Molecular Characterization And CRISPR/Cas9-mediated Editing Of Oncidesa OnEIN2 Gene

Oncidesa belongs to the Orchidaceae.It is one of the most important potted flowers and fresh cut flowers in the world and has high economic ornamental value.Ethylene produced by mechanical damage in the production and transportation of fresh cut flowers caused the petals faster senescence.This is a major factor that restricts its wider application.So far,only some chemical treatments can be used to postpone the senescence of the petals,which cannot be solved from the source.Therefore,the breeding of the ethylene insensitive type of Oncidesa is a major direction.Ethylene-insensitive 2(EIN2)is a key hub in the process of ethylene signal transduction and has an important impact on the completion of ethylene reaction mechanism.It has been confirmed that the EIN2 mutation can cause ethylene insensitivity and block the transduction process of the ethylene signal,so that the ethylene reaction cannot be completed.In this experiment,we used the orchid transcriptome data,by the RT-PCR combined with RACE strategy clone the EIN2 gene.The preliminary functional verification of the gene was carried out by real-time qPCR and transformation of PLBs,callus and protoplasts.The main results of this study were as follows:1.Cloning and bioinformatics analysis of OnEIN2 in OncidesaBased on the nucleic acid sequence of AtEIN2 as a reference and compared with the Oncidesa Gower Ramsey transcriptome database to obtain a cDNA sequence with high similarity.Primers were designed for RT-PCR combined with RACE to clone the OnEIN2 gene.The OnEIN2 cDNA is 4177 bp and its ORF is 3476 bp and encodes 1292 amino acids,named OnEIN2(GenBank accession number:MH497388).Bioinformatics analysis showed that OnEIN2 contains a typical Nramp motif and is a transmembrane protein.The phylogenetic tree analyzed with the 22 different species amino acid sequences of EIN2,can be divided into two categories,with class I of monocotyledonous plants except carnation,and OnEIN2 is monocotyledon,belonging to class I,which corresponds to the species evolution.2.Determining expression levels of OnEIN2 in OncidesaThe expression of OnEIN2 gene in different plant tissues and organs of Oncidesa was carried out through qPCR.The results showed that OnEIN2 were expressed in roots,pseudobulbs,leaves and flowers.The expression of OnEIN2 was the highest in the flower,which is much higher than the other three tissue parts,indicating that the gene plays an important role in the whole development of flower;while the expression level in different flowering stages shows its expression in blooming stage is highest,followed by is the senescence period,while the expressions in the buds stage,bud burst stage and half-opened stage were similar.The differential expression patterns of OnEIN2 and its spatiotemporal specific expression suggest that it may be related to the senescence of flowers.The use of different hormone treatment and abiotic stress in Oncidesa healthy plants showed that OnEIN2 responded to IAA,GA,ABA,6-BA,SA,MeJA,ETH,NaCl and PEG.OnEIN2 expression showed downward trend as a whole under IAA,GA,ABA,6-BA,SA,MeJA,NaCl and PEG,only ETH treatment showed the upward trend first,and then downward.The expression trends under ABA and PEG treatment were similar,and they all decreased first,followed increased,decreased again and then increased.Under the treatment of 6-BA,GA,SA,MeJA and NaCl,the expression showed a trend of decreasing first,followed rising and then decreasing.Under the IAA treatment,it shows a trend of falling,rising and then falling,rising.Both exogenous hormones and abiotic stresses affect the expression of OnEIN2.This result may be caused by the effects of various hormones on ethylene synthesis and signal transduction processes.3.The function research by using CRISPR/Cas9 to editing the OnEIN2The leaves and lateral buds of Oncidesa healthy plants were used to prepare protoplasts and induce callus,transformation materials.The CRISPR/Cas9 vector of OnEIN2 was constructed and transferred into protoplasts,PLBs and callus by PEG method and Agrobacterium-mediated method,respectively.GUS and Cas9 histochemical staining of the transgenic materials was employed to detect the transformation result of the target vector.The HRM was used to detect whether there was a base mutation,and the transgenic material was used to qPCR of OnEIN2.The results of the test indicated that most of the Oncidesa tissue materials were positive transgenes by GUS and Cas9 detection.HRM analysis showed that most of the transgenic materials had base mutations.However,the transgenic protoplasts have fewer base mutations than the PLBs and callus.The results of qPCR showed that the expression level of OnEIN2 of transgenic materials was decreased.The results of various tests indicated that the CRISPR/Cas9 vector had an effect on OnEIN2 editing,which caused base mutation to a certain extent,its expression decreased.It may be that base mutation causes a difference in the expression of the gene.