Quick efficient plant manpower fine RNA expression vector construction method

Abstract

The invention provides a fast highly-efficient construction method for plant artificial micro RNA (amiRNAs) expression vectors, which comprises the two following steps:I) construction of donor plasmids containing amiRNAs expression units: 1) selecting and modifying donor skeleton vectors; 2) designing primers; 3) PCR amplification; and 4) homologous recombination, transformation and screening, and II) cloning the amiRNAs expression units onto plant binary vectors: 1) selection and modification of donee vectors; and 2) cloning the amiRNAs expression units onto the plant binary vectors with theconjugation-assisted genetic integration colonal method, verifying recombinants and selecting out plant amiRNAs expression vectors. Selective marker genes or reporter gene expression units and coli conditional lethal gene expression units are respectively cloned in the modification of donor skeleton vectors and donee vectors. The invention does not need restriction-ligation reaction as well as the special treatment of the vectors or target fragments, the operations of the whole process are convenient, the experimental period is short, the recombination efficiency is high, and the plant amiRNAs expression vectors can be quickly constructed with high efficiency, zero background and high flux.

Description

technical field [0001] The invention relates to biological gene cloning and expression technology, in particular to a method for quickly and efficiently constructing plant artificial microRNAs (artificial microRNAs, amiRNAs) expression vectors. It is especially suitable for large-scale construction of plant artificial microRNA expression units for different genes of plants, so as to study the functions of plant genes. Background technique [0002] With the completion of the whole genome sequence determination of more and more species, the current genomics research hotspot has shifted from structural genomics to functional genomics, so as to develop fast, efficient and high-throughput gene function research methods and strategies It has also naturally become a research hotspot. RNA interference (RNA interference, RNAi) technology has become one of the important methods in the study of animal and plant gene functions due to its high specificity, easy operation, and good repea...

AI Technical Summary

Problems solved by technology

This process not only takes time, but also increases the self-connected background of the carrier, complicating the subsequent screening process

The third method requires chemically synthesized two oligonucleotide single strands to be annealed in vitro, and the obtained product needs to be purified and then ligated with the processed carrier; since the fragment to be cloned is less than 100bp, it is difficult to recover and purify, and it requires It is difficult to identify the correct recombinant plasmid, and the false positive rate is high

[0005] 2. Not suitable for high-throughput operations

These operations may be effective for constructing single or a small number of plant amiRNAs expression vectors, but it is very difficult and inefficient to construct plant amiRNAs expression vectors on a large scale

In the first method, obtaining a complete amiRNA stem-loop structure sequence requires two pairs of specific primers, four independent PCR and recovery steps, which are not only cumbersome steps, time-consuming and labor-intensive, but also costly

The primers used in the second method are usually larger than 60nt, and the oligonucleotide single strands used in the third method are usually 80-100nt. Synthesizing these long primers or oligonucleotide single strands not only greatly increases the experimental cost, Moreover, the correct rate of base synthesis is greatly reduced, which increases the cost of subsequent screening work

[0006] 3. Not suitable for all types of plant miRNA

The first two methods are not suitable for the construction of amiRNAs based on miRNAs whose loop sequence length is too short

In the first method, if the loop length of amiRNAs is too short, the length of the first-round PCR product will be too short, which will eventually lead to difficult purification and low recovery efficiency

The second method is only suitable for plant miRNAs with a small number of bases in the flanking sequence and a long loop length

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