Prokaryotic Expressing And Functional Analysis Of C3HC4-type Zinc Finger Protein NbZFP1in Nicotiana Benthamiana

Zinc finger proteins are one of the most abundant types of transcription factors in eukaryoticgenomes. Zinc finger protein can bind DNA, RNA and lipid substrates, also this kind of proteins areinvolved in numerous cellular processes, including transcription, signal transduction, andrecombination.In this study, a C3HC4-type zinc finger gene was isolated from Nicotiana benthamiana.Sequence analysis indicated that the gene encodes a24-kDa protein with203amino acids containingone typical C3HC4-type zinc finger domain; this gene was named NbZFP1. Transient expression ofpGDG-NbZFP1demonstrated that NbZFP1was localized to the chloroplast, especially in thechloroplasts of cells surrounding leaf stomata. Virus-induced gene silencing (VIGS) analysisindicated that silencing of NbZFP1hampered fruit development. An overexpression construct wasthen designed and transferred into Nicotiana benthamiana, The transgenic lines showed typicalcompactness, with a short internode length and sturdy stems. This is the first report describing thefunction of a C3HC4-type RING finger protein in tobacco.The main research results are as summarized as follows:1. NbZFP1cloning and phylogenic analysisUsing a pair of specific primers, the gene we have designated as NbZFP1was cloned from N.benthamiana by RT-PCR. The length of the full open reading frame (ORF: GenBank accessionnumber KJ169550) for this gene is576bp. The maximum likelihood (ML) tree of the C3HC4-typeRING finger domain-containing motif suggested that C3HC4-type RING finger genes are highlyconserved in25species. In addition, the tree of full-length sequences of C3HC4-type RING fingergenes showed that the similarities between NbZFP1and the C3HC4-type RING finger genes in theother25species are not high.2. Purification and detection of recombinant NbZFP1proteinNbZFP1was cloned into the prokaryotic expression vector pGEX-6p-2, the recombinantplasmids was then transformed into E. coli BL21(DE3) to express the recombinant protein. Theexpressed protein was soluble in E. coli and was purified with a GSTrap HP column followed by aHiTrap desalting column and a HiTrap Q HP column. The molecular weight of the GST tag is26kDa,and SDS-PAGE analysis showed a single band corresponding to the purified recombinant proteinwith an approximate molecular mass of50kDa.3. Subcellular localization of the NbZFP1proteinTo confirm the subcellular localization of the NbZFP1protein, we constructed a recombinantvector, pGDG-NbZFP1, for transient expression. Agrobacterium GV3101containing pGDG,pGDG-Rac1, or pGDG-NbZFP1was used to infiltrate healthy tobacco plants. Laser scanningconfocal micrographs showed that the protein of interest, NbZFP1-GFP, was located in thechloroplast, especially in the chloroplasts of cells surrounding leaf stomata.4. Developmental phenotypes revealed by silencing NbZFP1 TRV-mediated VIGS is an effective tool for assessing the functions of genes in the reproductivetissues of plants. We then cloned two different fragments of NbZFP1gene separately into theTRV2-LIC vector for silencing. We infiltrated plants at the four-leaf stage with a1:1mixture ofTRV1and the TRV2-LIC-NbZFP1fragment for each of the clones, and we monitored the infiltratedplants throughout their entire lifespan. We then determined the degree of silencing of NbZFP1fragments by qPCR. The results revealed a greater than90%reduction in transcript levels in thesilenced plants. Silencing NbZFP1resulted in fruits that were smaller than those of the TRV-infectedcontrol, although no other phenotypic defects were observed.5. Generation of NbZFP1transgenic tobacco plantsThe plant expression vector pBI121containing NbZFP1was transformed into tobacco via theAgrobacterium-mediated method. Nine independent kanamycin-resistant NbZFP1transgenic tobaccolines were obtained. These transgenic tobacco plants were verified by PCR, and eight transgenic lineswere positive. We randomly selected3transgenic plants and performed a Southern blottingexperiment, which showed that NbZFP1was successfully integrated into all3transgenic tobaccoplants and one of the lines had a single copy of NbZFP1.6. Growth measurement and TMV-GFP resistance identification in transgenic linesWe selected T0plants with a single copy of NbZFP1and obtained their seeds. These seeds werethen sown on1/2MS medium with kanamycin selection, we found that transgenic lines wereshorter than wild type lines at day20after the four-leaf stage, and the regenerated plantswere compact with short internodes and sturdy stems.