Strategies for exogenous RNA delivery in RNAi-mediated pest management / 生物工程学报

Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

Dual screening for targeted gene replacement mutant in Magnaporthe oryzae with GUS as negative marker / 生物工程学报

To improve the efficiency of targeted gene replacement (TGR), a dual screen (DS) system with gusA gene as negative selective marker (GUS-DS) was developed in Magnaporthe oryzae. First, we tested the endogenous beta-glucuronidase (GUS) activities of 78 fungal strains. All tested strains were GUS-, only with 3 exceptions. Whereas, after the gusA being introduced in, M. oryzae, Fusarium oxysporum and Colletotrichum lagenarium acquired high GUS activities. The gusA is thus usable as a selective maker in fungal species. With gusA as the negative marker, HPH gene as the positive marker, and the peroxisomal targeting signal receptor genes MGPEX5 and MGPEX7 as 2 instances of target genes, we established the GUS-DS system. After transformation, we collected the transformants from hygromycin B screen media and then tested the GUS activities of them. The GUS- ones were selected as potential mutants and checked in succession by PCR and Southern blotting to identify the true mutants and calculate the efficiency of GUS-DS. As a result, GUS-DS improved the screen efficiency for delta mgpex5 from 65.8% to 90.6%, and for delta mgpex7 from 31.2% to 82.8%. In addition, we established a multiple PCR (M-PCR) method for mutant confirmation. By amplifying the different regions at the targeted locus, M-PCR differentiated the wild type, the ectopic transformants and the mutants effectively and rapidly, and had the same reliability as Southern blotting. In conclusion, GUS-DS and M-PCR are useful tools to improve the efficiency of TGR and would be helpful for fungal genomics.