The induction of CPP-mCherry expression was conducted by IPTG gradient, the results are shown in Fig. 2. The obvious pink mCherry protein was clearly visible in the bacterial precipitation (Fig. 2A). The bacterium was then broken by sonication to release the protein. The molecular weight of R9-cys-mCherry protein was about 30 kD as expected (Fig. 2B). With the increase of IPTG concentration, there was no significant difference in the expression of target protein (Fig. 2B).
In addition to R9-cys-mCherry and cys-mCherry vectors, we tried others for induction, such as penetratin-cys-mCherry, transportan-cys-mCherry and Map-cys-mCherry. R9-cys-mCherry is productive.
2 SDS-PAGE
The target protein with 6x histag was purified by Ni column. After SDS-PAGE electrophoresis, the results are shown in Fig. 3. Lane 1–3 were from BL21 (DE3) competent cell as negative control. Lane 4–6 are from CPP-mCherry transformants. The negative control had no target protein as expected. In contrast, the transformants contain large amount of CPP-mCherry fusion proteins. SDS-PAGE results suggest that the cys-mCherry and R9-cys-mCherry protein could be induced from 1.0 mM IPTG induction for 5 h. After Ni column purification, the non-target protein was removed by MWCO dialysis.
Protein concentration was detected by Bicinchoninic acid (BCA) method. The concentrations of R9-cys-mCherry and Cys-mCherry protein were about 0.13 mg/ml (Table 1), which is sufficient for the following experiments.
Table 1
concentration of isolated CPPs-mCherry fusion protein
CPP fusion protein | Concentration(mg/ml) |
R9-mCherry | 0.13 |
cys-mCherry | 0.13 |
3 R9-cys-mCherry transfected into Arabidopsis somatic cells
In the beginning, 7-days Arabidopsis mesophyll cells were found to be transfected by R9-cys-mCherry accidentally (Fig. 4A). Leaves were incubated with 30 µg/ml R9-cys-mCherry overnight at room temperature. In the second day, strong mCherry red fluorescence signal was observed in mesophyll cells, Fig. 4A. These results suggest that CPP-mCherry fusion protein was successfully transfected into the cytoplasm. Arabidopsis thaliana mesophyll can be used as the CPP mediated transfection recipient.
Later, Arabidopsis root tip were used as DNA-free transfection recipient. Root tips were incubated with 50 µg/ml R9-cys-mCherry overnight at room temperature Fig. 4B. Strong red fluorescence signal was found in root tip cells. These results suggest that the somatic cells of Arabidopsis can be used as the transfection recipient. Mesophyll and root cells were usually used for protoplast isolation (Ikeuchi et al., 2016), our result also provides a technical basis for protoplast DNA-free transfection and genome editing.
4 Dose dependent effect
Cell penetrating peptide mediated transfection normally shows dose dependent(Cardozo et al., 2007; Ru et al., 2013; Bilichak et al., 2015; Guidotti et al., 2017)In order to optimize the best transfection condition, the concentration gradient was set as 1, 10 and 100 µg / ml, and incubated overnight at room temperature Fig. 5.
There is no mCherry signal in low concentration treatment. When R9-cys-mCherry concentration increased to 100 µg/ml, strong mCherry signal was found in root tip cells (Fig. 5B). No fluorescence signal was found in negative control, as expected (Fig. 5A). The results suggest that the transfection shows dose-dependency, the concentration of fusion protein can be selected between 10–100 µ g / ml.
In order to further determine R9-cys-mCherry distribution in recipient cell, we used high resolution confocal microscopy, Fig. 6A. In the cys-mCherry negative control, no fluorescence signal was found in root tip cells Fig. 6B. In contrast, the strong red fluorescence signal was found in the R9-cys-mCherry treatment. These results indicate that R9-cys-mCherry can be transfected into root tip cells in the range of 10–100 µg/ml, with 100% transfection efficiency, Table 2.
