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     5990 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.

References

[ 1 ] Aguilera-Arreola MG, González-Cardel AM, Tenorio AM, Curiel-Quezada E, Castro-Escarpulli G (2014) Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. BMC research notes 7 (1):433
[ 2 ] Apostolidis AP, Apostolou PK, Georgiadis A, Sandaltzopoulos R (2006) Rapid identification of Salmo trutta lineages by multiplex PCR utilizing primers tailored to discriminate single nucleotide polymorphisms (SNPs) of the mitochondrial control region. Conservation Genetics 8 (5):1025-1028. doi:10.1007/s10592-006-9239-1
[ 3 ] Ben M'hadheb M, Harrabi M, Souii A, Jrad-Battikh N, Gharbi J (2015) Multiplex RT-PCR and indirect immunofluorescence assays for detection and subtyping of human influenza virus in Tunisia. Current microbiology 70 (3):324-329. doi:10.1007/s00284-014-0719-0
[ 4 ] Chen X, Jia S, Yue S, Wang N, Li C, Wang Y (2010) Effect of solid bed-materials on vegetative cells of Nostoc flagelliforme. Journal of applied phycology 22 (3):341-347
[ 5 ] Dalmasso A, Fontanella E, Piatti P, Civera T, Rosati S, Bottero M (2004) A multiplex PCR assay for the identification of animal species in feedstuffs. Molecular and Cellular Probes 18 (2):81-87
[ 6 ] Gao K (1998) Chinese studies on the edible blue-green alga, Nostoc flagelliforme: a review. Journal of Applied Phycology 10 (1):37-49
[ 7 ] Jia S-R, Su J-Y, Qiao C-S (2005) Nostoc flagelliforme cells cultivation and its products. Chinese patent ZL031191010
[ 8 ] Kanekiyo K, Lee J-B, Hayashi K, Takenaka H, Hayakawa Y, Endo S, Hayashi T (2005) Isolation of an Antiviral Polysaccharide, Nostoflan, from a Terrestrial Cyanobacterium, Nostoc f lagelliforme. Journal of natural products 68 (7):1037-1041
[ 9 ] Lü J, Jin X, Mao P-H, Ma X-D, Ling H-Q, Fan Y-H, Yu L, Wu B-S, Ouyang P-K (2009) Transfer of Ephedra genomic DNA to yeasts by ion implantation. Applied biochemistry and biotechnology 158 (3):571-581
[ 10 ] Landete JM, de las Rivas B, Marcobal A, Muñoz R (2010) PCR methods for the detection of biogenic amine-producing bacteria on wine. Annals of Microbiology 61 (1):159-166. doi:10.1007/s13213-010-0068-6
[ 11 ] Lu I-J, Lin C-H, Pan T-M (2010) Establishment of a system based on universal multiplex-PCR for screening genetically modified crops. Anal Bioanal Chen 396:2055-2064
[ 12 ] Qian W-D, Zhao D-Z, Fu Y-F, Mao P-H, Zhou Y-X (2014) The construction of Gentiopicroside-producing recombinant yeast using one-step recombinant method. Journal of Shaanxi University of Science ＆ Technology 5 (32):123-128.(in Chinese with English abstract)
[ 13 ] Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard R (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences 81 (24):8014-8018
[ 14 ] Su J, Jia S, Chen X, Yu H (2007) Morphology, cell growth, and polysaccharide production of Nostoc flagelliforme in liquid suspension culture at different agitation rates. Journal of Applied Phycology 20 (3):213-217. doi:10.1007/s10811-007-9221-4
[ 15 ] Tsakogiannis D, Diamantidou V, Toska E, Kyriakopoulou Z, Dimitriou TG, Ruether IG, Gortsilas P, Markoulatos P (2015) Multiplex PCR assay for the rapid identification of human papillomavirus genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 in clinical samples. Archives of virology 160 (1):207-214. doi:10.1007/s00705-014-2261-6
[ 16 ] Tseng C (2001) Algal biotechnology industries and research activities in China. Journal of Applied Phycology 13 (4):375-380
[ 17 ] Vilaithong T, Yu L, Alisi C, Phanchaisri B, Apavatjrut P, Anuntalabhochai S (2000) A study of low-energy ion beam effects on outer plant cell structure for exogenous macromolecule transferring. Surf Coat Tech 128:133-138
[ 18 ] You Y (2000) Impact of the Nostoc flagelliforme trade on the ecoenvironment of the Inner Mongolia pasture and some policy suggestions. Arid Land Geogr 23 (1):279–283
[ 19 ] Yu Z (2000) Ion beam application in genetic modification. Plasma Science, IEEE Transactions on 28 (1):128-132