Method for increasing catharanthus roseus hairy root terpenes indole alkaloid content

[0019] Cloning of str and g10h genes of periwinkle

[0020] 1. Isolation

[0021] Soak periwinkle seeds in 75% ethanol for 1 min, then soak in 2% NaClO for 10 min, rinse with sterile water for 3-4 times, dry the surface with sterile absorbent paper, and inoculate them in hormone-free MS (Murashige and Skoog, 1962 ) in solid medium, 25°C, 12h / 12h light culture, to obtain sterile periwinkle seedlings, after the seedlings grow to about 5cm, cut leaves and stems for RNA extraction.

[0022] 2. RNA isolation (RNA isolation)

[0023] Weigh 0.5 g of the periwinkle aseptic test-tube seedlings, quickly freeze them with liquid nitrogen, grind them quickly with a mortar, add them to a 1.5 mL Eppendorf tube filled with 1 mL TRIzol (TRIzol Reagents, GIBCO BRL, USA), shake them fully Afterwards, stand at room temperature for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 sec, place at room temperature for 2-3 minutes, then centrifuge at 12,000 g for 15 minutes at 4°C; pipette

[0030] Construction of Plant Binary Expression Vector Containing str and g10h Genes

[0031] 1. Intermediate vector pCAMBIA1304 + build

[0032] Select pBI121 and pCAMBIA1304 as the basic elements to construct the binary plant expression vector pCAMBIA1304+ . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII and EcoRI; the GUS expression cassette of pBI121 and the large fragment of pCAMBIA1304 were recovered; the recovered products were ligated, transformed and screened, and verified by plasmid digestion.

[0033] 2. Plant expression vector pCAMBIA1304 + Construction of +str

[0034] With the described pCAMBIA1304 + For the expression vector, replace the gus gene on it with str in Example 1. Specifically, BamHI / SacI double enzyme cut pGEM T-easy+str and pCAMBIA1304 + , recycling str and pCAMBIA1304 + Large fragments, ligated transformation, picking single clones, extracting plasmids for PCR detection and enzyme digestion verification.

[0035] 3. Plant exp

[0039] Agrobacterium rhizogenes mediated str and g10h gene genetic transformation of Vinca to obtain transgenic hairy roots

[0040] 1. Obtaining the engineering bacteria of Agrobacterium rhizogenes containing the binary plant expression vector of str and g10h genes

[0041] The plant binary expression vector containing str and g10h genes in Example 2 was transformed into Agrobacterium rhizogenes (such as C58C1) and verified by PCR. The results showed that the plant binary expression vector containing str and g10h genes had been successfully constructed into the strain of Agrobacterium rhizogenes.

[0042] 2. Agrobacterium rhizogenes mediates str and g10h gene transformation of periwinkle

[0043] 2.1. Preculture of explants

[0044] Cut periwinkle aseptic seedling blade (0.5cm * 0.5cm) and stem segment (0.5cm) explant in embodiment 1, inoculate on the pre-cultivation medium (MS+AS 100 μ mol / L), 25 ℃ of dark cultures 2d.

[0045] 2.2. Co-cultivation of Agrobacterium and exp