In Vitro Mutagenesis Conditions And Genetic Transformation Of AUX1/AP2L1

Marchantia polymorpha L.belongs to bryophytes and is an emerging model plant.There are two types of reproduction methods,one is sexual reproduction: spores;the other is asexual reproduction: thallus or gemma.The M.polymorpha have the advantages of easy cultivation,short cycle and wide distribution.It can be used as a model to reveal the development mechanism of asexual reproduction,but the molecular regulation mechanism of gemma development is still unclear.In order to further understand the molecular regulation mechanism of gemma development,this experiment used gemma of M.polymorpha as the research object.The gemma were disinfected for different times by using 70% alcohol and 0.1% NaClO solution and compared with outdoor culture gemma to obtain the best disinfection treatment for the gemma and suitable tissue culture conditions for the gemma,and an in vitro culture system of M.polymorpha was established.Two mutagens,EMS and 5-BrU,were used to treat the gemma,and the best combination of mutagenic concentration and treatment time was screened to establish an in vitro mutagenesis system of M.polymorpha.The use of overexpression technology and CRISPR/Cas9 genome editing technology to obtain transgenic plants were used to explore the functions of MpAUX1 and MpAP2L1 genes in M.polymorpha.This study provides a reference for the use of chemical mutagens to mutagenize gemma and a scientific basis for elucidating the molecular mechanism of gemma development,and at the same time further lays the foundation for the similarities and differences for exploring the regulation mechanism of asexual reproduction in advanced plants and lower plants.The main research results are as follows:(1)70% alcohol and 0.1%NaClO solution were used to disinfect the gemma for different times.Finally,the best disinfection treatment was obtained as follows: 0.1%NaClO solution for 60 s.Compared with outdoor culture,gemma were more suitable to be cultured in 1/2 B5 medium(p H5.5-5.8),temperature 22?,photoperiod 16h/8h(day/night).(2)Two chemical mutagens,EMS and 5-BrU,were used to treat gemma.The germination of gemma was inhibited to varying degrees under EMS mutagenesis.The higher the EMS concentration and the longer the treatment time,the stronger the inhibitory effect.Finally,it was determined that the best combination of screening concentration and time for EMS mutagenic gemma was 0.30% for 4 hours,and mutant plants with inconsistent phenotypes with control plants were obtained.Under 5-BrU mutagenesis,the growth of gemma was inhibited to varying degrees.The higher the concentration,the stronger the inhibitory effect,but it was not enough to cause gemma death.(3)The MpAUX1 and MpAP2L1 genes were successfully cloned and inserted into the plant expression vector p CAMBIA1300 to construct the p1300-GUS-MpAUX1 and p1300-GUS-MpAP2L1 overexpression vectors.The MpAUX1 gene was inserted into the plant expression vector p BI121,and the p BI121-GUS-MpAUX1 overexpression vector was constructed.(4)According to the CDS sequence information of the target genes MpAUX1 and MpAP2L1,a pair of target sites were designed respectively,and the designed g RNA fragments were inserted into the CRISPR/Cas9 vector to successfully construct p1300-Cas9-MpAUX1 and p1300-Cas9-MpAP2L1 knock-out vector.(5)The cefotaxiene and hygromycin tolerance test was carried out on the gemma using pouring method and mixed culture method.In the pouring method,the change of cefotaxiene concentration has no obvious effect on the gemma.The best screening intensity of hygromycin for gemma is 100 ?g/m L for 2-3 weeks.In the mixed culture method,when the concentration of cefotaxiene is 2 mg/m L,all gemma die,indicating that the concentration cannot be exceeded for screening during the mixed culture stage.The best screening intensity for hygromycin to gemma gemma is 5 ?g/m L for 7-8 weeks or 10 ?g/m L for 2-3 weeks.Compared with the pouring stage,cefotaxiene and hygromycin have a greater impact on the gemma in the mixed culture stage.(6)The successfully constructed overexpression vectors p1300-GUS-MpAUX1,p1300-GUS-MpAP2L1,p BI121-GUS-MpAUX1 and the knock-out vector p1300-Cas9-MpAUX1,p1300-Cas9-MpAP2L1 were introduced into the gemma of M.polymorpha.Tissue culture obtained 10 p1300-GUS-MpAUX1 transformed plants;7 p1300-GUS-MpAP2L1 transformed plants;4 p1300-Cas9-MpAUX1 transformed plants,and 15 p1300-Cas9-MpAP2L1 transformed plants.After identification,they are all false positives.The overexpression vector p BI121-GUS-MpAUX1 was introduced into M.polymorpha,and transgenic plants were successfully obtained.