Cloning And Stress Tolerance Analysis Of A ThPrx1 Gene From Tamarix Androssowii

In this paper, a ThPrx1 gene was cloned from Tamarix androssowii, which belong to an active oxygen scavenging enzyme peroxidase protein (ThPrx1) family. The length of the ThPrxl gene was 851 bp. The open reading frame of ThPrxl is 489bp in length that was from 165 of ATG to 653 of the TAA, and encoded 162 amino acids.The molecular weight of ThPrx1 protein is 17.5KD, and its theoretical isoelectric point is 6.08. The promoter sequence of ThPrxl gene was amplified by using Tail-PCR, which is 1768bp in length.The ThPrxl gene was inserted into pYES2 and transformed into yeast cells (Saccharomyces cerevisiae).Yeast cells transformed with pYES2 empty vector were also generated as a control. The ThPrxl yeast transformants and pYES2 empty vector yeast were stressed by 5 mol/1 NaCl,5 mol/1 KCl,6% Na2CO3,3% CuSO4,3% CdCl2,53℃high temperature,-20℃freezing,2 mol/1 sorbitol and 3% methyl viologen.The result showed that the ThPrxl yeast transformants exhibited a higher tolerance to the stresses of NaCl, KCl, Na2CO3, CuSO4, CdCl2, high temperature, freezing, sorbitol and methyl viologen compared with the control transformants cells, indicating that the ThPrxl gene is tolerant to the above abiotic stresses.In this paper, a Tamarix cDNA library used for yeast two-hybrid was constructed with yeast strain Y187 as host. The lengths of cDNA library inserts were from 750 bp to 3500 bp, and the transformation efficiency was 1.89×107 transformants/3μg pGADT7-Rec. Primary titer of the cinstructed library was 2.13×107pfu/ml with the rate of recombination of 80%.The ThPrxl gene was constructed into the bait expression vector PGBKT7-ThPrx1. Autoactivation and toxicity testing showed that the ThPrxl do not have the autoactivation ability and it is non-toxic to host strain, indicating that the PGBKT7-ThPrx1 can be used in yeast two-hybrid analysis.The yeast two-hybrid cDNA library was sereened with the bait of PGBKT7-ThPrx1. The bait plasmid and the library plasmid were co-transformed into the competent yeast Y2H with acetic acid lithium catalysis. All the clones were harvested from the TDO platessupplied with X-α-Gal and AbA(Aureobasidin A). After further confirmation, two proteins interacting with ThPrx1 proteion were obtained, which are calcium lipid binding protein and CONSTANS interacting protein.