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A gene silencing and leaf technology, applied in the field of peach gene silencing, can solve the problems of instability and low efficiency, and achieve good stability, high silencing efficiency and good effect
Active Publication Date: 2021-11-16
HUAZHONG AGRI UNIV
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However, in the actual application process of these two sets of vectors, there are problems such as low efficiency and instability in the use of peach leaves as materials and the use of Agrobacterium tumefaciens to mediate recombinant plasmid transfection. A system for efficiently inducing plant endogenous gene silencing in peach seedlings
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Embodiment 1
[0036] In the following, the phytoene dehydrogenase (PDS) gene was used as the target gene to construct the silencing vectors pTRV2-PpPDS and pCaRNA3-PpPDS, using the method mediated by Agrobacterium tumefaciens, and peach leaves were used as the injection material to explore the temperature, inoculation Effects of seedling age and concentration of inoculum on the silencing efficiency of peach TRV-VIGS and PNRSV-VIGS.
[0037] 1. Construction of silencing vectors pTRV2-PpPDS and pCaRNA3-PpPDS
[0038] (1) Using Peach leaves as materials, total RNA was extracted using EASY spin Plus Plant RNA Rapid Extraction Kit (Aidlab, Beijing), and the RNA concentration and quality were tested by Nanodrop one (Thermo, USA) and gel electrophoresis. reverse transcription kit RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used for reverse transcription to obtain cDNA;
[0039] (2) Search for the mRNA sequence of PpPDS in the peach genome, and use primer5 to design primers (prim...
Embodiment 2
[0076] In the following, PpERF98 was used as the target gene, and the same method as in Example 1 was used to conduct a silencing effect experiment.
[0077] Such as Figure 9 As shown, the leaves of peach seedlings were infected by leaf injection. After 25 days of infection, the pCaRNA3 empty vector plants and pCaRNA3-PpERF98 plants were analyzed by qRT-PCR with the pCaRNA3 empty vector plants as the control. Five biological replicates were set up in this experiment, named pCaRNA3-PpERF98#1, pCaRNA3-PpERF98#2, pCaRNA3-PpERF98#3, pCaRNA3-PpERF98#4 and pCaRNA3-PpERF98#5. Using PpTEF2 (Translationenlongation factor2) (Tong et al 2009) as an internal reference gene, the expression level of the target gene PpERF98 was analyzed by qRT-PCR. The results showed that, compared with pCaRNA3 control, the expression levels of pCaRNA3-PpERF98 plants were significantly reduced.
[0078] The sequence list of the above-mentioned genes involved is as follows:
[0079] 1. The CDS of PpERF98...
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Abstract
The invention relates to a VIGS-based efficient peach leaf gene silencing method, which comprises the following steps: 1, constructing a pCaRNA3-target gene recombinant plasmid, and transferring into agrobacterium tumefaciens GV3101 to obtain positive agrobacterium tumefaciens transferred into the recombinant plasmid; 2, preparing a VIGS transformed bacterium solution by utilizing the positive agrobacterium with the transferred recombinant plasmid, and injecting the VIGS transformed bacterium solution into all completely expanded leaves of a peach seedling with the seedling age of 5-6 completely expanded leaves; and 3, culturing the injected peach seedlings in weak light for 2 days, continuously culturing the peach seedlings in a plant growth room under the conditions of (22 + / -1) DEG C and 16h illumination / 8h darkness, and irrigating a Hoagland nutrient solution during the peach seedling culture period. The practical application of the VIGS gene silencing technology in peaches is optimized, a set of efficient peach leaf gene silencing system is established, the silencing efficiency is high, and the stability is good.
Description
technical field [0001] The invention relates to the field of peach gene silencing methods, in particular to a high-efficiency peach leaf gene silencing method based on VIGS. Background technique [0002] VIGS (Virus-induced gene silencing) is a virus-induced post-transcriptional gene silencing technology. This technology uses a virus to introduce a recombinant virus vector with a target gene into the host plant. After the virus is successfully infected, it induces the silencing of the endogenous gene in the plant, resulting in a change in the host phenotype, and further realizing the functional analysis of the target gene. VIGS technology has the advantages of short time, quick effect, simple operation, etc., and can obtain gene silenced plants without genetic transformation. It has become one of the powerful technical means to study gene function. VIGS technology has been successfully applied to gene silencing in many plants. However, at present, peach (Prunus persica) ha...
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