Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 5, Cd 2+ : Cd 2+ ΛCd 2+
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- Ἰωακείμ Διαμαντόπουλος
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1 ISSN CN gQ 16 5 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 5, ΑΑ-cD NA Cd 2+ 3 (,, ), Β, Α Α Β, ΑΑ PCR ΑΑ2cDNA A T G AA CA, CaM V 35S ΑΑ2cDNA pgptv d35s2 ΑΑ, (ba r) N C89 Sou thern W estern ΑΑ2cDNA N o rthern ΑΑ2cDNA Cd 2+,, Cd 2+, Cd 2+ Cd 2+ ΑΑ2cDNA Cd 2+ : 200 m o lgl CdC l2, 400 m o lgcdc l2 100 m o lgl CdC l2 ΑΑ, cdna, Cd 2+, Q 78 Expression of the M ouse M eta lloth ione in M utan t ΑΑ-cD NA Im prov ing Cadm ium Resistance in Tran sgen ic Tobacco ZHAN G X iao2yu, ZUO X iao2feng, X IAO Chuan2ying, SHAN L ong, RU B ing2gen 3 3 Tel: (010) , E2m ail: Rulab@pku. edu. cn,, , (T he N ational L aboratory of P rotein E ng ineering and P lant Genetic E ng ineering, : , : Colleg e of L if e S ciences, P ek ing U niversity, B eij ing , Ch ina) Abstract M etallo th ionein is a m etal b inding p ro tein w ith sm all m o lecu lar w eigh t, w h ich has Β and Α tw o dom ain ṡ M ou se M etallo th ionein m u tan t ΑΑ has been con structed by rep lacing the Β dom ain of the m etallo th ionein w ith the o ther Α dom ain. Som e nucleo tides beside A T G w ere changed to AA CAA T G by PCR m ethod, w h ich w ere favo red in p lan tṡ CaM V 35S235SgΑΑcDNA exp ression vecto r pgptv d35s2ααw as con structed w h ich had herb icide (PPT ) 2resistance gene ba r as a selectab le m arkeṙ T he ch im eric gene via A grobacterium m ediated tran sfo rm ation w as in troduced in to the tobacco (N icotiana tabacum L. c. v. N C89). A cco rding to Sou thern b lo t and W estern b lo t analysis of som e putative tran sfo rm an ts, it w as found that the in troduced ΑΑcDNA w as in tegrated in to tobacco genom e and cou ld be exp ressed. N o rthern b lo t analysis indicated that the tran scrip ts of ΑΑ cdna in roo ts w ere stronger than these in leaveṡ D eterm ination of Cd 2+ concen tration and its distribu tion in side the p lan ts dem on strated that the tran sgen ic tobacco cou ld accum u late m o re Cd 2+ in its roo ts than the con tro l,w h ich cou ld reduce the con ten t of Cd 2+ in the leaveṡ T he experim en t of the resistance to Cd 2+ p roved that the exp ression of ΑΑcDNA in tobacco enhanced the p lan t to lerance tow ards Cd 2+. U nder 200 m o lgl CdC l2, p lan ts w ith no rm al grow th w ere ob served. T ran sgen ic tobacco cou ld even live in the m edium con tain ing 400 m o lgl CdC l2,
