acrysta ll iferous stra in Btk BE20

J ou rnal of N anj ing A g ricu ltu ral U niversity S18-4 - Btk BE20 3 1 2 2 2 2 ( 1, 321004; 2, 510275) S1824, PH T 3101, DNA P stl, B tk BE 20 SD S2PA GE, 2,, S1824 ; 2 ; ; S48217 Clon ing and expression of -endotox in gene of a new B acillus thu r ing iensis isola te S18-4 in the acrysta ll iferous stra in Btk BE20 J iang Donghua 1, D eng R iqiang 2, Pang Y i 2, Yu J ianx iu 2 and L ong Q ingx in 2 ( 1 Zhejiang N o rm al U n iv, J inhua 321004; 2 State Key L ab fo r B iocon tro l, Zhong shan U n iv, Guangzhou 510275) ABSTRACT A new B acillus thu ring iensis iso late (S1824) from so il w as used as the dono r of 2endo toxin gene. A shuttle vec2 to r (PH T 3101) w as used as an exp ression vecto r in the acrystalliferous strain B tk BE 20. T he purified p lasm id DNA are comp letely digested w ith P stl restriction enzym e and ligated w ith T 4 ligase. T he recom binant p lasm id w as transfered into B tk BE 20 by electropo ration. SD S2PA GE and scanning electron m icro scopy show ed that the 2endo toxin gene w ere ex2 p ressed in h igh level in B tk BE 20. T he recom binant strain show ed h igher toxicity than the dono r strain S1824 to P rod enia litu ra larvae. Key words B acillus thu ring iensis; 2endo toxin; gene cloning; exp ression ( B acillus thu ring iensis, B t) 2 [1, 2 ],, B t, B t, S1824, 48 h 80% 100% [3, 4 ] B t S1824 B t B tk BE 20 1 111 S1824 ; HD 21 ; B tk BE 20 ; PH T 3101 (Em ), 1 3 (940124) : 1998203210

34 22 1 PH T 3101 F ig. 1 Physical m ap of the shuttle vecto r PH T 3101 112 ( P rod en ia litu ra) (S p od op tera ex ig ua)(h elith is a rm ig era) (A rg y rog ram m a ag na ta) 3, 113 LB, SOC, T ETX [ 5 ] (A p ) 100 ΛggmL, (Em ) 150 ΛggmL 114 T 4DNA P rom ega, Serva, K Boeh ringer M annhein 115 D NA S1824 DNA [ 6 ], PH T 3101 ( [ 5 ]) 116 Bt B rian [7 ] L uke [8 ] 117 D NA DNA [ 7 ] 118 Bt D NA 10 ml DNA,, 10 m in (012 mm ), 1 500V 400 800 8, 7 8 m s 10 m in, 800 ml SOC, 28 1 2 h, Em LB 119 SD S-PAGE L aemm li [9 ] [ 5 ] 1110 5 d, T ETX 3,, 1111 LB 5 d B t, 410 510 m ggml, 200 ΛL 1 g 48 h 30, 3,, LD 50 2 211 S18-4 2, S1824 7, DNA, HD 21 DNA, S1824 120 10 6, 110 10 6, 52 10 6, 44 10 6,

1 : S1824 2 B tk BE 20 35 29 10 6, 9 10 6 415 10 6, 4, S1824 DNA 1 2 1 2 3 4 5 6 120 10 6 110 10 6 97 10 3 52 10 6 44 10 6 29 10 6 66 10 3 43 10 3 9 10 6 31 10 3 415 10 6 22 10 3 11S1824 strain; 2 5. R ecom binant strain; 11S1824 strain; 21HD 21 strain 2 S1824 DNA F ig. 2 T he electropho resis pattern of S1824 DNA in agaro se gel 61Standard p ro tein m arker 3 S1824 SD S2PA GE F ig. 3 T he SD S2PA GE pattern of p ro teins from S1824 and recom binant strains 212 S18-4 SD S-PAGE S1824 SD S2PA GE 135 000 65 000, 120 000 ( 3) B tk BE 20 SD S2PA GE, 2 B tk BE 20 213 S18-4 S1824 2 ( 4), ( 4) B tk BE 20 S1824 strain R ecom binant strain 4 S1824 ( 6 000) F ig. 4 T he paraspo ral crystals of S1824 and recom binant strain ( 6 000)

