Optimization Of The Regeneration System Of Cerasus Humilis And Analysis Of Its VDE Gene Promoter

Cerasus humilis(Bge.)Sok is a perennial shrub in China,which belongs to the genus Cerasus in the family Rosaceae.Because of its high calcium content,strong plant resistance to stress,and wide adaptability to soil and environment,it has attracted more and more attention.With the in-depth research of Cerasus humilis and the continuous development of transgenic technology,the establishment of a stable genetic transformation system has become the basis for studying the molecular mechanism of Cerasus humilis’ resistance to stress.The establishment of a rapid and efficient regeneration system of Cerasus humilis and the construction of plant expression vectors are the prerequisites for exploring a stable genetic transformation system.The main research results of this paper are as follows:1.Using two-year-old Cerasus humilis((Nongda No.4))newly born young leaves and stems as explants to study the conditions of tissue culture of Cerasus humilis,the regeneration system of Cerasus humilis was optimized.(1)Optimal disinfection conditions for explants:Rinse the stems and young leaves under running water for 1 h,then move them to a benchtop and rinse them with 75%alcohol for 1 min.The stems were disinfected with 2%NaClO solution for 4 min,and the leaves were disinfected with 2%NaClO solution for 1 min.Finally,rinse with sterile water more than 5 times,1 minute each time.(2)The content of agar powder added to the medium at different stages of tissue culture is 7.5-8 g/L,the amount of sucrose added is 30 g/L,and the pH is 5.7-5.8.The optimal medium formula selected is shown below.Obtain sterile materials:MS+0.2 mg/L 6-BA;Stem segment to induce callus:MS+1.0 mg/L 6-BA+0.8 mg/L NAA;Leaf induced callus:MS+1.0 mg/L 6-BA+1.0 mg/L NAA;Proliferating callus:MS+0.5 mg/L 6-BA+0.5 mg/L NAA(or 0.6 mg/L 6-BA+0.6 mg/L NAA);Induce callus differentiation:MS+1.0 mg/L 6-BA+0.5 mg/L NAA;Induce differentiation of seedlings to take root:1/2MS(or WPM)+1.2 mg/L IBA.(3)The optimal conditions for domestication and transplantation of sterile seedlings:After the temperature stabilizes in May,the plum seedlings cultured in the rooting medium for 1 month are selected for domestication and transplantation according to the following steps.Closed bottle cultivation of seedlings:Move the tissue culture bottle to normal sunlight in a constant temperature room,cover it with a shading net,and cultivate the seedlings for 5 days;Open the bottle to exercise seedlings:Open the cap of the tissue culture bottle and move it to a ventilated and constant temperature place,cover it with a shade net,and refining the seedlings for 3 days;Domestication and transplanting:Wash the root medium,soak it with 0.1%carbendazim solution for 5 minutes,and transplant it into a flower pot with vermiculite and flower soil(ratio1:2)in the greenhouse.Cover with shading nets,and transplant the domesticated seedlings to the field one week later.2.According to the Cerasus humilis genome database,specific primers were designed at 1978 bp upstream of the start codon of the Cerasus humilis VED(Violaxanthin de-epoxidase)gene for PCR amplification.The Plant CARE promoter analysis website was used to analyze the upstream promoter of the Ch VDE gene,and the 5’ end deletion design was carried out on the full length of the promoter according to the regulatory elements.Finally,the expression-vectors of Cerasus humilis VDE promoter and 5’ deletion promoter were successfully constructed by homologous recombination.