2 w h ile 100 m o lgl CdC l2 becam e tox ic du ring 30 days grow th period. Key words M etallo th ionein m u tan t ΑΑ, cdna, Cd 2+, R esistance (Cd 2+ ) : ΑΑ2cDNA Cd 2+, Cd 2+ Cd 2+, Cd 2+,,, Cd 2+ Cd 2+ [ 1 1, ] Cd 2+, Cd 2+, M T ΑΑ2 pu C18, LBA 4404, helper pr K2013, 1987, D. D. L efebvre [ 2 ] 35S (M T ), pb I426, pgptv 2BA R Cd 2+ M T M T, E cor I H indg X ba g Cd 2+ G. J. W agner [ 3 ], M T PCR D IG (digox igen in) D IG Cd 2+, (Boeh ringer M annheim ), RNA Q IA GEN (M etallo th ionein, M T ) 112 M T PCR Α Β, Α Cd 2+ ΑΑ2cDNA pu C18, H g 2+, Β Cu 2+ [ 4 ] 35S ΑΑ2cDNA, M T 2cDNA (ba r) Β Α,, [7 ] M T ΑΑ ΑΑ2cDNA ΑΑ M T E. coli,, Cd 2+ m ggl (ba r) 200 m o lgl CdC l2 M S M T ΑΑ Sou thern SD S [ 8 ] Cd 2+, DNA, 15 g DNA ΑΑ2cDNA 112%,, Cd 2+ Sou thern, PCR digox igen in (D IG), ;, D IG CD P2Star TM Cd 2+,, Cd 2+, 111, Cd 2+, cdna pbsαα(pb luescrip tks + ΑΑ), S acg P rom ega, N C89, 6 Cd 2+ M T, N o rthern 200 m o lgl CdC l2 M T Cu 2+ Fe 2+, 100 m g, [ 5 ] Q IA GEN (Germ any) RNA, Phytochelatin ( PC s ), RNA D IG [ 6 ] M arker g PCR D IG M T cdna, D IG CD P2Star TM
![1987, D. D. L efebvre [ 2 ] 35S (M T ), pb I426, pgptv 2BA R Cd 2+ M T M T, 11112 E cor I H indg X ba g Cd 2+ G. J.](/docs-images/45/23275586/images/page_2.jpg "W agner [ 3 ], M T PCR D IG (digox igen in) D IG Cd 2+, (Boeh ringer M annheim ), RNA Q IA GEN (M etallo th ionein, M T ) 112 M T 11211 PCR Α Β, Α Cd 2+ ΑΑ2cDNA pu C18, H g 2+, Β Cu 2+ [ 4 ] 35S")
3 5 : ΑΑ2cDNA Cd W estern Cd 100 m o lgl CdC l2,, rg 4 N C89 ΑΑ m in, 18 % SD S2PA GE ΑΑ M T 80,,, 100 m g 500, 1 m o lgl HC l [ 9 N C, ] 5 m l, Cd, M T, 3 ΑΑcDNA M T cdna (99 bp ), M T M T 2 ΑΑ2cDNA 211 (F ig. 1) F ig. 1 Sketch m ap of p lant exp ression vecto r of mouse M T m utant gene ΑΑ2cDNA F ig. 1, p GPTV d35s2αα CaM V 35S, m RNA RNA, ΑΑcDNA AM V 5 2 N o s F ig. 3 ba r ( ) LBA 4404, 5 E cor g E cor g, E cor g 500 bp 212 Southern Cd 2+, PCR DNA, 15 g, Sou thern, PCR D IG M T cdna M T M T cdna ΑΑcDNA 100 bp, ΑΑ cdna, F ig. 2 : E cor g ( 1 4 ) 500 bp X ba g gs acg 215 bp ( 2 ) X bag H indg DNA ( 5 7 ) : 213 Northern F ig. 3 RNA F ig. 2 Southern blo t analysis of transgenic p lants w ith the p robe of M T cdna 1, 2, 3: T ransfo rm ant N o. 1 digested w ith E cor g (1), X ba g gs ac g ( 2 ) and H ind g ( 3 ) ; 4: T ransfo rm ant N o. 2 digested w ith E cor g ; 5, 7: T ransfo rm ant N o. 3 digested w ith X bag (5) o r H indg (7) ; 6: N on2transfo rm ant contro l; 8: Po sitive contro l of M T cdna (206 bp) 112 RNA 214 W estern 10 g, 112%, Sou thern, F ig. 3
4 634 16, M T ΑΑ M T, Cd 2+,,, Cd 2+ SD S 6 500, E. coli ΑΑ 6 583, : M T SD S ( ΑΑ, ΑΑ2cDNA ) M T Cd 2+ 47% 62% M T M T cdna, Cd 2+ M T, 47% 6012% F ig. 5 F ig. 6 F ig. 4 : 7 M T, 6 1, 5, 8 F ig. 5 Comparative Cd 2+ tobacco and contro l seedlings content analysis of transgenic F ig. 3 N o rthern blo t analysis of transgenic p lant ( to tal RNA iso lated from two transgenic p lants) 1: RNA m arker; 2, 5: F rom leaves; 3, 4: F rom roo ts F ig. 6 T issue Cd 2+ tobacco seedlings distribution in transgenic and contro l F ig. 4 W estern blo t analysis of transgenic p lant 1, 2, 4, 5, 6: Po sitive transgenic tobacco samp les; 3: N egative transgenic tobacco samp le; 7: Rabbit2liver M T p ro tein; 8: Contro l of N C Cd 2+, Cd 2+, 75 m o lgl CdC l2, CdC l2 100 m o lgl N C89 : 200 m o lgl CdC l2,,,, CdC l2 300 m o lgl,, CdC l2 400 m o lgl, 215 Cd, Bo rne [ 10 ], M T 300 m o lgl CdC l2 Cd 2+ [ 11, ]
