The application of long-fragment fusion PCR in the construction of rho1-compelement strain of Aspergillus fumigatus

Abstract

Abstract: Objective To evaluate the effect of primer design and the amplification condition on long-fragment fusion PCR.Methods With the example of 6.5 kb-long fragment amplification by fusion-PCR during construction of rho 1 gene complement in Aspergillus fumigatus, primer design with diverse homologous regions and different condition for amplification were evaluated.Results The 6.5 kb-long fragment was successfully amplified by fusion-PCR under following conditions: the primers with homologous region of 35 bp; DNA polymerase with high amplification efficiency and high fidelity; the concentration of template fragments higher than 15 ng/μL in whole reaction system.Conclusion With the appropriate primer, DNA polymerase and concentration of templates, the fusion PCR for the long-fragment might be a highly-efficient tool in gene-manufacturing of filamentous fungi.

Key words: fusion PCR, Aspergillus fumigatus, rho1, gene complement

CLC Number:

R379.6

HAN Gai-ge, JIA Xiao-dong, HAN Xue-lin, ZHAO shuai, HAN li. The application of long-fragment fusion PCR in the construction of rho1-compelement strain of Aspergillus fumigatus[J]. Chinese Journal of Mycology, 2013, 8(3): 129-133.

References

[1] Beauvais A, Bruneau JM, Mol PC, et al.Glucan synthase complex of Aspergillus fumigatus[J]. J Bacteriol,2001,183(7): 2273-2279.
[2] Abad A, Fernandez-Molina JV, Bikandi J, et al.What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis[J]. Rev Iberoam Micol,2010,27(4): 155-182.
[3] Dichtl K, Helmschrott C, Dirr F, et al.Deciphering cell wall integrity signalling in Aspergillus fumigatus: identification and functional characterization of cell wall stress sensors and relevant Rho GTPases[J]. Mol Microbiol,2012, 83(3):506-519.
[4] Cardon CM, Beck T, Hall MN, et al.PAS kinase promotes cell survival and growth through activation of Rho1[J]. Sci Signal,2012, 5(209):ra9.
[5] Guest GM., Lin X, Momany M. Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis[J]. Fungal Genet Biol,2004, 41(1):13-22.
[6] Walther A, Wendland J.PCR-based gene targeting in Candida albicans[J]. Nat Protoc,2008, 3(9):1414-1421.
[7] 李敏,杨谦.一种高效构建同源重组DNA片段的方法--融合PCR[J]. 中国生物工程杂志,2007, 27(8):53-58.
[8] Yu JH, Hamari Z, Han KH, et al.Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi[J]. Fungal Genet Biol,2004, 41(11):973-981.
[9] Szewczyk E, Nayak T, Oakley CE, et al.Fusion PCR and gene targeting in Aspergillus nidulans[J]. Nat Protoc,2007,1(6):3111-3120.
[10] Li X, Gao M, Han X, et al.Disruption of the phospholipase D gene attenuates the virulence of Aspergillus fumigatus[J]. Infect Immun,2012, 80(1):429-440.
[11] Li Y, Fang W, Zhang L, et al.Class IIC α-mannosidase AfAms1 is required for morphogenesis and cellular function in Aspergillus fumigatus[J]. Glycobiology,2009, 19(6):624-632.
[12] Frandsen RJ, Andersson JA, Kristensen MB, et al.Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi[J]. BMC Mol Biol,2008, 1(9):70.
[13] Gravelat FN, Ejzykowicz DE, Chiang LY et al. Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence[J]. Cell Microbiol,2010,12(4): 473-488.
[14] Ejzykowicz DE, Cunha MM, Rozental S, et al.The Aspergillus fumigatus transcription factor ace2 governs pigment production, conidiation and virulence[J]. Mol Microbiol,2009,72(1):155-169.
[15] 白耀霞, 杨毅, 王玉飞, 等.利用T载体克隆快速构建布鲁氏菌缺失突变株[J]. 中国生物工程杂志,2010, 30(9):62-67.
[16] Xiao P, Shin KS, Wang T, et al. Aspergillus fumigatus flbB encodes two basic leucine zipper domain (bZIP) proteins required for proper asexual development and gliotoxin production[J]. Eukaryot cell,2010, 9(11):1711-1723.