Efficient Methods For The Construction And Application Of Artificial MicroRNA Expression Vectors In Plants

AmiRNAs (artificial miRNAs) are exogenous small RNAs generated by endogenous miRNA precursors, which substitites miRNA and its complementary sequence miRNA* for amiRNA and amiRNA*. The advantages of amiRNAs ---- efficiency, specificity and no off-target, have been confirmed in many plants, What’s more, AmiRNA vectors are recognized as second-generation tools for gene function research in plants followed by hairpinRNA interference (hpRNAi).For the specific stem-loop structure of the amiRNA precursor, it is always fussy and expensive to construct amiRNA expression vectors. To solve the problem, we developed three novel, simple, efficient, low-cost and high-throughput approaches to generate plant amiRNA expression vectors in the research:1. Construction of plant amiRNA expression vectors through enzyme-free cloning based on one-step PCR and MAGIC.Three universal donor plasmids pD319a-gfp, pD159a-gfp and pD164a-gfp and a recipient plasmid p1301-gfp were constructed. Three specific amiRNA precursors were obtained by one-step PCR and then cloned into universal donor plasmids and three special donor plasmids pDonor-amiR319aFLC1, pDonor-amiR159aPDS and pDonor-amiR164aEIN2 were generated. Finally, the amiRNA binary expression vectors p1301-amiR319aFLC1, p1301-amiR159aPDS and p1301-amiR164aEIN2 were generated through MAGIC. The phenotypes were all appeared as expected in T1 seedlings containing amiRNA expression cassettes. The results of Real-time PCR analysis showed that the relative expression level of both flcl and ein2 declined dramatically; amiR319aFLC1 and amiR164aEIN2 were detected by splinted ligation method in the corresponding transgenic lines. The success rate generating amiRNA expression cassettes through this method is above 70% according to the first sequencing results of more than 180 amiRNA expression vectors for rape.2. Construction of plant amiRNA expression vectors based on blue-white screening for lacO reconstitution and MAGIC.Firstly, amiR319aCHS precursor carrying 7bp-long lacO site, was inserted into pD319a-gfp (T) carrying the rest of 13bp-long lacO site and two Bful sites, and obtaining the positive blue recombinants containing the donor plasmid pD(T)-miR319aCHS through observation. Finally, the binary expression vector containing amiR319aCHS expression cassettes, p1301 (T)-miR319aCHS was generated through MAGIC. The color of T1 trangenic seeds was thinner than wild type and the result of Real-time PCR analysis showed that the relative expression level of chs was declined to vary degrees. This method is visal and rapid to screen recombinants for lacO reconstitution. The success rate generating amiRNA expression cassettes through this method is above 90% according to the first sequencing results of more than 40 amiRNA expression vectors for rice.3. Construction of plant amiRNA expression vectors based on multi-primers annealing.The precursors of miR528PDS, miR528SpⅢand miR528Euil, were designed and three sets of primers 40-50nt long, were synthesized and annealed one another respectively. Then they were mixed with linear amiRNA expression vector, pYH1301-gfp digested by HindⅢ, NcoI and T4 DNA pol. Finally, the mixtures were transformed into E.coli directly and three amiRNA expression vectors, pYH1301-miR528PDS, pYH1301-miR528SpⅢand pYH1301- miR528Euil were generated. The success rate generating amiRNA expression cassettes through this method is acheive 100% according to the first sequencing results of 20 amiRNA expression vectors for rice. This novel method emerges the highest efficiency, the simpliest operation and the lowest cost than all known methods for amiRNA expression vectors construction.