The Mechanism of Loss of Germination Ability of A. flavus Spore with Citral

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 18 (2) : ),2) 2) 1) 3,, ( 1), ; 2), ) CCD(charge coupled device) DEPHI SCGE( single2cell gel electro2 phoresis), CCD DNA, DNA Olive, 115 mgπl,dna,, DNA, DNA,,,, Q ) The Mechanism of Loss of Germination Ability of A flavus Spore with Citral LUO Man 1),2), J IANGLi2ke 2), ZOU Guo2lin 1) 3 ( 1) College of Life Sciences, Wuhan University, Wuhan , China ; Department of Biological Engineering, Anhui Agriculture University, Hefei , China) Abstract A fast multi2channel micro2spectrophotometer and a single2cell gel2electrophoresis imaging device were used for studying the mechanism of loss of germination ability of A flavus spore with citral The former device was composed of a inverted microscope,a differential grating and a linear array charge coupled device ( CCD),while the latter was operated by a computer with a DEPHI program A photo was taken under the fluo2 rescence microscope via CCD and the image signal was transmitted to a computer via a frame grabber The im2 ages of A flavus plasma membrane and the DNA damage were displayed, stored,handled and analyzed Four parameters,i e comet length,fluorescence intensity,moment and the ratio of head DNA % to tail DNA % were determined It was found that there existed positive correlation between citral concentration and the above pa2 rameters When the citral concentration reached above 115 mgπl,dna damage became lethal and not repairable in cell It was also found that after citral entered into cell and damaged the plasma membrane,damaged DNA became irreversible with the loss of germination ability of the spore of A flavus Compared with other methods the method is rapid,sensitive,informative,quantitative and free of disturbance Key words citral, Aspergillus flavus, germination ability : , : ( ) 3 Tel : (027) ,E2mail : edu cn,,1963,, Received :July 9,2001 ;Accepted :October 24,2001 Supported by Department of National Archives (No 9032protect202) 3 Corresponding author Tel : (027) ,E2mail : edu cn

2 ( Litsea cubeba oil) 1 [1 ], 72 % 95 %, [2 ] [3 ] 111, ( Aspergillus flavus), 24 h,, DYY2 26B ( ),, IMI1 0 ( ),Nikon Eclipse TE300( ) A : 1 molπl DNA 011 molπl Tris2HCl, ph 913 ; B 1 molπl [4 6 ] 0102 molπl 2NaH 2 PO 4,pH 518 ; C 215, mmolπl NaCl, 10 mmolπl Na 2 2EDTA, 10 mmolπl Tris2 HCl, 1 % Na2,1 % Triton X2100,10 %, DMSO (, ) ; D 30 :300 mmolπl NaOH,1 mmolπl Na 2 2EDTA,pH 10 ;, E : 014 molπl Tris2HCl, ph 715 ; F 2, gπml ; G 015 % ; H 016 %,, Yakult Co [7 9 ] 112, (1) :, [3 ] CCD (2) :, CCD 350 nm 800 nm, ( 1 ms, 012 nm) [10 12 ],, nm,,, [13, ] ; DEPHI, DNA (1) SCGE DNA 4 : ( Fig 1), DNA, : CCD Olive DNA % Π, CCD DNA DNA,,, DNA : Inprise Borland Delphi5, Microsoft Windows98 DNA Windows NT Fig12 Fig 1 IMI 1 0 comet assay analysis composition setup