Table 2
CPPs-mCherry transfection efficiency in Arabidopsis root tip
CPP-mCherry Fusion protein 50 µg/ml | mCherry positive root tip | total number of root tip | Transfection efficiency |
R9-mCherry | 98 | 98 | 100%** |
Negative control cys-mCherry | 0 | 93 | 0% |
Note: Student’s t test (*indicate difference p < 0.05; ** indicate significant difference p < 0.01) |
5 R9-cys-mCherry transfected into microspores of Brassica rapa.
DNA-free transfection was also carried out in Chinese cabbage microspores. The microspores are nearly spherical cells with thick exine, different orientation would interfere laser scanning. Therefore, the “face-up” microspores were selected for observation, whereby germination furrows are vertical to the glass slide. The thick exine was stained by Renaissance dye. The concentration gradient of R9-cys-mCherry and cys-mCherry was set as 1, 10, 50, 100 µ g / ml, respectively, Fig. 6. No cys-mCherry signal was detected at any concentration as expected (Fig. 7A). In contrast, the positive results were found in R9-cys-mCherry transfection. With the increase of concentration, strong red fluorescence signals were found at 50 and 100 µg/ml (Fig. 7B). These results suggest that 'FT' microspore can be used as DNA-free transfection recipient cell. The concentration can be selected between 10–100 µg/ml. The transfection efficiency was about 8.13% (Table 3).
Table 3
CPPs-mCherry transfection efficiency in B. rapa 'FT' microspores
CPP-mCherry Fusion protein 50 µg/ml | mCherry positive microspores | Observed microspores | Transfection efficiency |
R9-mCherry total | 27 | 332 | 8.13%** |
Negative control cys-mCherry total | 0 | 312 | 0.00% |
Note: Student’s t test (*indicate difference p < 0.05; ** indicate significant difference p < 0.01) |
Table 4
CPPs-mCherry transfection efficiency in B. rapa 'FT' MDE
CPP-mCherry Fusion protein 100 µg/ml | mCherry positive MDE | total number of MDE | Transfection efficiency |
R9-mCherry | 91 | 96 | 94.79%** |
Negative control cys-mCherry | 0 | 102 | 0.00% |
Note: Student’s t test (*indicate difference p < 0.05; ** indicate significant difference p < 0.01) |
CRISPR RNP protein complex needs to enter the nucleus, then performing gene editing. In order to clarify the distribution of R9-cys-mCherry in microspores, we observed transfected microspores by high resolution confocal, Fig. 8. Strong fluorescence was clearly visible in the nucleus of microspore, indicating that CPP-mCherry was successfully transfected into microspore nucleus.
6 R9-cys-mCherry transfected into MDE of Brassica rapa.
DNA-free transfection of microspore derived embryos (MDE) was carried out in this study. The concentration of CPP-mCherry was 100 µg / ml. After incubation overnight at room temperature, confocal microscopy was performed, Fig. 9.
With 100 µg / ml of R9-cys-mCherry incubation, strong red fluorescence signal was found in MDE epidermal cell, as the medium section is too thick for the laser went though. MDE treated with cys-mCherry had no fluorescence signal as expected. The transfection frequency was over 94.79%. The results showed that, 21-day microspore embryos of 'FT' could be used as the DNA-free transfection recipient.
Many successful attempts have been made to transfect Chinese Cabbage (Boulter et al., 1990; Zhang et al., 1998; 2000; Tang et al., 2003; Yang et al., 2004; Baskar et al., 2016; Li et al., 2018). However, these techniques showed difficult to regenerate after transfection. Microspore embryo is an important haploid transfection recipient, whereby pure homozygous Lines DH line would be obtained. In this study, we found that, transfected MDEs are able to regenerate into plantlets due to low cytotoxicity (Chugh et al., 2010; Bilichak et al., 2015; Huang et al., 2015; Derakhshankhah et al., 2018)
In general, CPP-mCherry can be used as a DNA-free transfection tool in crops. Microspore and 3-week-old MDEs, as well as Arabidopsis somatic cells, could be used as transfection recipient.