Method for improving currant tomato endogenous gene silencing efficiency by viruses through induction
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A technology of gooseberry tomato and endogenous gene, which is applied in the field of bioengineering, can solve the problems of different, large influence of silencing efficiency, interference with the observation of silencing phenotype, etc., and achieves the effect of high speed and high efficiency.
Inactive Publication Date: 2013-06-12
NORTHWEST A & F UNIV
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Although VIGS technology has many advantages, it also has its inherent limitations: VIGS can rarely completely silence or inhibit the expression of target genes, and the level of silencing varies with different plant species and experimental methods, therefore, it should be combined with plant Species use the correct method for maximum silencing efficiency
Studies have shown that the initial accumulation of the virus has a greater impact on the silencing efficiency, so different growth stages may have a greater impact on the infection and proliferation of the virus, thus affecting the silencing efficiency of VIGS
4. The plant introduction method of the VIGS vector, the plant introduction method of the VIGS vector directly affects the initial accumulation of the virus, and then affects the initiation of gene silencing, thus significantly affecting the VIGS silencing efficiency
[0003] Based on the superiority of VIGS technology, VIGS has developed into one of the main technologies for rapid and high-throughput study of plant gene functions and has been widely used, but VIGS can rarely completely silence or inhibit the expression of target genes, even if there are few target genes The transcripts may also produce functional proteins, thereby interfering with the observation of the silencing phenotype, and the silencing efficiency varies with different plant species and experimental methods, so the evaluation of silencing efficiency and the improvement of silencing efficiency become the key to VIGS technology
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Embodiment 1
[0032] 1. Preparation before the test:
[0033] (1) Test material: gooseberry tomato L03708
[0037] Induction medium: 475ml distilled water, MES 4.88g, glucose 2.5g, NaH 2 PO 40.156g, 25ml ABsalt.
[0038] AB salt: 1L distilled water, NH 4 Cl 20g, MgSO 4 ·7H 2 O 6g, KCl 3g, CaCl 2 0.2g, FeSO 4 ·7H 2 O 0.05g. Take 25ml and add it to the induction medium.
[0041] (1) Construction and transformation of recombinant viral plasmid TRV2-PDS into Agrobacterium: the pTRV2 vector and the PDS gene fragment were connected through Sac I and Xho I restr...
Embodiment 2
[0046] The steps of the experiment are the same as in Example 1, the concentration of Agrobacterium infusion solution OD 600 25-35 days after inoculation, the overall evaluation of VIGS silencing efficiency was made.
[0047] Experimental results
[0048] Different concentrations of liquid infusion and different inoculation methods can induce target gene silencing, but the silencing efficiency is different. The specific evaluation index values are shown in Table 1.
[0049] Table 1 Evaluation index values of each experiment
[0050]
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Abstract
The invention discloses a method for improving currant tomato endogenous gene silencing efficiency by viruses through induction. According to the method, multi-aspect elements affecting virus induced gene silencing (VIGS) efficiency are comprehensively considered, through different inoculation methods and inoculation agrobacterium bacterium concentration, through combination of the silencing efficiency, namely silencing frequency, silencing effectiveness and silencing effects, an evaluation method obtains the optimal system that currant tomato viruses induce the gene silencing, and an appropriate inoculation method and concentration improve the silencing efficiency. Evaluation for the VIGS efficiency by means of the method is short in time, high in speed and high in efficiency, the method can be applied to evaluation for an optimization system that other plant viruses induce gene silencing, and an effective method is provided for rapid verification of a gene function.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for improving the efficiency of virus-induced endogenous gene silencing in gooseberry tomato. Background technique [0002] Compared with the traditional method of studying plant gene function, virus-induced gene silencing has the characteristics of simple and efficient, short cycle, no need to transform plants, and gene family can be silenced, and can be used in different genetic backgrounds between species and within species. Comparison of gene function among different plants. Since Kumagai et al. first used recombinant tobacco mosaic virus (tobaccomosaic virus, TMV) to successfully silence plant endogenous phytoene dehydrogenase gene in Nicotiana benthamiana in 1995, various VIGS vectors have been developed and applied successively. TRV is currently the most widely used VIGS vector. It is composed of two RNA virus strands, TRV1 and TRV2. TRV1 is used to assist T...
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