Research On Cell Penetrating Peptides Mediated DNA-free Transfection Technology In Arabidopsis And Chinese Cabbage

Arabidopsis thaliana L.is an important model plant,and its genome research has achieved fruitful results.Chinese cabbage(Brassica rapa L.)is an important vegetable crop which belongs to Cruciferae.The research results of Arabidopsis genome and related technologies provide a good reference for Chinese cabbage.Due to genotype dependence,regeneration after transfection in some varieties is difficult,some occasional results can not be widely repeated.In recent years,the third-generation genome editing system represented by CRISPR/Cas has shown strong application prospects in gene function analysis and crop genetic improvement.However,this technology largely depends on the genetic transformation.Public concerns on GMO(genetically modified organisms)safety,as well as related regulations,have restricted the application.Therefore,to establish DNA-free transfection system has important theoretical and practical significance for promoting the industrialization of CRISPR/Cas genome editing.In this paper,we built somatic embryogenesis system in Arabidopsis thaliana and microspore embryogenesis system in Chinese cabbage,prepared the DNA-free transfection complex,and initially established the DNA-free transfection system in Arabidopsis thaliana and Chinese cabbage.The main results are as follows:1.A 2,4-D-induced somatic embryogenesis system was established in Arabidopsis seedlings.The effects of seed physiological age,ABA content and storage environment on somatic embryogenesis were studiedThe somatic embryogenesis of Arabidopsis seedlings could be effectively induced by adding 1?M 2,4-D to 1/2MS liquid medium.Experiments on parent plant growth conditions,seed physiological age and seed storage showed that the somatic embryogenesis rate decreased with the increase of seed physiological age.The somatic embryogenesis rate of Col-0 seeds harvested from fresh yellow silique was about 49.6%,while that of seeds harvested from brown old silique was only about 24.7%.The SE rate decreased to about 28.3% after 60 days storage at 4 C,which was significantly lower than control.The ABA content of ABA biosynthetic mutant aba2-1 is 20% of wild type Col-0,and the SE rate in fresh seeds is only about 3.85%.Seed storage temperature and time significantly affected somatic embryogenesis rate.The SE rate of Arabidopsis thaliana seeds stored at-80 ? for 5 years was still 43.6%,which was significantly higher than the seeds stored at room temperature for 5 years.The SE rate could drop to about 33.9% within 2 weeks after storage in aerobic environment.2.The Chinese cabbage microspore and MDE receptor system was prepared,and isolated microspore culture system was optimizedIn this paper,the effects of different genotypes on microspore embryogenesis in Chinese cabbage were studied.The microspore embryogenesis and seedling formation systems of 'FT' with high embryogenesis rate and 'Chun Da-jiang' and 'Duo Kang Xin 3' with lower embryogenesis rate were optimized.The results showed that low temperature pretreatment for 4 hours had no significant effect on microspore embryogenesis of 'Chun Da-jiang' and 'Duo Kang Xin 3'.Heat shock 24 hours promoted microspore embryogenesis in ' Chun Da-jiang 'and 'Duo kang Xin 3'.The suspensor bearing MDE of Chinese cabbage 'FT' were induced by 32? heat shock for precise 24 hours.The induction rate was 0.01%.Activated charcoal did not boost 'Chun Da-jiang' and 'Duo Kang Xin 3' embryogenesis rate.The MDE seedling rates in MS medium containing 0.8% agar were significantly higher than those in MS containing 1.2% agar.In B5 and MS medium,the seedling rate and callus induction rate of 'Chun Da-jiang' and 'Duo Kang Xin 3' were similar,the difference was not significant.The bud size,culture density of 'FT' microspore were systematically optimized.The results showed that when the bud length was 2.5-2.75 mm and the culture density was 20,000-40,000,the microspore embryogenesis rate was the highest.In this study,Chinese cabbage 'FT' microspore culture system was used as a haploid transfection receptor system.3.DNA-free transfection complex: preparation of CPP-m Cherry fusion proteinIn this paper,CPP-m Cherry fusion protein was found to be an effective DNA-free transfection complex.DNA sequences of nine tandem arginine(R9),one cysteine(cys),reporter protein m Cherry and histidine label His-tag were sequentially constructed into p ET 45B+ expression vector and transformed into Escherichia coli BL21(DE3)strain.CPP-m Cherry fusion protein can be produced in BL21(DE3)by exogenous 1m M IPTG for at least 1 hour in 28?.High purity CPP-m Cherry fusion protein can be obtained by 200 W ultrasonication,then purified by Ni column and MWCO dialysis membrane.The purified protein concentration was above 1 mg/ml,which could be used for downstream transfection experiment.4.DNA-free transfection system of diploid Arabidopsis thaliana cells,using CPP-m Cherry fusion protein.A DNA-free transfection system in Arabidopsis thaliana diploid somatic cells was successfully built,which was mediated by CPP-m Cherry fusion protein.The root tip and mesophyll cells of Arabidopsis thaliana wild type Col-0 can be used as R9-m Cherry transfection receptors.Concentration can be selected between 10-100?g/ml and incubated overnight at room temperature.Cell penetrating peptide fusion protein R9-m Cherry can be used as an ideal transfection tool to infect the root tip and mesophyll cells in Col-0.The transfection efficiency of root tips reached 100%.These results lay a technical foundation of DNA-free genome editing.5.DNA-free transfection system of Chinese cabbage microspores and MDEs,Using CPP-m Cherry fusion protein.In this study,a CPP-m Cherry fusion protein-mediated DNA-free transfection system was built.The Chinese cabbage 'FT 'microspores and 3-week-old microspore embryo can be used as transfected receptors.Concentration can be selected between 50-100?g/ml and incubated overnight at room temperature.R9-m Cherry protein can be transfected into the nucleus of microspore cells.The transfection efficiency of microspore and MDE was 8.13% and 94.79%,respectively.The microspore embryo could regenerate after transfection.These results lay a technical foundation for DNA-free haploid genome editing.