Preliminary Study On The Function Of Plant OPA3-like Gene

Optic atrophy type 3(OPA3),also known as optic atrophy syndrome,is an autosomal recessive neuroencephalopathy syndrome characterized by early-onset bilateral optic atrophy and late developmental paralysis.Type 3 optic atrophy is caused by loss of OPA3 protein function.OPA3 is a protein encoded by the nuclear genome that localizes to mitochondria.Homologous genes of OPA3 are also present in fungi and plants.In plants,Potato virus Y(PVY)helper component proteinase(HC-Pro)can help potato virus to invade plants and affect chloroplast division.Tomato OPA3-like protein and HC-Pro protein can interact in yeast cells,and HC-Pro protein and tomato OPA3-like protein can also interact on the plasma membrane of onion epidermal cells.It is therefore suspected that there may be a correlation between the two proteins.In tobacco epidermal cells,tomato OPA3-like protein localizes to chloroplasts,suggesting that this protein may be involved in chloroplasts.Objective: Through the study of tomato OPA3-like(SlOPA3L)gene and tobacco OPA3-like(NtOPA3L)gene,the function of plant OPA3-like gene was preliminarily explored.Methods: Analysis of protein physicochemical properties,signal peptides,secondary structures and transmembrane domains using the online software ProtParam,SignalP,SOPMA,ProtScale,TMHMM and WoLFPSORT;overexpression of the NtOPA3 L gene pBP-Nt-OPA3L1,pBP-Nt-OPA3L2 And interference vectors pBRi-Nt-OPA3L1,pBRi-Nt-OPA3L2,transformed into tobacco by Agrobacterium-mediated transformation;construction of SlOPA3 L gene CRISPR/Cas9 vector pKAN-sgRNA-Cas9-OPA3,transformed into tomato by Agrobacterium;The over-expressed vector SlpBP-OPA3-like and the interfering vector SlpBRi-OPA3-like of the constructed SlOPA3 L gene were transformed into tomato by Agrobacterium tumefaciens;the transgenic tobacco T2 generation seeds overexpressing the SlOPA3 L gene were used for analytical statistical experiments and salt stress resistance experiments.Results:(1)Bioinformatics predicts that plant OPA3-like is an unstable protein with no transmembrane domain,localized to chloroplast and mitochondria.The N-terminal signal anchoring sequence of Arabidopsis OPA3-like protein has a signal peptide cleavage at position 24 of the N-terminal signal anchoring sequence.Cut the site and secrete the protein.(2)Transgenic tomato plants overexpressing the S1OPA3 L gene in T1 generation were dwarf,with many branches and easy to vitrify;transgenic tomato plants that interfered with the Sl OPA3-like gene in T1 generation were tall,with few branches,and the top branches were easy to be yellow.(3)Some tissues of transgenic tobacco cotyledons overexpressing the S1OPA3-like gene in T2 generation were chlorotic,and the leaves were mosaic-like,while the true leaves of most transgenic plants were not significantly different from wild-type tobacco,and a few early true leaves were mosaic.The phenotype of the transgenic plants,the later growing leaves returned to normal.(4)The overexpression vectors pBP-Nt-OPA3L1 and pBP-Nt-OPA3L2,the interfering vectors pBRi-Nt-OPA3L1 and pBRi-NtOPA3 L of NtOPA3 L gene were successfully constructed;the SlOPA3 L gene CRISPR/Cas9 vector pKANsgRNA-Cas9-OPA3 was successfully constructed.(5)The transgenic tobacco pBP-Nt-OPA3L1 overexpressing the NtOPA3-like gene was obtained;the transgenic tomato pKAN-sgRNA-Cas9-OPA3 expressed by the CRISPR/Cas9 vector of the SlOPA3-like gene was obtained.