CRISPR/Cas9-mediated Gene Editing Of Fd And FNR In Oncidium

Oncidium hybridum belongs to Orchidaceae,and it is a valuable tropical flower.Both Fd(ferredoxin)and FNR(ferredoxin-NADP+ oxidoreductase)are electron transfer proteins widely found in higher plants and are involved in many physiological and biochemical processes in plant cells.There were many types of Fd and FNR in plants and all of them are related to redox energy and tolerance to various stresses.In this study,the Fd and FNR genes of O.hybridum were cloned and analyzed,and the FdC2 and RFNR genes were further studied with overexpression and CRISPR/Cas9 editing technologies based on an protocorm-like bodies(PLBs)in O.hybridum.The results were as follows:1.Cloning and expression analysis of Fd genes in O.hybridumBased on 7 Fd gene sequences obtained from tanscriptome data,the members of Fd gene family in O.hybridum were cloned from the mixed samples of PLBs and PLBs-derived seedlings.The encoded amino acids of Fd genes were less than 200 each.The bioinformatics analysis showed that there were a mitochondrial MFDX and six chloroplast-type Fd in O.hybridum,which were closely related to Fd in Phalaenopsis amabilis.During the evolution of the Fd gene family,they showed condon biase of ending with A/T(U),and the biase was influenced by mutational stress,natural selection and other factors.The results of Fd genes expression showed tissue-specific expression patterns,and those expressions played an important role during the development of O.hybridum and were related to the plant responding to soft rot and high temperature stresses.2.Cloning and expression analysis of FNR genes in O.hybridumThe RFNR gene was cloned from the mixed samples of PLBs and PLBs-derived seedlings,which encoded 379 amino acids.The sequence of LFNR was obtained from the NCBI data to analyze the FNR genes of O.hybridum.The bioinformatics analysis showed that LFNR and RFNR belonged to an FNR-like super-family with flavin adenine dinucleotide(FAD)and nicotinamide adenine dinucleotide(NAD)binding domains and these domains were conservative in the evolution process,but exhibited significant differences in the composition of amino acids,protein structure and configurations.The RFNR was more conservative than LFNR.The results of codon usage bias of FNR in Orchidaceae showed there existed a significant T(U)bias between A and T(U)and a significant C bias between C and G in the codons.NADP binding domain showed less codon bias than that of the non-binding domain,and the codon usage bias of FNR was mainly influenced by the natural selection in Orchidaceae.The gene expression analysis showed that expressions of LFNR and RFNR exhibited organ-specific distributions,i.e.LFNR was mainly in the leaf tissue and RFNR in the root tissue.Both of them took part in the growth and development of O.hybridum and RFNR showed more rapidly and higher expression levels of responses to high temperature and soft rot stresses.3.Subcellular localization of FdC2 and RFNR and overexpressed study in O.hybridum PLBsThe subcellular localization of FdC2 and RFNR were checked in tobacco and O.hybridum leaves,and the results showed both of FdC2 and RFNR expressed mainly in the chloroplasts.In the case of overexpression,the PLBs that overexpressed the FdC2 formed a longer hypocotyl,while the PLBs that overexpressed RFNR formed leaves earlier.Therefore,it was speculated that FdC2 and RFNR in O.hybridum participated in the morphogenesis of PLBs.4.CRISPR/Cas9-mediated gene editing and functional study of FdC2 and RFNR in O.hybridumThe gDNA sequences of FdC2 and RFNR were cloned.The results showed there was not any intron in FdC2,while the RFNR gene had 5 introns and 6 exons.The CRISPR/Cas9 technology was used to edit the FdC2 and RFNR in O.hybridum and the results were primarily detected by HRM technology.It was found that CRISPR/Cas9 mediated gene edition changed the gene sequences both in protoplasts and in PLBs of O.hybridum.In protoplast,the 296 th base of FdC2 coding region was edited and caused the replacement of arginine by Lysine,which further led to the configurational change of the activity center and tertiary structure of the protein.In RFNR,after edition,a base in 5'UTR was changed.The morphological observation showed that the differentiation of PLBs with edited FdC2 gene was weakened,and PLBs became partially yellowed in the RFNR-edited PLBs.It was speculated that FdC2 and RFNR genes played an important role in the morphogenesis of PLBs.5.Morphological observation of Fd3 and RFNR transformed PLBs of O.hybridumTo study the effects of Fd and FNR genes on the development of O.hybridum PLBs,we observed the morphology of Fd3 and LFNR transformed PLBs.The results showed that the deviation of Fd3 and LFNR expression from the normal expression levels affected the differentiation and development of PLBs,in which lower expression levels of Fd3 and LFNR genes inhibited the development of PLBs and higher expression levels led to dedifferentiation of the PLBs.In summary,the function of Fd and FNR genes was studied by means of bioinformatic analysis,gene cloning,gene expression analysis,genetic transformation and CRISPR/Cas9 editing technologies.The results showed that Fd and FNR not only responsed to the soft rot and high temperature stresses,but also participated and highly affected the morphogenesis of PLBs in O.hybridum.