3 2 : 229 Fig 2 Software analytical flowchart of SCGE : CCD ( H(i),i = k a, a =, - ) k = = ( ( H(i) (i - k) )Π H(i) ),, ; H(i),i = k a, a =, [1 2 2 (2) ] k = Excel = ( ( H(i) (i - k) 2 )Π H(i) ), H(i),i = k a, a =, Delphi5 Server SCGE, Office DNA % = [ Π( + ) ], WordΠExcel % : :CPU Celeron 800 MHz, 40 G, 128 M,600 DNA % Olive 800, 150 dpi, : Windows98 Windows2000 (2) SCGE = (Right Edge) c - (Left Edge) c 4 (Left Edge) c (Right Edge) c [12 ], SCGE P t = T (i), T (i), 19 : = ( ( T (i) i)πp t ) T (i), : i = k a,a =,k = : = (Right Edge) c - (Left Edge) c = ( ( C(i) (i - k) )Π ( C(i) ) = ( ( C(i) (i - k) 2 )Π C(i) ) : Π Π C(i),i = k a, a =, Π k = DNA (1) : [3 ] 48 h, DNA % : 1 % Tween220, = (Right Edge) h - (Left Edge) h, ( (Left Edge) h (Right Edge) h, P h = H(i) ), g 20 min, ; A H(i) = ( ( H(i) i)πp h ) k = = ( ( T (i) (i - k)π T (i) ) T (i),i = k a, a =, (Left Edge) c (Right Edge) c k = P c = C(i), C(i) = ( ( T (i) (i - k) 2 )Π T (i) ) = ( ( C(i) i)πp c ), C(i), T (i),i = k a, a =, i = k a, a =,k = k = DNA % = DNA % C(i),i = k a, a =,k = Olive = ( - ) DNA 2 3, g 20 min ; 4 ml B ( 20 mgπml

4 mgπml 20 mgπml ), 30,,, 15 %,, ( 50 min ), 30 min,, g 10 min, 4 ml A (2) (Fig13) ; ( C D G H, nm ) [14,15 ], ( E F) [16 ] (3) 100 W nm 590 nm, ( 015 mgπl), 1818 %, ;, CCD 1213 %, 3218 % 2, CCD, mgπl, 40 %,, FISH,, (4), 2 mgπl IMI110, 19, DNA, 212 SCGE 2,, nm SCGE ( Fig14),, 211,, DNA,, CCD DNA Fig 3 Observation on A flavus membrane damaged by citral via fast multi2channel micro2spectrophotometer A Control ; B 1 mgπl citral ; C 2 mgπl citral Fig 4 SCGE image of A flavus damaged by citral 213 SCGE SCGE, Table 1 : Olive Π ;, DNA %, (Fig15) 2 mgπl, DNA,, DNA

5 2 : 231 Table 1 The relativity of comet parameter with citral dosage Citral Πmg L - 1 Cells Tail length Π m Tail gravity centerπ m Olive tail momentπ m Tail inertiaπ m Dispersed moment of tailπ m Ratio of tailπhead Tail DNA ( %) Fig15,Olive ( Y 5, R 2 = 0197) ( Y 2, R 2 = 0192) ( Y 4, R 2 = 0190),, 214 ( Olive DNA % ) Table 2 DNA %, , Olive, P < DNA, DNA Y, DNA X ( Fig16) DNA, DNA ;,DNA Table 2 Relativity among comet parameters Fig 5 Relativity of tail length,olive tail moment and so on with citral dosage Tail DNA ( %) ; Tail length ; Tail gravity centre ; Dispersed moment of tail ; Olive tail moment ; The ra2 tio of tailπhead Y 1 = 15119X , R 2 = 0157 ; Y 2 = 15157X , R 2 = 0192 ; Y 3 = 6107X , R 2 = 0189 ; Y 4 = 6163X , R 2 = 0190 ; Y 5 = 6186X , R 2 = 0197 ; Y 6 = 0185X , R 2 = 0187 Test item Tail lengthπ m Light density of tail Tail gravity centerπ m Olive tail momentπ m Tail inertiaπ m Dispersed moment of tailπ m Tail DNA % Ratio of tailπhead Tail lengthπ m Light density of tail Tail gravity centreπ m Olive tail momentπ m Tail inertiaπ m Dispersed moment of tailπ m Tail DNA % Ratio of tailπhead Average value

