Cloning And Functional Analysis Of MtCYP450 In Medicago Truncatula

The monooxygenase encoding gene cytochrome P450(CYP450)plays an important role in plant defense against abiotic stresses by involving in a variety of catalytic reactions in plant.In this study,we identified and characterized Mt CYP450 from Medicago truncatula in order to provide reference for improving the salt tolerance of leguminous pasture.The main results are as follows:1.Cloning of Mt CYP450 from M.truncatula and bioinformatics analysis of the putative protein.Mt CYP450 encodes a peptide of 508 amino acids with molecular weight of 58.31 KDa.The secondary structure of the peptide contains alpha helix(44%)and beta sheets,beta turn and random coil about 20% individually.Sequences alignment of Mt CYP450 homologs displayed that Mt CYP450 shared high sequence identity with CYP94 amino acid sequences from Lupinus angustifolius(75.7%)and Glycine max(73.9%).Hence,Mt CYP450 belongs to the CYP94 subfamily of cytochrome P450.2.Analysis of the expression pattern of Mt CYP450 in different tissues and its response to abiotic treatments.According to q RT-PCR results,Mt CYP450 predominantly expressed in leaves and with the lowest level detected in root,which accounts for 1.2% of the amount in leaves.The expression level of Mt CYP450 transcript in the leaves was increased significantly by the treatments of 200 m M Na Cl,5% PEG,100 ?M ABA or 100 ?M Me JA.Therefore,Mt CYP450 is inducible by the above mentioned treatments.3.Enhanced stress tolerance of the transgenic Arabidopsis over-expressing Mt CYP450.The over-expression vector for Mt CYP450(p BI121-Mt CYP450)was constructed and transformed into Arabidopsis.q RT-PCR proved the expression of Mt CYP450 in 8 transgenic lines,indicating that exogenous Mt CYP450 was successfully transcribed in the transgenic Arabidopsis.The expression of Mt CYP450 in the transgenic plants was up-regulated by the treatment of PEG,ABA or Me JA.Morphologically,the Mt CYP450 overexpression Arabidopsis was observed with root length around 1.63 times of that of the wild type under Na Cl(125 m M)treatment,suggesting introduction of Mt CYP450 into Arabidopsis improved plant tolerance to salt.Thus,Mt CYP450 plays a vital role in plant tolerance to abiotic stress.4 Obtained transgenic M.truncatula plants over-expressing Mt CYP450 or with Mt CYP450 knocked-out.The vector p BI121-Mt CYP450 was transformed into M.truncatula.The expression of Mt CYP450 in 32 lines with the vector integrated into the M.truncatula genome was confirmed by RT-PCR.For knockout of Mt CYP450,vector p RGEB31-Mt CYP450 was constructed using CRISPR/Cas9 technology.Sequence analysis of the M.truncatula transformants confirmed 12 positive lines containing Mt CYP450 knockout construct.The transgenic plants will be used to study the salt tolerance of Mt CYP450 in Medicago truncatula.