Selection Of Reference Genes And Identification Of MsWRKY33Gene In Medicago Sativa L

Medicago sativa.an excellent leguminous forage grass,play an important role in the development of animal husbandry and aquaculture. Gene expression patterns of a target gene in different tissues and at different processing conditions can provide clues towards the understanding of its biological function. The accuracy of gene expression strongly depends on the transcript normalization using appropriate reference genes via quantitative real-time reverse transcription-PCR (qRT-PCR) analysis. WRKY which is one of the most important transcription factors during diverse physiological and developmental processes of plants, play an important role in stress response and plant growth. We screened reference genes for using in real-time fluorescence quantitative PCR in Medicago sativa and conducted the analysis of MsWRKY33transcription factor. The main results of this study are as following:1.The full length of18S rRNA one of reference genes, was cloned from Medicago sativa.Furthermore, the stability of eight traditionl reference genes (UBC2、EF-1α、β-tubulin、 β-actin、Actin2、MSC27、GAPDH7) was analyzed in different tissues and stress treatments including ABA,salt,drought and dark by using the real-time fluorescence quantitative PCR.In order to screen more stable reference genes and make sure the number of reference genes, the geNorm analysis show that:MSC27、18S rRNA、EF-1αand Actin2is relatively stable reference genes in different organs and under different stress conditions in Medicago sativa.2. A unigene sequence of WRKY transcription factors was selected as candidated gene using high-throughput screening method.A WRKY gene,MsWRKY33,was cloned from the leaves of Medicago sativa by using RT-PCR and RACE.The full-length cDNA of Ms WRKY33was1891bp.It contained a1536bp open reading frame and encoded511amino acids. The deduced amino acid sequence of MsWRKY33protein shared high identity with Arabidopsis and rice via multiple alignment.MsWRKY33contained two highly conserved amino acid sequence WRKYGQK and two structures of Cys2His2zinc finger,which belonged to group I subfamily.3. Acording to the reference genes of18S rRNA that was screened, Real-time fluorescence quantitative PCR was performed to reveal the transcript levels of Ms WRKY33under different tissues and stresses.The expression level of MsWRKY33transcript was increased by PEG.cold and ABA.However,the expression level was decreased after4hours by salt treatment. The expression of Ms WRKY3’3briefly increased in the first twelve hours under heavy metal treatment and then it decreased.4.The coding region of MsWRKY33from Medicago sativa was inserted into the binary expression vector pCAMBIA3301to construct the recombined vector pCAMBIA3301-MsWRKY33.The recombinant vector was transformed into Arabidopsis by floral dip method. Transgenic Arabidopsis was btained by generations of selection,propagation and molecular detection.The transgenic Arabidopsis increased the elongation of root compared with wild-type under4℃and it was suggested that Ms WRKY33was associated with the cool resistance.