6 Fig 6 Integral curve of comet image from A flavusπprotoplasts (A) SCGE image from A flavus ; (B) Integral curve of SCGE image from A flavus 3 (ph ), ph,,,,, 63 68,,, 5, DNA, 7, DNA, ( 4 mgπl ) [17 ] SCGE, DNA SCGE,Olive, Olive, DNA DNA,, (comet assay) DNA DNA, DNA, ( ),, DNA, ( References) 1 ( Zhou Jian Progress in study on the pharmacology and clinical medicine of Litsea cubeba oil Combin Trad Chin Western Med), 1991,11 (8) : ( Yu Bo2liang Antibiotic activities of Litsea cubeba oil on moulds and effects of Litsea cubeba oil on aflatoxin production Mi2 crobiology), 1998,25(3) : ,, ( Wu Zhen2san, Jiang Li2ke, Zhou Shu2xiang Study on Litsea cubeba oil resistance mould Chin J Appl Environ Biol), 1997, 9 (2) : 4, (Zhou Jian, Li Pei2tao Study on the mechanism of Litsea cubeba oil resisting to Staphylococcus aureus Bull Hunan Med Univ), 1992,17(4) : ,, ( Zhou Jian, Li Pei2tao, Cheng Wen Study on the mechanism of Litsea cubeba oil resisting to Bull Hunan Med Univ), 1993,18 (1) :33 35 Pseudomonas pyocyanea 6,, ( Xia Zhong2di, Yang Jun2xiang, Li Pei2tao Study of antifungal mechanism of Litsea cubeba oil in Candida albicans Bull Hunan Med Univ), 1995, 20 (2) : ,, (Li Pei2tao,Zhou Jian, Zha Guo2zhang A research on antibacterial mechanism of Litsea cubeba oil emulsion Bull Hunan Med Univ), 1994,19(6) : ,, ( Zhou Yong, Tao Jun2di, Zhang Jia2jun Study on the antifungal role by Litsea cubeba oil and its primary compo2 nent the citral Combin Trad Chin Western Med), 1984,4 (9) : ,,, ( Tu Xin2yi, Liu Ju2ying, Xu Xiao2 qing, Zheng Ji2xia Comparison with Litsea cubeba oilπs antifungal role

7 2 : 233 and citralπs J Chin Hosp Pharmacol),1987,7 (9) : Wojcik E M, Miller M C, O Dowd G J Value of computer2assisted quantitative nuclear grading in differentiation of normal urothelial cells from low and high grade transitional cell carcinoma Anal Quant Cytol Histol, 1998, 20(1) : Yokoyama H, Hoyer P H, Hansen P M Immunohistochemical quantifi2 cation of heparan sulfate proteoglycan and collagen IV in skeletal muscle capillary basement membranes of patients with diabetic nephropathy Diabetes 1997,46(11) : Meyer B, Schaller C, Frenkel C Distributions of local oxygen satura2 tion and its response to changes of mean arterial blood pressure in the cerebral cortex adjacent to arteriovenous malformations Stroke 1999, 30 (12) : Brown I F, Sisler H D Mechanisms of fungutoxic action of N2dodecyl2 guanidine acetate Phytopathology, 1960,150 : Rojas E, Lopez M C, Valverde M Single cell electrophoresis assay : Methodology and applications J Chrom B, 1999,722 : Lee J G, Madden M C, Reed W The use of the single cell gel electro2 phoresis assay in detecting DNA single strand breaks in lung cells in vitro Toxic Appl Pharmacol, 1996, 141 : Muller Pillet V, Joyeux M, Ambroise D Genotoxic activity of five halo2 acetonitriles: comparative investigations in the single cell gel electro2 phoresis (comet assay) and the amesfluctuation test Environ Mol Mut, 2000,36(1) : ( Zhuang zhi2xiong Some development in technology of single cell gel electro2 phoresis Health Res), 1998, 27(supplementary issue) : 6 8,, 5 (LRP5) Cell, LRP5,, LRP5 LRP5,, LRP5, LRP5,, LRP5,,,, LRP5,,,LRP5 DNA ( J Travis : Science News, Vol 160,Oct 27,2001,p 261)