Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.

Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization.Because of their simple protocols,they have become some of the most frequently used methods.However,standard fluorescent proteins present several drawbacks for sophisticated microscopy.With the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands.Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods.Therefore,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand.Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous actin.In addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated imaging.Split-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods.Therefore,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.