Cloning, Prokaryotic Expression And Study On The Biological Function Of AtGEN1in Arabidopsis Thaliana L.

The integrity of genomes of all living organisms is continuously being challenged bydifferent environmental agents and metabolic by-products. Consequently, evolution hasprovided organisms with several DNA repair and recombination pathways to remove or totolerate lesions in their genetic material and maintain the integrity of genome. Resolving ofthe intermediate Holliday junction (Hj) is the key step during DNA repair and recombination,and the enzyme involved in this process was known as resolvase. Previous studies haveshown that the features of resolvase in yeast and human, however, little is known about plantresolvase. Our study first acquired At1g01880(AtGEN1)and At3g48900which are speculatedas candidate genes of resolvase in Arabidopsis thaliana L. by reverse transcription PCR. Thetissue-specific expression of AtGEN1was confirmed using quantitative RT-PCR. The targetprotein－AtGEN1which contained599amino acid residues was obtained in prokaryoticexpression system, as well as we tested the enzyme activity of AtGEN1in different ion buffer,temperature and substrates. In addition, we screened Arabidopsis thaliana L. mutants withT-DNA insertion in exons or introns, and seeds of homozygotes and heterozygotes wereharvested in different mutants. Results obtained in this paper as follows:1. Cloned the candidate genes of resolvase in Arabidopsis thaliana L.(At1g01880,At3g48900), the protein they encoded contains three conservative blocks: XPG-N, XPG-I,helix-hairpin-helix, which is characteristic of Rad2/XPG protein family. Homology analysisshowed that they belonged to the class Ⅳ of Rad2/XPG just as the resolvase branch inyeast and human. Phylogenetic tree of GEN1-like in plant was divided into3branches:monocotyledonous plants, dicotyledonous plants, Selaginellales and Bryopsida.2. Detected the tissue-specific expression of At1g01880(AtGEN1) by RT-PCR in fourdifferent tissues: the roots, the leaves, the stems and the flowers in Arabidopsis thaliana L..The results showed that constitutive expression of AtGEN1in these tissues, but there wasa significantly rise in flowers.3. Prokaryotic expression analysis suggested that low amount of AtGEN1with His tag,what was the worse that almost all was inclusion bodies. We successfully purified full-length recombinant AtGEN1-His protein from soluble fractions by using a lowtemperature (20℃) for cell growth and induction in the bacterial pET expression system.Western blot analysis revealed the protein we getted just was the target protein.4. Results about the resolving activity of AtGEN1-His were that it could work at30℃withMg2+(5mmol/L). Its specific substrates were5′-flap and replication fork. It has noactivity to liner duplex DNA. Both of these were normal and consistent with articlespublished, but unfortunately it was noneffective to Holliday junction structure(J3andX0).We tried to truncate the C-terminus of AtGEN1which was disorder sequences andfused MBP to N-terminus of ΔcAtGEN1, then analyzed the resolving activity of AtGEN1once more, but no enzyme activity desired.5. Using PCR, we screened4kinds of Arabidopsis thaliana L. mutants with T-DNAinsertion. Harvested homozygous mutants in SALK-027797and SALK-106839,heterozygous mutants in SALK-099116and SALK-135735. Studies have shown thatdeficiency of OsGEN-L (the analogous gene of GEN1in rice) led to male sterility. So weinfluced mutants with no homozygous mutants may because of the mutation of AtGEN1which caused reduction of pollen fertility.Our study first expressed and purified AGEN1in Arabidopsis thaliana L. and analysedits catalytic characteristics. This work have laided the foundation for GEN1's functionalillustrate in plants' DNA recombination and repair.