Site-directed mutagenesis has become an indispensable technique for deciphering protein structure and function. To serve new development for protein structural biology, simultaneous construction for batch of recombinant expression vector with multiple sites mutations is required. Here, we describe a procedure for the unidirectional insertion of site-directed mutagenized DNA into an expression vector via overlap extension and sticky-end PCR. This method takes advantage of two classical PCR-based techniques and has improved the efficiency of the construction of recombinant plasmid with multiple points mutation.

Brown rot is a devastating fungal disease on stone and pome fruits worldwide. Remarkable progresses have been made to understand this disease and its causal agents based on researches for more than one hundred years. In this article, it has been reviewed on various aspects including pathogen populations, life cycle and symptoms, distributions and host ranges, biological characteristics (colony morphology, conidial size and shape, conidial germination profile and virulence), molecular detection methods, fungicide resistance and mana-gement strategies. Prospects were also provided for further researches on brown rot of fruits. Such information are crucial for better understanding of both brown rot disease and its causal agents and improving the outcomes of brown rot management, thus to keep safe production of stone and pome fruits.