Genome Editing And Expression Analysis Of AcrSOC1c And AcrSVP1 In Kiwifruit

As an important economic crop,kiwifruit is loved by people for its rich nutritional value and good taste,so its market demand and planting scale are also expanding day by day.However,in the early stage of kiwifruit breeding,it takes up to 3-4 years to bloom,and when spring buds enter a dormant state,they will develop into inflorescence buds.SOC1(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)and SVP(SHORT VEGETATIVE PHASE)have regulatory effects on the germination and dormancy of kiwifruit buds,and play an important role in regulating the flowering process of plants.Although they have made many progress in the vernalization and environmental temperature pathways,there are few reports on how to respond to environmental temperature in kiwifruit to regulate flowering period.Exploring the functions of SOC1 and SVP in kiwifruit can help to better analyze the kiwifruit flowering regulation network and provide solutions for early flowering of kiwifruit.We selected Hongyang kiwifruit as the experimental material in this project,then the expression and transcriptional characteristics response to temperature of AcrSOC1 c and AcrSVP1 genes were explored by bioinformatics analysis,gene cloning,gene editing and real-time fluorescence quantitative PCR in kiwifruit.Firstly,sequence alignment was performed on the SOC1 and SVP genes,and identified 8 AcrSOC1 s and6 AcrSVPs homologous genes in kiwifruit.The results of phylogenetic tree showed that the homology of kiwifruit AcrSVP1 with At SVP and Ac SVP2 was as high as 86%,and the homology of AcrSOC1 c with At SOC1,Ac SOC1 e and Ac SOC1 i was all above 92%.Subsequently,amino acid sequence alignment was performed on AcrSOC1 s and AcrSVPs,and the results showed that all AcrSOC1 s and AcrSVPs proteins contained the typical and conserved MADS-box and K-box regions of the MADS family,indicating their functional structures domain are conserved.According to the results of promoter sequence analysis,in addition to the classic cis-elements,AcrSOC1 s and AcrSVPs also contained various cis-elements related to abiotic stress response,such as AcrSVP1 containing the LTR(low-temperature response motif),and AcrSOC1 c containing the GARE-motif(gibberellin response motif),etc.RT-q PCR results showed that the most AcrSOC1 s and AcrSVPs were highly expressed in flower buds,but lower in flowers.Secondly,the DNA sequences of the SRF domain,K-box domain and promoter region of AcrSOC1 c and AcrSVP1 genes were cloned,respectively.Subsequently,based on the sequencing results,the target sites of the above sequences were predicted,and a total of 120 sg RNA sequences were designed.These primers were mixed and complemented directly,and further connected to the PTG-Cas9-TV vector,successfully constructing the sg RNA mixed vector library.Multi-site editing of kiwifruit target sequence by the mixed sg RNA,which can ensure that the target gene is mutated efficiently,and finally multiple mutants with edited promoter regions of AcrSOC1 c and AcrSVP1 were obtained,including soc1c-6,soc1c-7 and svp1-4.Combined with RT-q PCR analysis,we found that the transcript levels of target genes in these mutants were changed to varying degrees compared with wild-type or unedited plants,and the transcript levels would exhibit different changes with different temperature and times of treatment.Collectively,this study identified AcrSOC1 s and AcrSVPs of kiwifruit by bioinformatics analysis,and successfully edited the promoter regions of AcrSOC1 c and AcrSVP1 by transforming mixed sg RNA,so that the transcription level showed different changes from the wild type under different temperature treatments.Combined with the effects of AcrSOC1 c and AcrSVP1 on the flowering in other plants,the results of this study not only successfully verified the feasibility of mixed sg RNA for genome editing in kiwifruit,but also expanded the genome editing technology of kiwifruit.In addition,the mutants provide foundation for exploring the regulation of environmental temperature on the flowering of kiwifruit.The precise regulation of kiwifruit flowering makes further research on the key regulators involved in kiwifruit flowering have important scientific significance and commercial value.