and vector alone.After IPTG induce,wegot AnnAT1after purification and then obtain the antibody of AnnAT1.(2) Expression of AnnAT1improve survival ability of prokaryotic cell in stress.E. coli cells were grown on LB basal plates or supplemented with NaCl, KCl orMannitol. The recombinant E. coli cells showed good growth under NaCl, KCl andmannitol stress compared to the vector alone. The growth was also analyzed in the LBliquid medium and OD600was recorded. The recombinant E. coli cells showed bettergrowth against all the treatments compared to vector alone.The result indicate thatexpression of AnnAT1improve survival ability of prokaryotic cell in stress.(3) Arabidopsis VKOR affected the expression of AnnAT1on transcription levelin stress. Arabidopsis leaves of wide type(WT), knockout (lto1-2), and compensation (lto1-2C) plants were treatment with salt and drought. For salt stress treatment, leaveswere laid onto the surface of150mM NaCl solution in the growth chambers for0h,12h,24h,36h and48h. And for drought, fully expanded leaves were detached andlaid in petri dishes for0h,1.5h,3h,4.5h and6h. The qRT-PCR results showed thatmRNA expression of lto1-2was obviously less than WT and lto1-2C. The latter twowere similar. These indicated that VKOR affected the expression of AnnAT1onmRNA level in stress.(4) Arabidopsis VKOR affected the expression of AnnAT1on transcription levelin stress. Arabidopsis leaves of wide type(WT), knockout (lto1-2), and compensation(lto1-2C) plants were treatment with salt and drought. For salt stress treatment, leaveswere laid onto the surface of150mM NaCl solution in the growth chambers for0h,24h and48h. And for drought, fully expanded leaves were detached and laid in petridishes for0h,3h and6h. The results of Western Bloting have no changes with theabove result. These indicated that VKOR affected the expression of AnnAT1onprotein level in stress.(5) VKOR affected the accumulation of negative oxygen ionand hydrogen peroxide of Arabidopsis in stress. Accumulation of negative oxygen ionand hydrogen peroxide were examined from leaves of wide type(WT), knockout(lto1-2), and compensation (lto1-2C) plants treatment with salt for24h and droughtfor3h. The two plants stress time point were sorted out on the basis of the biggest gapin expression between WT and lto1-2. Result revealed that accumulationof negative oxygen ion and hydrogen peroxide increased much more in lto1-2becauseof knockout of VKOR,comparing with WT and lto1-2C. That is impossibly becausethat VKOR affected the expression of AnnAT1on mRNA and protein level.