Functional Analysis Of Small GTP-binding Proteins Activating Protein MtArfGAP In Medicago Truncatula

ADP ribosylation factor belongs to the Arf subfamily of the small G protein superfamily,which is commonly found in eukaryotic organisms.Arf plays an important role in the transport of vesicles between endoplasmic reticulum?ER?and the Golgi apparatus for maintaining the structure of the Golgi and ER,and in the lysosomal/vacuole transport pathways.Like the other small G proteins,Arf has a weak background GTPase activity.GTPase-activating protein can efficiently hydrolyze GTP to GDP and become the inactive form.The ADP-ribosylation factor GTPase-activating proteins promote the hydrolysis of Arfs by GTPase,which converts Arf from the active form combined with GTP into an inactive form that binds to GDP,and play a key role in the regulation of Arfs activity and function.Two ArfGAP genes were investigated based on our previous study on the the gene transcriptome of Medicago truncatula in response to low temperature.MtArfGAP1?Mt1g069000?and MtArfGAP2?Mt7g099830?encode proteins consisting of 481 and 443amino acids,respectively.The all contain a conserved group of zinc finger domains belonging to the CX₂CX₁₆CX₂C,ArfGAP-specific domain.MtArfGAP1 and MtArfGAP2proteins are similar in structure and have 65%homology.MtArfGAP1 and MtArfGAP2proteins were more similar to AtAGD6 and AtAGD7 among fifteen Arf GAP proteins in Arabidopsis and were classified to class II ArfGAP.Mt ArfGAP1 and MtArfGAP2 proteins were found to be distributed in the nucleus observed by laser confocal microscopy.P_MtArfGAP1::GUS and P_MtArfGAP2::GUS expression vectors were constructed and transformed into Arabidopsis,and P_MtArfGAP1::GUS transgenic plants were obtained,but we faild to obtaine P_MtArfGAP2::GUS plants.MtArfGAP1 was found to be mainly expressed in flowers and leaves.Real-time fluorescence quantitative PCR analysis showed that both light and low temperature influence expression of MtArfGAP1 and MtArfGAP2.Both MtArfGAP1 and MtArfGAP2 expression was induced by light,but down-regulated by low temperature.MtArfGAP1 and MtArf GAP2 intact ORF regions were cloned into overexpressing vectors to construct Overexpression vectors of MtArfGAP1 and MtArfGAP2,respectively,and the CRISPR/Cas9 vectors of MtArfGAP1 and MtArfGAP2 were constructed by using the genome editing technique by designing two targets in the ORF 5'region and the functional domain of MtArfGAP1 and MtArfGAP2.The vectors were transformed into M.truncatula,and transgenic plants were selected by basta-resistance.Using PCR detection,nine transgenic M.truncatula overexpressing MtArfGAP1,eight transgenic M.truncatula with knockout MtArfGAP1,two transgenic M.truncatula with overexpression of MtArfGAP2,and one transgenic M.truncatula with knockout MtArfGAP2 were identified.Two novel knockout vectors of MtArfGAP1 and MtArfGAP2were constructed by the simultaneous design of MtArfGAP1 and MtArf GAP2 primers on CRISPR/Cas9 vector and transformed into M.truncatula to obtain four transgenic plants editing both MtArfGAP1 and MtArf GAP2.More transgenic plants will be generated within next months.Although phenotyping of the transgenic plants has not been conducted,our results are important provided plant materials for further study of MtArfGAP1 and MtArfGAP2functions in plant development and abiotic stress adaptation.