36 22 214 1, 4, LD 50 6152 13140 Λg, S1824; B tk BE 20 1 S18-4 4 (LD 50) Table 1 The tox ic ity of S18-4 and recombinan t stra in to larvae of four differen t Nocto idae (LD 50) Λg Strains P 1litu ra S 1ex ig ua H 1arm ig era A 1ag na ta S1824 2812 2515 6132 1013 R ecom binant strain 6152 13140 6158 8166 B tk BE 20 3, S1824 2B tk BE 20 S1824,,, [10, 11 ], [3, 4 ], S1824 B tk BE 20, B tk BE 20, Sh ivakum ar [12 ],,,, DNA,,,, B t, B t,,,, ( B t),,, B t,, [13 ], [14 ] 10 3 gm g,, 10 5 g m g [15 ] B t,,, 10 6 gm g, 10 7 g m g, B rian [7 ] L uke [8 ],, 1 L ereclus D, L ecdet M M, R ih ier J, et al. M o lecular relationsh ip s among p lasm ids of B acillus thu ring iensis: Conserved sequence th rough 11 C rystalliferous strain. M GG, 1982, 186: 391 398 2 Carlton B C, Gonzdae J M. P lasm ids and D elta2endo toxin p roduction in different subspecies of B acillud s thu ring iensis. In: M o lecular B io logy of M icrobial D ifferentiation. W ash ington D C: Am erican Society fo r M icrobio lgy, 19851246 252 3,,,.., 1997, 13 (2): 82 85 4,,,. B t., 1997, 24 (5): 262 265 5 Sam brook J, F ritsch E F,M aniatis T. M o lecular C loning2a L abo rato rym anual. N ew Yo rk: Co ld Sp ring H arbo r L abo ra2 to ry P ress, 19891320 680 6 Kalm an Sue, K ristine L K, John L L, et a l1co loning of a novel crylc from a strain bacillus thu ring iensis subsp g alleriae. A pp l Enviro M icrobio, 1993, 56 (9): 1131 1372 7 B rian H B, Jack T T. T ransfo rm ation of B acillus cereus vegetative cells by electropo ration. A pp l Enviro M icrobio, 1989, 55 (6): 1649 1652

1 : S1824 2 B tk BE 20 37 8 L uke M, Gabrielle P, Ro land B. T ransfo rm ation of B acillus thu ring iensis vegetative cells by electropo ration1fem S M icrobio l L ett, 1989, 60: 273 278 9 L aemm li U K. C leavagae of structural p ro teins during the assem bly of the head of bacteriphage T 4. N ature, 1970, 227: 600 685 10 L ereclus D, A rantes S D, L ecadert M M. T ransfo rm ation and exp ression of a cloned delta2endo toxin gene in B acillus thu ring iensis. FEM S M icrobio l L ett, 1989, 60: 211 218 11 Jam es A B, Do lo resm C, PearceM G, et a l. N ovel cloning vecto r fo r B acillus thu ring iensis. A pp l Enviro M icro, 1990, 56 (11): 3420 3428 12 Sh ivakum ar A G, Gundling G J, Benson T A, et al. V egetative exp ression of 2endo toxin genes of B acillus thu ring iensis subsp ku rstak i in B acillus subtilis1j Bacterio l, 1986, 166 (1): 194 204 13 A likhanian S Z, R yabchenko N F, Bukanon N O, et al. T ransfo rm ation of B acillus thu ring iensis subsp g alleria p ro to2 p lasts by p lasm id PBC16. J Bacterio l, 1981, 146: 7 9 14 H eierson A L, L ovgroen A, D alhamm ar G, et a l. T ransfo rm ation of vegetative cells of B acillus thu ring iensis by p lasm id DNA. J Bacterio l, 1987, 169: 1147 1152 15 C raw fo rd I T, Greis K D, Streip s V İ Facile autop last generation and transfo rm ation in B acillus thu ring iensis subsp ku rstak i. J Bacterio l, 1987, 169: 5423 5428 ( ) 1998 1998 38, 1 ; 1998 15, 45%, 83%, ; 5 ; 8, 14 1998 (), ; 16 1998, : ; ; ; ; ; ; DH ; ; ; ; ; ; TR 15 ; ; ; ( )