Application and cloning method of gene NtFLS2 with function of increasing rutin content of tobacco leaves
A cloning method, tobacco technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of low accumulation of rutin and difficulty in meeting the requirements of industrial production, etc., and achieve the effect of increasing the content of rutin
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Embodiment 1
[0048] Example 1: Cloning of gene NtFLS2
[0049] (1) Extraction of total RNA from tobacco
[0050] Take about 1g of tobacco leaf and grind it into powder in liquid nitrogen; pick a little powder and transfer it to a 1.5mL EP tube, add 1mL Trizol, shake vigorously to completely lyse the cells, mix well with Trizol and let stand for 10min; Add 0.25mL of chloroform to the tube, vibrate vigorously for 15s, and let it stand at room temperature for 10min; Centrifuge at 7500rpm / min for 10min at 4°C; discard the supernatant carefully, and be careful not to RNA was discarded, and the EP tube was placed in a clean bench and left to air dry for 10 minutes; 35 μL RNase free water was added to dissolve the precipitate in the EP tube, and stored in a -80°C refrigerator;
[0051] (2) cDNA first-strand synthesis
[0052] Using TaKaRa's PrimeScript TM RT reagent Kit with gDNA Eraser (Perfectreal Time) kit was used to synthesize template cDNA.
[0053] Table 1 Genomic DNA removal
[005...
Embodiment 2
[0063] Example 2: Preparation of plant expression vector pK2-NtFLS2
[0064] Digest pTOPO-NtFLS2 and pENTR with BamHI and XhoI TM 2B, the target fragment NtFLS2 and the vector fragment pENTR were recovered separately TM 2B, the entry vector pENTR was obtained after ligation with T4 DNA Ligase TM 2B-NtFLS2 ( figure 2 ), and the target vector pK2GW7 was reacted by LR to obtain the plant expression vector pK2-NtFLS2 ( image 3 ).
Embodiment 3
[0065] Embodiment 3: Preparation of the Agrobacterium tumefaciens bacterial strain containing plant expression vector pK2-NtFLS2
[0066] Pick up a single colony of Agrobacterium and inoculate it in 5 ml liquid LB medium, culture at 28°C with shaking at 250 rpm until the OD600 is 0.5, transfer to 50 ml fresh liquid medium at a ratio of 1:10 for expansion, at 28°C, 250 Shake culture at rpm until OD600 is 0.5. Cool in ice for 30 min, centrifuge at 5000 rpm for 5 min, discard the supernatant, add 10 ml CaCl 2 (20 mM) resuspend the cells, centrifuge again, add 350 µl CaCl 2 (20 mM) suspended cells, add 150 µl 50% sterilized glycerol, mix well, aliquot 100 µl / tube, and store competent cells at -80°C.
[0067] Take 0.5-2 µl of the recombinant plasmid pK2-NtFLS2, add it to 50 µl of competent cells of Agrobacterium C58C1 strain thawed on ice, mix gently, place in ice bath for 5 min, and rapidly freeze in liquid nitrogen for 5 min, then place at 37°C Bath in water for 5 min, add 1 m...
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