Nucleotide sequence and vector for directional cloning

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a nucleotide sequence for directional cloning and a vector constructed by using the sequence and capable of realizing directional cloning by single enzyme digestion. The vector is in operable connection with a corn ubiquitin promoter sequence and an NOS (nitric oxide synthase) terminator sequence. The invention also relates to application of the nucleotide sequence for the directional cloning in constructing the vector, and application of the vector in plant gene expression, in particular monocotyledon or dicotyledon gene expression. Compared with a dual enzyme digestion vector, the single enzyme digestion vector of the invention reduces the cost, and saves the time.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a nucleotide sequence for directional cloning and a plant gene expression vector constructed by using the sequence. Background technique [0002] At present, when constructing plasmids or viral vectors through genetic recombination, the method of double digestion is generally used, that is, two restriction endonucleases with different restriction sites are used to simultaneously digest the vector to insert the exogenous target fragment. This method has the advantage of directional cloning, which can ensure the direction of inserting foreign fragments. After the vector and inserting fragments are double-enzymatically digested and ligated, there is no need to identify the directionality of the inserting fragments; and it can prevent the self-ligation of the vector and increase the recombination rate. However, the disadvantage of the double enzyme digestion method is tha...

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Problems solved by technology

However, the disadvantage of the double enzyme digestion method is that the cost is high, and two enzymes need to be used at the same time; When multiple foreign fragments are inserted into the multiple cloning site, a suitable enzyme cutting site may not be found; in addition, if the optimum reaction temperature or reaction buffer (Buffer) of the two enzymes are different, or the two enzymes cannot be When reacting in the same Buffer, it will involve the choice of reaction temperature and Buffer and even step-by-step enzyme digestion, which increases the complexity of the operation

[0003] For the method of constructing a recombinant vector by single enzyme digestion, only one restriction endonuclease is used for digestion and ligation, which costs less and requires less technology, and when multiple fragments are inserted into the multi-cloning site, it is relatively In terms of double enzyme digestion, there are more enzyme cutting sites to choose from; however, the disadvantage of this method is that directional cloning is not possible. After enzyme digestion and ligation, sequencing is required to determine whether there is an insert fragment and whether the direction of the insert fragment is correct, so as to construct a correct recombinant vector. The probability is less than 50%. Therefore, the sample size for sequencing is at least twice that of the double-enzyme digestion method, and the cost of sequencing is higher than that of the double-enzyme digestion method.

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