Cloning And Function Identification Of SKIP Homologous Genes In Medicago Sativa L.

In this study,the cDNA sequence of SKIP homologous gene from Medicago sativa L.was cloned and identified.Medicago sativa L.was cultured in 1/2 MS medium which contains 20%PEG 6000 to simulated drought stress.The total RNA was extract from the leaves of Medicago sativa L.which was treated with 6h drought stress.The total RNA was used as the template of reverse transcription.The full-length cDNA sequence of splicing factor SKIP in Medicago sativa L.was isolated by RT-PCR and named MsSKIP(GeneBank:KX240083.1).The sequence analysis of the MsSKIP showed the full lenghth cDNA is 2256 bp,and containes a1836 bp open reading frame which encodes 611 amino acids.The molecular weight of Ms SKIP is 69.08 kDa and the isoelectric point is 8.78.The MsSKIP contains a SNW_SKIP domain with the S-N-W-K-N polypeptide that is a typical domain of SKIP family.Sequence analysis of MsSKIP in the genome of Medicago sativa L.showed that the MsSKIP has no intron.To study the function of MsSKIP and MfSKIP,we using the technology ofGateWay to construct the tasiRNA vector of pKGWRR-tasi-MsSKIP and pKGWRR-tasi-MfSKIP.The plasmid of pKGWRR-MsSKIP was used as control.All of the recombinant plasmids were transferred into Agrobacterium rhizogenes Arqua1 with freezing-thawing transformation method,and transferred into the A17 root cultured for 2 weeks with the hair roots transformation method.The experiment was repeated three times.Detection of explants with truncated clam plants transformed with exogenous genes in root tissue under fluorescent microscope.Thirty seedlings with the same growth potential were selected from each transformant and subjected to different stress treatments(0° C,300 mM Mannitol,200 mM NaCl)for 10 days.The length and weight of the root tissue were measured and statistically processed.The total RNA was extracted from each plantroot and Q-PCR was used to detect the relative expression of stress-resistance related gene MtDREB1 c.The results showed that the length and the weight of the roots which transformed with pKGWRR-tasi-MsSKIP and pKGWRR-tasi-MfSKIP under three stresses were significantly lower than the roots of the seedlings transformed with pKGWRR-MsSKIP,and the relative expression of MtDREB1 c was significantly reduced.These results showed that the MsSKIP and Mf SKIP could enhance the ability of plants to resist abiotic stress.The DREB1 c may be regulated by the SKIP.