Preliminary Study On The Tissue Culture And Regulation Mechanism Of Fruit Color Of Ailanthus Altissima ’Liaohong’

Ailanthus altissima ‘liaohong’ is a new cultivar of Ailanthus altissima cultivated by Liaocheng University after many years of cultivation,which has high ornamental value.In order to promote the new variety Ailanthus altissima ‘liaohong’ quickly and clarify the mechanism of the samara color forming and regulating,the tissue culture and rapid propagation system of Ailanthus altissima ‘liaohong’ were studied by us,also the the key regulatory factors of tissue-specific expression of anthocyanins in fruit pods of Ailanthus altissima ‘liaohong’ was analysis through transcriptome sequencing,and then an key regulatory factors was selected to construct the Cas9 knockout vector.The main research contents and conclusions are as follows:1.Establishment of tissue culture and rapid propagation system of Ailanthus altissima ‘liaohong’.(1)disinfection: The best disinfection method for axillary buds and terminal buds(single buds)as explants was soaking in 75% alcohol for 30 s,15% sodium hypochlorite solution for 15 min,0.1% mercury chloride solution for 6 min,respectively.The callus contamination rate was 20.00%,and callus induction rate was 40.00%.(2)Callus induction and subgeneration: In MS medium for dedifferentiation induction and subculture,the ratio of hormones was 0.1 mg/L 6-BA,0.1 mg/L NAA and 2,4-D,and the induction rate of callus was 40.00%.The color of callus was yellowish green and the growth was good.(3)adventitious bud induction: The optimal ratio of hormones in MS medium was 3.0 mg/L 6-BA,0.05 mg/L NAA and 2,4-D,and the induction rate of adventitious buds reached 82.33%.(4)Adventitious root generation: The optimal rooting medium MS contained NAA 2.0 mg/L,and the adventitious root induction rate was58.33%.(5)Test seedling transplanting: The survival rate of tissue culture plantlet was65.63% when the temperature was 25℃ and the humidity was 75%.2.Transcriptome sequencing and analysis of Ailanthus altissima ‘liaohong’ and Ailanthus altissima.In this study,transcriptome sequencing,splicing and functional annotation were performed on the pod samples of Ailanthus altissima ‘liaohong’ and Ailanthus altissima.And then to |log2FC|>1 and FDR<0.05 was used as the screening criterion to screen out differentially expressed genes,and related differentially expressed genes were analyzed by GO annotation and KEGG enrichment.Finally,the functional genes related to anthocyanin synthesis were selected for comparison and analysis,and three key regulatory genes with significant differences were identified,namely genes DN15707,DN16609 and DN16720.3.Construction of Cas9 knockout vector.According to transcriptome sequencing results,DN15707 gene of Ailanthus altissima ‘liaohong’ was targeted by CRISPR/Cas9 system.Two sg RNA was designed for the target gene,and two sg RNA expression box was connected to the same CRISPR vector skeleton(hygromycin resistance)to construct the knockout vector.