Application of hypovirulence CMV vector in expression of pest-resistant gene and enhancement of pest resistance of plant
An insect-resistant gene and virulence technology, which is applied to the application field of cucumber mosaic virus attenuated virulence vector in enhancing plant insect resistance, can solve problems such as no report on enhancing plant insect resistance, insect RNA interference and the like
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[0142] Example 1,
[0143] 1. Transform the virus CMV into a weak virus expression vector that lacks 2b gene sequence; specifically includes the following steps:
[0144] (1) Design forward and reverse primers with CMV sequence as template.
[0145] Add Nco I restriction site primer NcoIF2:CATG Ccatgg ctgagtttgcctg;
[0146] Primer 2aORF1R:ga with Stu I restriction site AGGCCT T CTA aattctttcgctgtttgttgg.
[0147] Using plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the PCR product, and digests the PCR product with Nco I and Stu I.
[0148] Purify and spare.
[0149] The PCR amplification system is:
[0150]
[0151] The PCR amplification program is:
[0152]
[0153]
[0154] Note: 1. Plasmid pCB-CMVF209 is an invasive clone of CMVF209 sequence. For the specific construction method, please refer to Chinese Agricultural Science 2011,44(14):3060-3068).
[0155] (2) Design the forward primer 2bORF333F:GA with restriction sites Stu I, Mlu I, and BamH I AGGCCT GGGATCCAACctccc...
Example Embodiment
[0178] Example 2,
[0179] 1. Transform the virus CMV into a weak virus expression vector with deletion of 2b gene sequence.
[0180] (1) Design forward and reverse primers with CMV sequence as template.
[0181] Add the forward primer NcoIF2 of restriction site Nco I: CATGCcatggctgagtttgcctg;
[0182] Reverse primer 2aORF1R:gaAGGCCTT with restriction site Stu I CTA aattctttcgctgtttgttg.
[0183] Using plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the PCR product, digests the PCR product with Nco I and Stu I, purifies it, and prepares it for use.
[0184] (2) Design the forward primer 2bORF333F:GA with restriction sites Stu I, Spe I, Apa I, BamH I AGGCCT GGACTAGT gggcccG GGATCCAACctccccttccgcatct; reverse primer 2bAvrIIR: Cttccgaagaaacctaggag. Using plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the PCR product, digests the PCR product with Stu I and AvrII, purifies it, and prepares it for use.
[0185] (3) The plasmid pCB-CMVF209 was digested with ...
Example Embodiment
[0200] Example 3.
[0201] 1. Transform the virus CMV into a weak virus expression vector with deletion of 2b gene sequence.
[0202] (1) Design forward and reverse primers with CMV sequence as template.
[0203] Add the forward primer NcoIF2 of restriction site Nco I: CATGCcatggctgagtttgcctg;
[0204] 2aORF1R with restriction site Stu I: gaAGGCCTT CTA aattctttcgctgtttgttg.
[0205] Using plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the PCR product, digests the PCR product with Nco I and Stu I, purifies it, and prepares it for use.
[0206] (2) Design the forward primer 2bORF333F:GA with restriction sites Stu I, Mlu I, Spe I, Apa I, BamH I, Sac II AGGCCT G ACGCGT GACTAGT gggcccG GGATCCccgcggAACctccccttccgcatct; reverse primer 2bAvrIIR: Cttccgaagaaacctaggag. Using plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the PCR product, digests the PCR product with Stu I and Avr II, purifies it, and prepares it for use.
[0207] (3) The plasmid pCB-CMVF209 w...
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