QC cloning is a ligation-independent cloning procedure that relies on the exonuclease. ![]() dahliae to adverse environmental conditions and is involved in a transition to a dormant form for prolonged survival. cloning procedure was developed to clone specific products only. In conclusion, our results show that VdASP F2 plays an important role in the response of V. However, after inducing microsclerotial formation and incubation at low temperatures, cotton infected with the VdASP F2 deletion mutant did not exhibit wilt symptoms. Cotton inoculated with the VdASP F2 deletion mutant wilted, demonstrating that VdASP F2 is not associated with pathogenicity under normal conditions. Further assessment revealed that VdASP F2 was required for the expression of VDH1 and VMK1, two genes involved in microsclerotial formation. However, on semisynthetic medium or under limited nutrient conditions at lower temperatures, the VdASP F2 deletion mutant exhibited vigorous mycelium growth, less branching, and a significant delay in melanized microsclerotial formation. dahliae growth on potato dextrose agar under various stress conditions. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). Phenotypic analysis suggests that VdASP F2 is not necessary for V. dahliae and deleted the VdASP F2 gene using the developed knockout system. We identified secretory factor VdASP F2 in a T-DNA insertion library of V. dahliae using ligation-independent cloning and fluorescent screening. In this study, we developed a fast and easy gene knockout system for V. However, the construction of gene-deletion vectors and screening of deletion mutants have remained challenging in V. dahliae has facilitated large-scale investigations of individual gene functions using gene-disruption strategies based on Agrobacterium tumefaciens-mediated transformation. The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. Conclusion: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform.Results: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning (LIC) method. Background: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in in China for chickens against Newcastle disease. ![]()
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