Preliminary Establishment Of Tissue Culture And Rapid Propagation System Of Flood-tolerant Mulberry And Functional Study Of MnCIPK2

Mulberry is one of the important economic tree species in the Yangtze River region of Anhui Province.In this paper,the mulberry trees with strong flood tolerance were screened by mulberry trees in Xinguanzhou Forest Farm of Anhui Province for many years,and the tissue culture research was carried out to establish a tissue culture and rapid propagation system.On this basis,the CIPK gene family related to waterlogging stress in mulberry trees was excavated,and the molecular characteristics of CIPK2 gene in mulberry were explored.In this study,we first analyzed the genome of mulberry using bioinformatics such as gene structure,evolution and transcriptome data,and analyzed the CIPK extracted from mulberry leaves by simulating flooding environment treatment of mulberry trees.The expression pattern was induced,and the MnCIPK2 gene was screened based on the experimental results.Subsequently,the MnCIPK2 gene was cloned from the mulberry,and the molecular sequence characteristics of MnCIPK2 were analyzed,including sequence characteristics,tissue expression patterns,subcellular localization and prokaryotic expression analysis.The main results of this study are as follows:1.In the mulberry tissue culture experiment,when the concentration of sodium hypochlorite is 1%,the disinfection effect of mulberry leaves for 5 minutes is most effective;when the concentration of PVPK-60 was 0.8 g/L in the medium,browning was significantly inhibited;the optimum callus medium for mulberry leaves is:MS+6-BA 1.5 mol/L+NAA 0.5 mol/L+PVPK-60 0.8 g/L.The mulberry tree leaf stem segment medium formula was 1/2 MS+IBA 0.8 mg/L+PVPK-60 0.8 g/L.2.42 MnCIPKs were identified from the mulberry genome database.Gene structure analysis indicated that 40%of MnCIPKs had only 1 intron.The introns of the seven MnCIPKs genes were all greater than 10,of which MnCIPK25 contained the most introns of 15.Conserved motif analysis indicated that most MnCIPKs include motif 1 and motif 7,and are arranged in the order of 1-7.By analysis of gene structure and conserved motif,it was found that the CIPK gene of mulberry were conserved to a certain extent.Similar conserved motifs were found among genes with high sequence similarity.3.qRT-PCR showed that MnCIPK2 was highly expressed after treatment with flooding stress.And tissue expression pattern showed that MnCIPK2 was highly expressed in roots,leaves and fruits.4.Subcellular localization revealed that MnCIPK2 localizes to the nucleus and cell membrane.Prokaryotic expression experiments showed that the prokaryotic expression vector of MnCIPK2 was successfully constructed,and its expression was induced and purified.