5 5 : ΑΑ2cDNA Cd m o lgl CdC l2, ΑΑ, M T ΑΑ 6 8 F ig. 7 M T N C89,, Cd 2+, 35S ΑΑcDNA 35S,, Cd 2+, 400 m o lgl CdC l2, 300 m o lgl CdC l2 ΑΑ, Cd 2+ Cd 2+ ΑΑcDNA,, ΑΑ F ig. 7 Grow th of transgenic and contro l tobacco seedlings in m edium w ith 300 mo lgl CdC l2 (A )Contro l seedlings of N C89; (B) Tobacco transfo rm ed w ith ΑΑ2cDNA : (References) 3 ΑΑ bar 35S AM V, Sou thern W estern, N o rthern, 100 m o lgl CdC l2, ΑΑ 6%, Cd 2+ 62%, 47% Cd 2+ M T 419%, Cd 2+ 7,,,, 6012% Cd 2+ Cd 2+, ΑΑ M T Cd 2+ Cd % 1611% ; Cd 2+ Cd 2+ 24% 1 Kubo i T, N ohuch i A, Yazak i J. Fam ily2dependent cadm ium accum ulation characteristics in higher p lants. P lant S oil, 1986, 92: L efebvre D D, M ik i B L, L aliberete J F. M amm alian m etallo thionein function in p lants. B iogt echnology, 1987, 5: 3 Yeargan R, M aiti I B, N ielsen M T, H unt A G, W agner G J. T issue partitioning of cadm ium in transgenic tobacco seedlings and field grow n p lants exp ressing the mouse m etallo th ionein I gene. T ransg enic R es, 1992, 1: V allee B L. Introduction on m etallo th ionein. M ethod s E nzym ol, 1991, 205: V liet C V, A ndersen C R, Cobbett C S. Copper2sensitive m utant of A rabid op is thaliana. P lant p hy siol, 1995, 109: Grill E,W innacker E L, Zenk M H. Phytoch ilatins: the p rincipal heavy2m etal comp lexing pep tides of h igh p lants. S cience, 1985, 230: (Zhang X iao2yu, L iw ei, Zhang J ing, H e D u2xiu, Ru B ing2gen. Construction of high efficient exp ression vecto r of m etallo thinoein m utant in p lant and its exp ression in tobacco. Ch in J B iochem M ol B iol), 1999, 15 (6) : ,,,, M T ΑΑ ( ) (Shi Su2hua, Zhang qun,
6 Chen Yue2qin, Tang Shao2qing, Q u L ian2hu. A simp le m ethod fo r iso lation of to tal RNA and DNA from silicagel2dried and fresh leaves of p lants. A cta S ci N atu r U niv S uny atseni), 1996, 35 (2) : Sam brook J, F ritsch E F, M aniatis T. M olecu lar C loning: A partitioning in transgenic tobacco p lants exp ressing a m amm alian m etallo th ionein gene. M ol B reed ing, 1998, 4: ,,, mm T2g Cd 2+. (M o hua, Feng Ya2jun, Xue yu, L aboratory M anul, 2nd ed. N ew L abo rato ry P ress, 1989 Yo rk: Co ld Sp ring H arbo r Ru B ing2gen. The effect of the mm T2g transgenic tobacco on Cd 2+ to lerence and its cyto logical study. A cta B otan S in), 1996, 10 Bo rne F D, E lm ayan T, Ro ton C, H ys L, T epfer M. Cadm ium 38: ,, 1985,, 1998 (, ), 70%, (CA ), , CA, , 18 IS I (SC I Expanded) ( ),, (160, 16 ), , ( ), : , : BM 957 : , 338 : (010) : shxb@m ail. b jm u. edu. cn : : :
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