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Rice potato leaf roll virus 2 infectious clone as well as construction method and application thereof

A leaf-roll virus and a construction method technology, applied in the field of plant genetic engineering, can solve the problems of inability to use rice, little research on positive single-stranded rice virus, and complex structure.

Pending Publication Date: 2022-07-01
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The genome structure of rice viruses reported so far is relatively complex, mostly in the form of double-strand and negative-strand, and a small number of positive-single-strand rice viruses have been found. The related research is very little and not complete and in-depth
Previously, only rice yellow mottle virus (Rice yellow mottle virus, RYMV) and rice baculi-form virus (Ricetungro bacilli-form virus, RTBV) were reported for the construction of rice virus invasive clones, and these two viruses are currently in It does not occur in rice in China, and it cannot be used for rice-related research
The recently reported infectious clone of rice stripe virus (Rice stipe virus, RSV) is only infectious in Nicotiana benthamiana, and cannot be used in rice. Reports of infectious clones

Method used

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  • Rice potato leaf roll virus 2 infectious clone as well as construction method and application thereof
  • Rice potato leaf roll virus 2 infectious clone as well as construction method and application thereof
  • Rice potato leaf roll virus 2 infectious clone as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Determination of the complete genome sequence of rice potato leaf roll virus 2 (RPV2)

[0032] 1. Extraction of plant total RNA

[0033] The total RNA of the leaves of wild rice with poison was extracted by the TRIzol method, and the extraction method was referred to the instructions of the plant RNA extraction kit.

[0034] Put about 0.1 g of leaves into a 2.0 centrifuge tube containing steel balls, grind them into powder under liquid nitrogen using a plant tissue grinder, then quickly add 1 mL of TRIzol, shake vigorously for 30 s, incubate at room temperature for 5 min, and then add 0.2 ml Chloroform, shake vigorously for 30 s, incubate at room temperature for 5 min, centrifuge at 12,000 g for 15 min at 4°C, take the upper aqueous phase into a new centrifuge tube, add an equal volume of isopropanol, invert and mix, and let stand at -20°C for 10 minutes. -30 min, 4°C, centrifuge at 12000g for 10 min, remove the supernatant, wash the precipitate twice with 75...

Embodiment 2

[0037] Example 2 Construction of wild rice virus infectious clone

[0038] Using the plasmids pTOPO-A1 and pTOPO-A2 correctly sequenced in Example 1 as templates, the complete nucleotide sequence of RPV2 was amplified in two sections with primer pairs XT001F / RPV2-3267R and RPV2-3248F / XT3R; then, CloneExpress®MultiS The One Step Cloning Kit Recombinant Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) combines the purified amplified target fragment with the Stu I and Bam The HI linearized pXT vector was used for the recombination reaction, and a 10 μL system was used for the reaction, including 2 μL of 5×CE MultiS Buffer, 1 μL of Exnase MultiS, 90 ng of the linearized cloning vector pXT, and primer XT001F / RPV2-3267R. The target fragment 2 obtained from fragment 1 and RPV2-3248F / XT3R is about 65 ng and 50 ng, respectively, ddH 2 O was adjusted to 10 μL, and the components were mixed gently and reacted at 37 °C for 30 min. After the reaction was completed, it was placed on...

Embodiment 3

[0039] Example 3 pXT-RPV2 invasive clone inoculation

[0040] 1. Bunsen cigarette vaccination

[0041] First, a single colony of Agrobacterium GV3101 containing plasmid pXT-RPV2 was picked and inoculated in LB medium containing resistance to kanamycin (Kan, 50 mg / L) and rifampicin (Rif, 50 mg / L) 28 Activated by shaking overnight at ℃; the next day, the activated bacterial solution was transferred to the desired liquid LB at 1:100 (except for containing 50 mg / L Kan+Rif, additionally added 10 mM MES pH 5.6, 45 mM acetosyringone), 28° C culture OD 600 to 0.8-1.0; finally, 4000 rpm / min, room temperature, centrifugation for 10 min to collect the bacteria; with a final concentration of 10 mM MgCl 2 , 10 mM MES (pH 5.6), and 150 µM acetosyringone (As) buffer to resuspend, adjust the bacterial concentration to OD 600 0.8-1.0 was used for infection after at least 3-4 h at room temperature in the dark. Use a 1 mL syringe to suck up the Agrobacterium solution to be used, remove the n...

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Abstract

The invention discloses a rice potato leaf roll virus 2 (RPV2) infectious cloning vector as well as a construction method and application thereof, and the rice potato leaf roll virus 2 (RPV2) infectious cloning vector is obtained by cloning the RPV2 genome complete sequence to a pXT binary vector containing cauliflower mosaic virus dual-35S promoters in a homologous recombination manner on the basis of obtaining the RPV2 genome complete sequence. The infectious clone which can be successfully inoculated and stably and efficiently infect nicotiana benthamiana and rice (indica rice and japonica rice) plants through an agrobacterium-mediated method is prepared for the first time, and a foundation is laid for researching the gene structure and function of the virus and the relationship between the virus and a host; the positive genetics direction is utilized to research the disease-resistant mechanism of the rice and determine more potential disease-resistant genes in the rice, theoretical and practical bases are provided for cultivating new disease-resistant varieties, and finally the yield and quality of the rice are improved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an infective clone of rice potato leaf roll virus 2 and a construction method and application thereof. Background technique [0002] Rice potato leafroll virus 2 (Rice polerovirus 2, RPV2) is a new virus of the genus Potato leafroll virus discovered in wild rice in this experiment. nucleotides, linked to VPg at the 5' end, encoding seven proteins through a variety of different translation strategies. The 5' end ORFs are sequentially translated through the genome to generate P0, P1 and P1-P2 proteins, while the 3' end ORFs are translated through a subgenomic strategy to generate P3, P4 and P3-P5 proteins, and the region between P1-P2 and P3 also exists. A non-ATG-initiated ORF that encodes a small protein, P3a. [0003] Viruses of this genus generally cannot be mechanically inoculated by sap, are exclusively transmitted by specific vector aphids in ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/66C12N1/21A01H5/00A01H6/82A01H6/46C12R1/01
CPCC12N15/8205C12N15/66Y02A50/30
Inventor 朱丽娟韩艳红吴建国李容柏赵珊珊张崇涛白雅妮解晓盈
Owner FUJIAN AGRI & FORESTRY UNIV
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