Establishment Of A System For Genetic Transformation Mediated By Cell Penetrating Peptides Under Isolated Microspore Culture In Chinese Cabbage

Chinese cabbage is a kind of important vegetable crops, originated in China, cultivation scope more extensive, cultivated area is also one of the largest vegetable crops. Modern genetic improvement of Chinese cabbage and functional genomics requires the establishment of a stable genetic transformation system. Previous studies showed that based on the traditional leaf disc method Agrobacterium Ti plasmid used in the Chinese cabbage is not ideal. Therefore, build efficient Chinese cabbage genetic transformation system is the primary premise. This study combined the Chinese cabbage isolated microspore culture technology with Cell penetrating peptides (CPPs) which could transfer between the cell membrane, the main research results are as follows:1. In order to establish high efficient regeneration system of microspore after transfection, isolated microspore culture technology was optimized through improving pretreatment method, medium formula and culture conditions, set up a high embryogenesis frequency and regeneration system.2. Selected plant binary expression vector pBI121 (contained report gene GUS, Kan resistance) and pB 1221-GFP (contained report gene GFP, Amp resistance) and transfered them into E. coli use the heat shock method. The complete sequence of pBI121 is 14758 bp, there is a Hind Ⅲ enzyme site at 495 1bp, 7983 bp with EcoR I enzyme site, the plasmid was double-digested with Hindlll and EcoRI (each restriction enzyme has only one recognition site within the plasmid), resulting in two fragments of 3kb and 12-13 kb. The 3.0-kb Hindlll-EcoRI fragment carrying the cauliflower mosaic virus(CaMV) 35S promoter (835 bp), GUS coding region (1812 bp) and nopaline synthase (NOS)terminator (253 bp), it is the total expression of GUS gene fragment which we needed. The complete sequence of pB 1221-GFP is 4530 bp, and was digested with Pst I(18bp), make its linearization, in order to get the full expression of GFP fragments. According to the two carrier segment sequence, respectively designed two pairs primers (product length 703 bp,509 bp and 523 bp,300 bp) in different regions of the PCR amplification reaction. Transformation was identified successfully by PCR amplification reaction and enzyme e reaction. The experimental results showed that, the PCR amplification reaction amplified out of the expected size fragments, enzyme fragment was also consistent with the expected fragment size. Recycling enzyme purpose fragment by Universal DNA Purification Kit (TIANGEN),the fragment stripe is clear and sharp, no degradation.3. Transfection experiment was done on the basis of Chinese cabbage isolated microspore culture system combined with the penetration ability of the membrane of Cell penetrating peptides. Before the formal transfection experiments, experiment must be carried out to study the influence of transfection reagent on Chinese cabbage microspore mbryogenesis, the results showed that microspore embryogenesis has been suppressed after adding Lipofectamine 2000. When its concentration reaches 4μg/mL, microspore embryogenesis was completely inhibited. Therefore, transfection reagent was not suitable for transfection experiment in this study. CPPs and purpose DNA combined according to the proportion of 4:1 can achieve the best combination ratio. Therefore, this study conducted the test in accordance with the proportion, identifying DNAcargos complexes by DNasel protection analysis experiments, due to the combination of CPPs and purpose gene fragments of DNAcargos from being digested by forming hydrogen bonds, so the results of fragments of DNA segments slightly greater than the purpose. After adding DNAcargos complexes, microspore embryo rate was 1.87-2.01 embryos per bud,2.10-2.33 embryos per bud in the controls, DNAcargos complex had little impact on the microspore embryogenesis. After adding DNAcargos complex,33℃ incubator 24h, after heat shock treatment most conducive to microspore embryogenesis, replaced and added new medium after 3 days was also beneficial to Chinese cabbage microspore embryogenesis.This study got 1088 GUS regeneration plants and 236 GFP filter regeneration plants finally.4. To identify the regeneration plant in this study, extract all of the regeneration plant genome DNA by small amounts of DNA extraction kit firstly, extraction of genomic DNA by agarose gel, electrophoresis detection, with clear and sharp banding, no degradation. Selected plasmid DNA were as positive control template, untransformation of Chinese cabbage genomic DNA as the negative control template, transformation plant genomic DNA for PCR amplification reaction, as a result,207 and 163 (repeated 151 plants, total 219 positive plants) plants of 1088 GUS regeneration plants are amplified to get the expected size respectively, about 703 and 509 bp fragments, PCR positive rate were 19.03% and 14.98% respectively, the positive control amplification segments of the same size, negative control did not get the stripe.121 and 115 plants (repeated 99 plants, total 137 positive plants) of 236 GFP regeneration plant are amplified to get the purpose of the expected size respectively, about 523 and 300 bp fragment, PCR positive rate were 51.27% and 48.73% respectively, the bandings were clearly visible, positive control amplification segments of the same size, negative control did not get the banding.5. In order to identify the positive plants furtherly, leaves were subjected to GUS histochemical analyses, the experimental results showed that 121 of the PCR positive plants exhibiting blue color on the leaves, the frequency of transformation was up to 11.1%, and the control test has failed to appear blue color. This confirmed that GUS gene had been integrate into Chinese cabbage genome. In addition to, the GFP positive plants were observed using Fluor Cam portable ChI/GFP fluorescence spectrometer, under the blue excitation light,80 leaves of PCR positive plants appeared green fluorescence, the frequency of transformation was up to 33.9%, contrast did not appear the green fluorescence, which proveed that the GFP gene had been integrate into Chinese cabbage and had been expressed in the genome.6. Southern Blot reflects the exogenous gene in Chinese cabbage genome on the DNA level. This study choosen roche DIG High Prime DNA Labeling and Detection Starter Kit Ⅱ.The probes had been carried on preparation firstly. In order to detect conveniently,20 positive plants were selected to do Southern Blot resppectivly. The plasmid DNA was used as a template, PCR amplification reaction products were used to make probes. Probes were diluted 20,50,100,200 times respectively, compared contrast DNA, thus calculated the probe quantity for later hybridization test. In view of the two plasmids carrier, respectively using restriction enzymes HindⅢ and Sph I digested genome DNA of positive transplants, the DNA presented a state of dispersion by agarose gel electrophoresis. After enzyme digestion of the DNA electrophoresis, then DNA were transferred to film, hybridization and coloration. The results showed that part of the exogenous gene inserted in the transformation Chinese cabbage plant genome in the form of single copy and part of the exogenous gene inserted in the transformation Chinese cabbage plant genome in the form of multiple copies, no hybridization signal in Chinese cabbage contrast treatment plants. Therefore, Southern hybridization indicated that the GUS and GFP gene were integrated into the Chinese cabbage genome.7. To get the positive plants ploidy identification in this study, flow cytometry was used to carried out the experiments, results demonstrated that haploid, double haploid and high body respectively accounted for 23.16%,69.49%,7.34% of the total number of GUS positive plants, haploid plants, double haploid and high body respectively accounted for 26.53%,66.33%,7.14% of the total number of GFP positive plants. This research established cell penetrating peptides mediated genetic transformation of Chinese cabbage microspore system firstly. Operation method is simple, can shorten the breeding period, and the transformation frequency (11.1%,33.9%) is much higher than the Agrobacterium mediated transformation method (< 3%). This method will provide foundation research platform for Chinese cabbage genetic transformation.