In this study, the yield and quality of total RNA extracted from whole course cultures of a 3′,5′-cyclic adenosine monophosphate (cAMP) - producing yeast strain G2 in yeast extract peptone dextrose medium was investigated and further optimization was carried out. The cells with culture time of 16, 36 and 60 h were selected, which corresponded to exponential, post-diauxic and stationary phase, respectively. Using a commercial RNAout kit, the RNA yields were obtained as 1 010.6, 171.5 and 91.3 ng/A600, respectively. The genes ACT1 and CYR1 were selected as the relatively high and low representatives, respectively, and used in quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR) and DNA pollution analysis. Agarose gel electrophoresis and RNA integrity number (RIN) test were used in RNA integrity assay. The results showed that 36 h and 60 h RNA extractions were polluted with DNA and only 16 h sample was qualified for RNA-Seq. Five cell wall disruption methods were further selected to investigate the disruption ratio and their effects on RNA extraction. The addition of bead-beating was proved to have no significant effect on the 16 h cells, while it increased the RNA yields of the 36 h and 60 h cells to 979.6 and 785.6 ng/A600, which were 5.71- and 8.60- fold of the value before modification, respectively. Further, all samples were qualified for qRT-PCR and 36 h isolations also met the requirement of RNA-Seq. This study helps to establish the RNA extraction protocol of yeast suitable for the whole course cultures and provides valuable insights into the mechanism relating to the cAMP pathway regulating.
LI Jiaman
,
ZHANG Xiaomao
,
GUO Jinghan
,
SUN Sifan
,
JIA Yudie
,
ZOU Shaolan
. Preliminary optimization of RNA extraction from whole course cultures of a cAMP-producing yeast strain[J]. Food and Fermentation Industries, 2020
, 46(24)
: 1
-8
.
DOI: 10.13995/j.cnki.11-1802/ts.024409
[1] 易弋,容元平,程谦伟,等.不同破壁方法提取酵母菌总RNA的比较[J].食品科学,2011,32(11):161-164.
YI G,RONG Y P,CHENG Q W,et al.Comparison of different cell wall disruption methods for yeast total RNA extraction[J].Food Science,2011,32(11):161-164.
[2] SASIDHARAN K,AMARIEI C,TOMITA M,et al.Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures[J].Yeast, 2012, 29(8):311-322.
[3] 吴思琪,宋静颐,秦倩,等.一种高效稳定的微生物总RNA提取方法[J].江苏农业学报,2017, 33(3):517-523.
WU S Q,SONG J Y,QIN Q,et al.A high-efficiency and stable method for extracting total RNA from micro-organisms[J].Jiangsu Journal of Agricultural Sciences, 2017,33(3):517-523.
[4] ANTONIO R,VANEECHOUTTE M.Comparison of the efficiency of different cell lysis methods and different commercial methods for RNA extraction from Candida albicans stored in RNAlater[J].Bmc Microbiology,2019,19(1):1-10.
[5] SCHMITT M E,BROWN T A,TRUMPOWER B L.A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae[J].Nuclc Acids Research,1990,18(10):3 091-3 092.
[6] 李维维,顿宝庆,王智,等.一种改良的热酚法高效快速提取酿酒酵母总RNA[J].生物技术通报,2012(12):163-166.
LI W W,DUN B Q,WANG Z,et al.A modified method for the efficient and fast extraction of total RNA from Saccharomyces cerevisiae with Hot-Phenol[J].Biotechnology Bulletin,2012(12):163-166.
[7] LI J,LIU J,WANG X,et al.A waterbath method for preparation of RNA from Saccharomyces cerevisiae[J].Analytical Biochemistry,2009,384(1):189-190.
[8] STEAD M B,AGRAWAL A,BOWDEN K E,et al.RNAsnapTM:A rapid, quantitative and inexpensive, method for isolating total RNA from bacteria[J].Nucleic Acids Res,2012,40(20):e156.
[9] SHEDLOVSKIY D,SHCHERBIK N,PESTOV D G.One-step hot formamide extraction of RNA from Saccharomyces cerevisiae[J].RNA Biology,2017,14(12):1 722-1 726.
[10] 田甜甜,孙军勇,蔡国林,等.基于转录组学的酿酒酵母耐酸机制解析[J].食品与发酵工业,2020,46(6):1-7.
TIAN T T,SUN J Y,CAI G L,et al.Transcriptomic profiling reveals the acid-tolerance mechanism of Saccharomyces cerevisiae[J].Food and Fermentation Industries,2020,46(6):1-7.
[11] MANNAN A U, SHARMA S, GANESAN K.Total RNA isolation from recalcitrant yeast cells[J].Analytical biochemistry,2009,389(1):77-79.
[12] HERMAN,PAUL K.Stationary phase in yeast[J].Current Opinion in Microbiology,2002,5(6):602-607.
[13] GRAY J V,PETSKO G A,JOHNSTON G C,et al.“Sleeping Beauty”:Quiescence in Saccharomyces cereviae[J].Microbiology and molecular biology reviews,2004,68(2):187-206.
[14] GANCEDO,JUANA M.Biological roles of cAMP:Variations on a theme in the different kingdoms of life[J].Biological Reviews,2013,88(3):645-668.
[15] 王凯,姬晓兵,徐欢欢,等.整合过表达嘌呤代谢途径关键酶基因提高酿酒酵母菌株环磷酸腺苷产量[J].食品与发酵工业,2016,42(8):31-36.
WANG K,JI X B,XU H H,et al.Over-expressing key enzyme genes in the purine synthesis pathway by integrating into genome improves cyclic adenosine monophosphate production by Saccharomyces cerevisiae[J].Food and Fermentation Industries, 2016,42(8):31-36.
[16] 徐欢欢,程丽娜,王凯,等.利用转录调控因子Bas1p和Bas2p协同作用提高酿酒酵母cAMP产量的研究[J].微生物学通报,2016,43(2):370-378.
XU H H,CHENG L N,WANG K,et al.Reinforced cooperative interaction between Bas1p and Bas2p improves cAMP production by Saccharomyces cerevisiae[J].Microbiology,2016,43(2):370-378.
[17] 仇申珅,丁娟娟,曲淑玲,等.发酵工艺初步优化提高酿酒酵母工程菌株环磷酸腺苷产量[J].食品与发酵工业,2018,44(5):104-108;114.
QIU S S,DING J J,QU S L,et al.Preliminary optimization of process to improve cAMP production by Saccharomyces cerevisiae[J].Food and Fermentation Industries,2018,44(5):104-108;114.
[18] WENZEL T J,THIBAUT A W,DE S H Y.PDA1 mRNA:A standard for quantitation of mRNA in Saccharomyces cerevisiae superior to ACT1 mRNA[J].Nucl Acids Res,1995,23(5):883-884.
[19] DEL A E M,DUTRA M B,SILVA J T,et al.Comparing protocols for preparation of DNA-free total yeast RNA suitable for RT-PCR[J].BMC Molecular Biology,2005,6(1):9-14.
