Establishment of High-throughput Screening System for Nostoc Flagelliforme Extracellular polysaccharide (EPS)－producing Recombinant Yeast Strains Based on Duplex PCR Reaction

Volume 5, Issue 1, February 2020     |     PP. 1-15      |     PDF (735 K)    |     Pub. Date: August 12, 2020
DOI:    221 Downloads     6003 Views  

Author(s)

WenJin Ma, The Institute of Gansu Province Light Industrial Scientific Research, Lanzhou 730000, People’s Republic of China
WenJin Lou, Gansu Provincial Academic Industrial for Medical Research, Lanzhou 620100, People’s Republic of China
Yanbing Zhou, The Institute of Gansu Province Light Industrial Scientific Research, Lanzhou 730000, People’s Republic of China
Bo Wang, The Institute of Gansu Province Light Industrial Scientific Research, Lanzhou 730000, People’s Republic of China
Meilin Li, The Institute of Gansu Province Light Industrial Scientific Research, Lanzhou 730000, People’s Republic of China
Huan Liu, School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an 710021, People’s Republic of China
XueFeng Chen, School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an 710021, People’s Republic of China

Abstract
A Common Primer Duplex PCR (CP-D-PCR) was developed to screen polysaccharide-producing recombinant yeast strains from the genotype. This method demonstrated higher sensitivity and efficiency than the conventional methods. In this study, two pairs of primers were designed according to the constitutive protein of photosynthesis from Nostoc punctiforme (N. punctiforme) PCC73120 which is similar to Nostoc flagelliforme (N.flagelliforme). Genomic DNA of N. flagelliforme and Rhodotorula mucilaginosa (R. mucilaginosa) were used as templates for amplification and to verify by PCR reaction. Duplex PCR screening system was established on the basis of the simplex PCR screening system and the products of PCR were analyzed by agorae gel electrophoresis. The results showed that both selected primers were able to correctly amplify the objective gene fragment (800 bp, 550 bp) with the N. flagelliforme genomic DNA. Electrophoresis showed clear and consistent bands after PCR amplification, respectively. No amplifications were obtained with R. mucilaginosa genomic DNA as template. And we have successfully screened five recombinant yeast strains from three hundred and seventy-five strains using duplex PCR screening method. So, the CP-D-PCR was used to screen recombinant yeast strains quickly, accurately and efficiently. This method provides a scientific basis to screen recombinant strains constructed by distant hybridization and no sufficient genetic information about the parent strains.

Keywords
Nostoc flagelliforme, R. mucilaginosa, Recombinant Yeast Strains, Duplex PCR, Screening System, Gel Electrophoresis

Cite this paper
WenJin Ma, WenJin Lou, Yanbing Zhou, Bo Wang, Meilin Li, Huan Liu, XueFeng Chen, Establishment of High-throughput Screening System for Nostoc Flagelliforme Extracellular polysaccharide (EPS)－producing Recombinant Yeast Strains Based on Duplex PCR Reaction , SCIREA Journal of Agriculture. Volume 5, Issue 1, February 2020 | PP. 1-15.

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