Cloning And Prokaryotic Expression Of RtF3H1and RtF3H2Genes From Reauria Trigyna

Reaumuria trigyna is a small shrub endemic to the Eastern Alxa-Western Ordos area in Inner Mongolia. R. trigyna has a vital ecological function due to its remarkable capability of salt and drought tolerance. Additionally, R. trigyna have been used as traditional herb for the treatment of eczema, dermatitis and other diseases. In the transcriptome sequencing database, a large number of genes were annotated with the synthesis of flavonoids, including CHS, F3H, FLS, DFR and LDOX, and the expression of these genes were upregulated under salt stress. F3H is a key enzyme in flavonoid biosynthetic pathway and plays an important role in plant responsed to abiotic stress. We isolated two F3H genes from R. trigyna and studied their function under the abiotic stress. The results of this study will lay a foundation to elucidate the relationship between the flavonoid biosynthetic and the abiotic stress of R. trigyna.The main results are as follow:1. The full-length cDNA of RtF3Hl and RtF3H2were obtained by means of RT-PCR and RACE (Rapid-amplification of cDNA ends) technologies. RtF3H1contains an open reading frame (ORF) with the length of1059bp and encodes a protein with353amino acids. The ORF length of RtF3H2is1074bp, which encodes a protein with358amino acids. 2. The studies of the expression patterns of RtF3H1and RtF3H2in different organs showed that RtF3H1was only expressed in stems, while RtF3H2was mainly expressed in roots and stems.3. RtF3H1showed the similar express patterns with RtF3H2under salt, cold and ABA stress conditions. Both of these two genes were unregulated their expressions in seedlings exposed by salt solution for12hours; the expression levels of RtF3H1and RtF3H2were upgraded in R. trigyna treated with cold and ABA for3h and12h. However, the expression of RtF3H1was significantly different from RtF3H2under drought stress, RtF3H1was upregulated at1h while RtF3H2was upregulated at1h and3h.4. The prokaryotic expression vectors pGEX-4T1-RtF3H1and pGEX-4T1-RtF3H2were constructed and transformed into E. coli. The fusion proteins in recombinant E. coli cells were expressed after IPTG inducing by SDS-PAGE electrophoresis analysis.5. To evaluate the protective properties of two RtF3H fusion proteins, Effects of abiotic stress on the growth of transformed E. coli and control strains were examined. RtF3Hl and RtF3H2fusion proteins enhanced the tolerance of transformed E. coli strain to salinity, drought, ABA and UV radiation. Furthermore, pGEX-4T1-RtF3H2transformed cells exhibited stronger survival rate than pGEX-4T1-RtF3H1recombinant cells under saltinity and drought treatments.