Novel rapid preparation methods for cDNA dimer and infectious clone of citrus viroid
A virus-infectious, citrus-like technology, applied in the field of molecular biology, can solve the problems of time-consuming, cumbersome steps, high cost, etc., and achieve the unique effect of the method
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Embodiment 1
[0028] Example 1 class of virus resources and one-step RT-PCR
[0029] The CEVd virus source was taken from the navel orange 'Meishan 9', and the CBCVd virus source was taken from the miscellaneous mandarin orange 'Zhujian'. Total RNA from plant leaves was extracted using a plant RNA rapid extraction kit (or trizol method). Design specific primers using PrimeScript TMOne-step RT-PCR kit (Catalog No.: RR055A, TaKaRa Company, Dalian, China) directly amplifies the full-length sequence of the required viroid dimer. The CEVd-specific primer pair used in the present invention is CEVd-For (5'-ggaaacctggaggaagtcgag-3') and CEVd-Rev (5'-ccggggatccctgaaggactt-3'), and the CBCVd-specific primer pair is CBCVd-For:(5'- ggggaaatctcttcagac-3') and CBCVd-Rev (5'-ggggatccctcttcaggt-3').
[0030] Using the obtained total RNA to directly amplify CEVd by one-step RT-PCR, the specific reaction system and amplification conditions are as follows:
[0031] 1. CEVd reaction system:
[0032]
...
Embodiment 2
[0042] Example 2 Construction and in vitro transcription of CEVd / CBCVd dimer cloning vector
[0043] Purify the CEVd / CBCVd dimer obtained by one-step RT-PCR in Example 1 and clone it into the pGEM-Teasy vector to obtain a ligation product comprising CEVd / CBCVd dimer. The principle is as follows: figure 2 As shown, T7 promoter site and SpeI site were used to synthesize CEVd or CBCVd dimer RNA. Transform into competent cells Escherichia coli DH5α for amplification, and the bacterial liquid sequencing determines the dominant sequence and insertion direction of the required CEVd / CBCVd dimer. Extract the plasmid, and purify it with ethanol after linearizing the plasmid with the restriction endonuclease SpeI. Steps such as dimer purification, cloning, transformation competence, and plasmid linearization can be performed according to the operating instructions of relevant reagents.
[0044] Purify with ethanol after SpeI linearized the plasmid:
[0045] First, the obtained mixed ...
Embodiment 3
[0050] Example 3 In vitro transcription product activity verification
[0051] The CEVd / CBCVd dimer RNA obtained by in vitro transcription was diluted to 500ng / ul with diluent (100mM Tris-HCl, 10mM EDTA, pH 7.5), and the in vitro transcription products of CEVd and CBCVd were inoculated with the woody indicator plant citron and the The in vitro transcription product of CEVd was rubbed inoculated with the herbaceous host tomato, and the symptoms of the infected citron were observed after four months (results see image 3 ) and RT-PCR detection (results see Figure 4 ), and extract the total RNA from the leaves of the herbaceous host tomato, and use the digoxin-labeled probe method to carry out Northern blot experiments to further verify the expression activity of CEVd (results in Figure 5 ).
[0052] The results showed that the CEVd-inoculated citron showed typical symptoms of CEVd infection, and the northern hybridization signal of CEVd-infected tomato was obvious, and the r...
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