Lithospermum erythrorhizon LeEIL-1 (L. erythrorhizon EIN3-like protein 1) transgenic hairy root strain line capable of producing medicinal natural products in high yield
A technology for a natural product, comfrey, which is applied in the field of plant genetic engineering, achieves broad development and application prospects and commercial value, and improves the transgenic efficiency of comfrey
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Embodiment 1
[0027] Embodiment 1, the induction of the hairy root of transsclerosiae LeEIL-1 gene
[0028](1) Cultivation of aseptic seedlings of Sclera sclerophylla: select plump Sclera sclerophyllum seeds, wash them with sterile water, and culture them in the dark at 4°C for 1-2 months until the seeds germinate; pick freshly germinated seeds, wash them with clean water Wash it; soak it in 6% sodium hypochlorite for 5 minutes, and then wash it several times with sterile water; place it in MS basic medium, cultivate it under light at 26°C-28°C, and obtain a certain number of aseptic seedlings of comfrey. One month later, when the aseptic seedlings of the comfrey grew to 8-10 cm, they were used as explants for inducing hairy roots.
[0029] (2) Preparation of transgenic infection bacterial liquid: replace the GUS gene fragment in the commercially available pBI121 vector with the enhanced green fluorescent protein eGFP gene fragment, and transform it into a plant eukaryotic expression vector...
Embodiment 2
[0031] Embodiment 2, the cultivation of the hairy root of transscleroderma LeEIL-1 gene
[0032] (1) Cultivation of hairy eradicating bacteria: the induced hairy roots grow for a week, cut the hairy roots with a length of about 1cm, and transfer them to the solid sterilization medium of MS+500mg / L cephalosporin, and then Change into MS+250mg / L cephalosporin in one week, until removing residual Agrobacterium rhizogenes; The hairy root that thoroughly sterilizes is changed into B5 solid medium culture ( figure 2 );
[0033] (2) hairy root expansion cultivation: the hairy root of solid expansion cultivation is transferred to B5 liquid medium, and light expansion cultivation in the shaker of 100rpm ( image 3 ).
Embodiment 3
[0034] Embodiment 3, the identification of the hairy root of the transformed comfrey LeEIL-1 gene
[0035] It is divided into two parts: molecular detection and fluorescence detection. Take a certain amount of hairy roots, extract hairy root DNA as a template by CTAB method, design primers rolc-F: 5'-ACAAGCCACTTCTGTTTCCC-3'; rolC-R: 5'-CAGCGACTGCAACCAGTTTA-3', and amplify by PCR Comfrey Genomic DNA was used as a control, and the amplified products were sequenced and compared to detect the presence or absence of the RolC gene in the Ri plasmid of Agrobacterium in transgenic hairy roots. RolC was detected in the three randomly selected expanded cultured Sclerophyllum hairy root lines the presence of genes ( Figure 4 ), indicating that the obtained hairy roots are all hairy roots; at the same time, the hairy roots with a length of about 2-3mm in the above strains were made into slices, and the fluorescence was detected with a laser confocal microscope, and the results showed th...
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