Lithospermum erythrorhizon sieb.et zucc transgenic hairy root strain highly producing medicinal natural products
A natural product, the technology of hard comfrey, applied in the field of plant genetic engineering, to achieve the effect of improving transgenic efficiency of comfrey, broad development and application prospects and commercial value
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Embodiment 1
[0026] Embodiment 1, the induction of the hairy root of transmutation Sclerophyllum LeACS-1 gene
[0027] (1) Cultivation of aseptic seedlings of Sclera sclerophyllum: select plump Sclera sclerophyllum seeds, wash them with sterile water and place them in a dark environment 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, and culture it under light at 26°C-28°C to 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.
[0028](2) Preparation of transgenic infection bacterial liquid: use the modified vector after replacing the GUS gene fragment in the pBI121 vector with the green fluorescent protein eGFP gene fragment; design the primer ACS according to the...
Embodiment 2
[0030] Embodiment 2, the cultivation of the hairy root of transmutation Comfrey LeACS-1 gene
[0031] (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 );
[0032] (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
[0033] Embodiment 3, the identification of the hairy root of the transformed comfrey LeACS-1 gene
[0034] 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', amplify by PCR method to 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 sho...
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