Rapid construction of Arabidopsis artificial miRNA gene interference vector
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A construction method and an Arabidopsis technology are applied in the field of rapid construction of artificial miRNA gene interference vectors in Arabidopsis, which can solve problems such as cumbersome content and so on.
Inactive Publication Date: 2011-12-07
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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[0007] The existing method requires four major steps to construct the carrier, and the operation content of each step is quite cumbersome, requiring the experimenter to have a certain basis for molecular biology operations
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Embodiment 1
[0095] Example 1 Modification of pCAMBIA1300 vector for rapid silencing vector construction
[0096] 1. Preparation of transformation vector
[0097] In order to improve the applicability of this patent, it is particularly emphasized to explain the method of modification of the target vector, so as to provide users of the present invention for reference, and can be used to modify other target vectors when necessary.
[0098] In order to put the first and last batches into the vector and be able to carry out restriction digestion linearization, we first used the modified pCAMBIA1300 vector as the initial vector, which is connected with the 35s promoter and rbcs terminator, which can be used to start and stop Arabidopsis. This vector is referred to as the 1300SR vector for short.
[0099] First, it was digested with SacI and KpnI endonucleases and digested overnight at 37°C. The digested DNA was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and then cut under a UV ...
Embodiment 2
[0119] Example 2 Rapid construction of 5 Arabidopsis gene silencing vectors using the patented method
[0120] 1. Preparations for the rapid construction of silent vectors
[0121] (1) Plasmid preparation and linearization
[0122] The vector obtained according to Example 1 was amplified by shaking bacteria, and 1300URA plasmid DNA was extracted using a plasmid extraction kit, and digested with AfeI. The digestion system is as follows:
[0123]
[0124] After digestion at 37°C overnight, the digested DNA was electrophoresed on a 1% agarose gel, stained with ethidium bromide and cut under ultraviolet light, and the digested product was recovered using a gel recovery kit. The linearized vector is a universal vector fragment, which can be used for the construction of all rapid silencing vectors and can be stored at -20°C for a long time for later use.
[0125] (2) Synthesis of bridge fragments
[0126] Two primers for bridging fragments were synthesized according to the method of this pate...
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Abstract
The invention relates to a method for quickly constructing an Arabidopsis thaliana artificial miRNA gene interference vector, which completes the construction of the Arabidopsis thaliana silencing vector, mainly for modifying the silencing segment for different genes, and the other parts are the same, expanding the unchanged DNA on the target carrier In the interval, the invariant regions at both ends of the silencing-related fragments are put into the transgenic target vector in advance, and then the core silencing fragments are connected into the transformed vector to directly complete the vector construction. When selecting the head and tail fragments, the DNA sequence was analyzed, so that after the head and tail fragments were connected by a bridge fragment, a StuI restriction site and a SnaBI restriction site could be obtained. After transforming E. coli, the vector could be prepared in large quantities and used with StuI and The SnaBI enzyme endonuclease linearizes it, and directly connects the core silencing fragment synthesized by this patent method to complete the construction of the Arabidopsis thaliana silencing vector. The beneficial effects of the present invention are: simple and easy to operate, convenient operation, high benefit, can greatly shorten the experiment time, and reduce the transformation cost of a single silencing carrier.
Description
Technical field [0001] The invention relates to the field of biotechnology, and is mainly a method for quickly constructing an Arabidopsis artificial miRNA gene interference vector. Background technique [0002] The existing methods for constructing Arabidopsis gene silencing vectors take Overlapping PCR as the core, and the Arabidopsis gene silencing vectors are constructed by PCR replacing the miR319a of Arabidopsis (Schwab et al., 2006). The existing methods for the construction of a single silencing vector need to use four PCR primers with silencing-related sequences and two common primers to amplify the pRS300 vector, recover the three amplified DNA fragments, and then perform fusion PCR , Connect to the T vector, and then connect to the transformation vector after restriction enzyme cutting, and finally obtain the silence vector for transgene. The method is divided into four steps : [0003] The first step is to design and synthesize 4 primers with a length of 40bp for each ...
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