Antivirus plant expression vector constructed by utilizing pre-miR159a and application thereof
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An anti-virus and vector technology, applied in the field of molecular biology and biology, can solve problems such as questioning the safety of transgenic plants
Inactive Publication Date: 2011-08-24
SHANDONG AGRICULTURAL UNIVERSITY
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Third, there is a risk of recombination with other invading viruses or host plant genomes when the long viral cDNA fragments are transferred, and the safety of transgenic plants has been questioned to a certain extent
However, the transgenic plants obtained by this method can only be resistant to a single virus, and the use of this strategy to obtain transgenic plants resistant to multiple viruses has not been reported.
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Embodiment 1
[0020] Example 1: Acquisition of Arabidopsis pre-miR159a
[0021] The leaves of Arabidopsis thaliana grown under normal conditions were used as materials, and Arabidopsis genomic DNA was extracted by the method of sodium dodecyl sulfate (SDS). According to the nucleotide sequence of Arabidopsis pre-miR159a published in the National Center for Biotechnology Information (NCBI) database, the DNA-specific primers were designed as follows:
[0022] pre-miR159aF, its sequence is shown in Seq ID No: 5
[0023] pre-miR159aR, its sequence is shown in Seq ID No: 6
[0024] Using the extracted Arabidopsis DNA as a template, the pair of specific primers were used for routine RCR amplification. The amplification conditions are:
[0025] The PCR reaction volume is 50 μl and includes the following components:
[0026] 10×Reaction Buffer 5μl
[0027] Deoxynucleotide mixture (dNTP) 4μl
[0028] Forward primer (5μM) 4μl
[0029] Reverse primer (5μM) 4μl
[0030] Template DNA 4μl
[003...
Embodiment 2
[0046] Example 2: Homologous comparison of TMV, PVX, and PVY silencing suppressors in different regions
[0047] (1) The 126kDa nucleotides of TMV in different regions were homologously compared, and the conserved 21nt region was selected as the target sequence, marked with a rectangle.
[0048]
[0049] (2) The nucleotides of the 25kDa protein of PVX in different regions were homologously compared, and the conserved 21nt region was selected as the target sequence, marked with a rectangle.
[0050]
[0051] (3) The nucleotides of HC-Pro of PVY in different regions were homologously compared, and the conserved 21nt region was selected as the target sequence, marked with a rectangle.
[0052]
Embodiment 3
[0053] Example 3: Transformation of pre-amiR159a
[0054] (1) Design specific primers according to the conserved 21nt sequence of the 126kDa protein gene of the selected TMV extract:
[0055] pre-miR159a P126 F: its sequence is shown in Seq ID No: 7
[0056] The 5-10 base pair in the sequence is the Xho I restriction site, and the 12-32 base pair is amiR159a P126 * the sequence in the precursor (i.e. amiR159a P126 The embodiment of the characteristic target sequence in the precursor, the same below)
[0057] pre-miR159a P126 R: its sequence is shown in Seq ID No: 8
[0058] The 5-10 base pair in the sequence is the Xho I restriction site, and the 12-32 base pair is amiR159a P126 *Sequence in precursor.
[0059] (2) Design specific primers according to the conserved 21nt sequence of the 25kDa protein gene of the selected PVX extract:
[0060] pre-miR159a P25 F: its sequence is shown in Seq ID No: 9
[0061] The 5-10 base pair in the sequence is the Xba I restriction s...
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Abstract
The invention relates to clone, reconstruction and transformation of a precursor (pre-miR159a) of arabidopsis thaliana microRNA159a and antivirus analysis of a transgenic plant and belongs to the techThe invention relates to clone, reconstruction and transformation of a precursor (pre-miR159a) of arabidopsis thaliana microRNA159a and antivirus analysis of a transgenic plant and belongs to the technical field of molecular biology and biology. The technology clones a nucleotide sequence of pre-miR159a from the arabidopsis thaliana and carries out site-directed mutagenesis on the sequence producinical field of molecular biology and biology. The technology clones a nucleotide sequence of pre-miR159a from the arabidopsis thaliana and carries out site-directed mutagenesis on the sequence producing mature miR159a. 126kDa protein of tobacco mosaic virus, 25kDa protein of potato virus X and HC-Pro protein of potato virus Y are subjected to genetic mutation to obtain three sections of the mutating mature miR159a. 126kDa protein of tobacco mosaic virus, 25kDa protein of potato virus X and HC-Pro protein of potato virus Y are subjected to genetic mutation to obtain three sections of the mutation sequences. The three sections of the sequences are in series to construct the plant expression vector; and an agrobacterium-mediated method is adopted to transform nicotiana benthamiana to obtain ton sequences. The three sections of the sequences are in series to construct the plant expression vector; and an agrobacterium-mediated method is adopted to transform nicotiana benthamiana to obtain transgenic tobacco. The transgenic plant has high disease resistance on three viruses. The antivirus strategy has the advantages of strong disease resistance, durable disease resistance, biological safransgenic tobacco. The transgenic plant has high disease resistance on three viruses. The antivirus strategy has the advantages of strong disease resistance, durable disease resistance, biological safety and the like and has wide application prospect in the breeding field of plant antivirus genetic engineering.ety and the like and has wide application prospect in the breeding field of plant antivirus genetic engineering.
Description
technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to an antiviral vector prepared by cloning, transforming, transforming and transgenic Arabidopsis pre-miR159a. Background technique [0002] Plant virus diseases are the main diseases of crops. At present, more than 1000 plant virus diseases have been recognized by people. Every year, crops around the world lose up to 20 billion U.S. dollars due to virus damage, which poses a serious threat to agricultural production. Therefore, the prevention and treatment of plant virus diseases has long been an important object of agricultural workers' attention and research. Early prevention and control measures for viral diseases were mainly cultivation management and traditional disease-resistant breeding. To enhance plant disease resistance by improving cultivation management, this measure can only reduce the degree of plant disease but cannot fundamentally solve the prob...
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