Ternary shuttle vector and method for constructing CYVCV infectious cloning via ternary shuttle vector
A technology of shuttle vector and linearized vector, applied in the field of molecular biology, can solve problems such as instability, failure to obtain CYVCV infective clone, etc., and achieve the effects of high cost, high speed and high inoculation efficiency.
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Embodiment 1
[0031] Example 1 Construction of ternary shuttle vector pTY
[0032]The binary vector plasmid pTX1 was single-digested with restriction endonuclease Aor51H I (also known as Eco47III) to obtain a linearized vector. Enzyme digestion reaction system: 112 μL of plasmid pTX, 2.5 μL of restriction endonuclease Aor51H I (Eco47III), 5 μL of 10×NEB Buffer, and 23 μL of double distilled water. Enzyme digestion reaction conditions: react at 37°C for 0.5h.
[0033] by pYES1LVector as template, PYES2117F and PYES2117R as primers to amplify the fragment pYES1L-2117 containing the yeast-associated replication origin site: the reaction volume is 25 μL, including 8.5 μL of double distilled water, PrimerSTARMax Premix (2×) 12.5 μL, specific upstream and downstream 1 μL of each primer, template pYES1LVector 1 μL. Reaction conditions: 98°C for 1min, 98°C for 10s, 58°C for 15s, 72°C for 2min, 30 cycles; 72°C for 5min, store at 4°C. Then use the DNA gel purification kit to recover the target...
Embodiment 2
[0035] Embodiment 2CYVCV full-length cDNA amplification
[0036] All steps in this example can be referred to "Long-chain RT-PCR amplification, cloning and sequence analysis of the citrus yellow vein bright virus genome" (Cui Tiantian et al., Acta Horticultural Science, 2017, 44 (5): 944- 952.).
[0037] (1) The total RNA of citrus leaves was extracted by Trizol (Invitrogen) method.
[0038] (2) CYVCV 5′RACE amplification and cloning identification: For detailed steps, see “Long-chain RT-PCR Amplification, Cloning and Sequence Analysis of Citrus Yellowing Vein Virus Genome” (Cui Tiantian et al., Acta Horticultural Science, 2017, 44(5):944–952.).
[0039] (3) Primer design
[0040] According to the complete genome sequence of CYVCV reported in NCBI (GenBank accession number: KP313240) and combined with 5′RACE, the specific primers for amplifying the CYVCV gene sequence were designed using PrimerPriemer 5.0 software. When primers were designed, pTY-CYVCVF and pTY-CYVCVR prod...
Embodiment 3
[0058] Embodiment 3 constructs the invasive clone of CYVCV
[0059] (1) Restriction endonucleases Stu I and Sma I were used to digest plasmid pTY to obtain a linearized vector of pTY. Reaction system: plasmid pTY 13 μL, restriction enzyme Sma I 1.0 μL, 10×NEB Buffer 5 μL, double distilled water 31 μL. Enzyme digestion reaction conditions: react at 25°C for 0.5h, then add 1.0 μL of Stu I, and incubate at 37°C for 0.5h.
[0060] (2) Transformation of yeast by lithium acetate conversion method
[0061] Referring to Tuo et al. (Tuo D, Shen W, Yan P, Li X, Zhou P. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning. Viruses, 2015, 7(12): 6241- 6250) and Youssef et al. (YoussefF, Marais A, Faure C, Gentit P, Candresse T. Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Applechlorotic leafspot virus as a case study. Virology Journal, 2011, 8( 1): 1-12) using ...
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