花粉介导法获得玉米转基因植株_英文_

花粉介导法获得玉米转基因植株_英文_ () 植 物 学 报 2001 , 43 3:275 - 279 Acta B ota nica Si nica 花粉介导法获得玉米转基因植株 3毅 王景雪崔贵梅胡晶晶孙 ( )山西省农业生物技术研究中心 , 太原 030031 ( ) 利用花粉作为外源基因的载体进行遗传转化在玉米 Zea mays L . 上获得了成功 。以玉米自交系太 9101 、 摘要 : 综 31 等为受体 ,以 p GL ?- RC- 1 质粒为供体 ,在玉米开花期 ,用花粉与质粒 DNA 混合并附加超声波处理 ,然后辅以 人工授粉的方法将外源基因导入到受体中 。DNA 斑点杂交和 PCR 扩增以及 PCR- Southern blot 杂交检测结果证明 , 几丁质酶基因确已导入玉米自交系中 。所得结果表明 :玉米花粉可以介导外源基因的转化 。利用花粉作为载体介 导外源基因转化 , 避免了传统的基因枪法和土壤杆菌法转化所要求的组织培养技术 ,转化方法简单 ,易操作 , 具有 很强的实用性 。 关键词 : 玉米 ; 花粉介导法 ; 基因转化 () 文章编号 : 057727496 20010320275205 中图分类号 : Q943. 2 文献标识码 : A Transgenic Ma ize Plants Obta ined by Pollen- mediated Transf ormation 3 WANG J ing- Xue , SUN Yi , CUI Gui-Mei , HU J ing-J ing ( )Agricultural Biotechnology Center of S hanxi , Taiyuan 030031 , China ( )Abstract : The genetic transformation was achieved by pollen- mediated approach on maize Zea mays L . inbred lines Tai 9101 and Zong 31 . Plasmid DNA of p GL ?- RC- 1 was mixed with fresh pollen of maize in2 breds in sucrose solution. The pollens were treated by ultrasonication and collected , pollinated on silks of maize ears. Transformants were confirmed by dot blot hybridization , PCR amplification and PCR- Southern blot hybridization. The pollen- mediated transformation approach could circumvent the tedious tissue culture proce2 dures like in particle bombardment and A grobacterium infection , etc . This approach is simple , easy to oper2 ate , and could be widely used in practice . Key words : maize ; pollen mediated ; genetic transformation Maize is an important cereal crop . Genetically , ing inside plant tissues. Hence , attempts were made to transform the chitinase genes into tobacco and potato , transformed maize plants could be obtained by various ap2 1etc . , and highly resistant plants to Trichdermy harzianum proaches , such as ovary injection , ultrasonication treat2 7 2 3 ,4were obtained. ment , particle bombardment , A grobacterium infec2 5 6 Most of maize diseases are fungal diseases , such as , tion, and electroporation, etc . Plant tissue culture ( ) ( procedures are prerequisites for most of the approaches leaf blight Setosphaeria turcica, leaf spot Cochliobolus above except for the ovary injection. Calli or even proto2 ) ( smuts heterostrophus , Ustilago maydis and ) plasts as receiptors are needed in these approaches. Mu2 Sphacelotheca reiliana . Introducing the chitinase gene into maize inbred lines may result in selection of plant tations are often induced during the process of callus in2 duction and plant regeneration , resulting in causing the lines resistant to above diseases. The goals of this study transformed plants to be difficult to transplant from test are to develop fungal disease- resistant maize lines as well ( ) tube to field greenhousedue to easy death of plantlets as the pollen- mediated transformation method. and production of aberrant sterile plants. Significantly 1 Materials and Methods these approaches are limited in application. Chitinase hydrolyses chitin existing in hyphae of fun2 1 . 1 Materials gi , and prevents fungi from infecting plants and propagat2 Maize inbred lines , Tai 9101 and Zong 31 were Received : 2000206222 Accepted : 2000210217 () ( Supported by the National Plant Genetic Transfer Study and Industrialization Item J 99- A- 010; the Science Research Project of Shanxi Province Co- sponsored by ) () National Science Research Project , 971002; the Nature Science Foundation of Shanxi Province 961026; the Agricultural Science and Technology Program by () () Shanxi Finance Department 980006; Research Project for Returned Over- Sea Students 1997- 7. 3 Author for correspondence . kindly provided by Mr . SU Shu- Wen , Crop Genetics In2 ?for 2 h. DNA dot blot hybridization was carried using stitute , Shanxi Academy of Agricultural Sciences. Plas2 labeled chitinase gene fragment as probe at 68 ?, and the membrane was probed with NBT and X-phosphate . mid p GL ?- RC- 1 was kindly gifted by Prof . S. Muthukr2 1 . 2 . 4 PCR a mplif ication Two pairs of primers were ishnan , Biochemistry Department , Kansas State Universi2 designed based on the sequence of the chitinase gene and ty , USA. synthesized by Sangon Biotechnology Company of Shang2 1 . 2 Methods hai. The primers , numbered as 8341/ 8342 and 49040/ 1 . 2 . 1 Preparation of pla smid D NA The plasmid 49041 sequenced as follows. 8341 : 5′- GCGGTGGTGGC2 p GL ?- RC- 1 contains the chitinase gene and hygromycin ′;′- GGGCCTCTGGTTGTAGC-CATGGCGGT- 38342 : 5 gene for resistant- selection. The chitinase gene fragment AAT- 3′; 49040 : 5′- GCTCCACCTCCGATTACTGC- 3′; ( ) is 1 . 1 kb Fig. 1. The plasmid DNA was prepared with 8 49041 : 5′- GCGTTGCCGTTGTTCTCCTC- 3′. The frag2 alkaline lysisand purified with PCR Fragment Recovery ment sizes between the two pair primers were 923 bp and ( () ) Kit Ta KaRa Biotechnology DalianCo . , Ltd. 376 bp , respectively. The PCR conditions were as follow2 ing : program 1 : 94 ?, 5 min ; 94 ?, 0 . 5 min ; 50 ?, 0 . 5 min ; 72 ?, 1 . 5 min ; 30 cycles ; 72 ?, 10 min. Program 2 : 94 ?, 4 min ; 94 ?, 0 . 75 min ; 45 ?, 0 . 75 min ; 72 ?, 1 . 75 min ; 30 cycles ; 72 ?, 10 min. PCR amplification was completed using Ta KaDa TM TaqKit and PTC- 200 Thermal Cycler . 1 . 2 . 5 PCR- Southern blot hybridization Following 10 the method of Wang and Fang, the 376 bp fragment of the chitinase gene as probe was labeled with DIG- dUTP Fig. 1. Physical map of plasmid p GL ?- RC- 1. using PCR DIG Probe Synthesis Kit . PCR products of to2 ( ) 35S , CaMV 35S promoter ; A , poly A; Chit , chitinase coding tal DNA as temperate isolated from transformed plants region ; d , adaptor ; hph , hygromycine resistant gene . were fractionated by electrophoresis in 1 . 2 % agarose gel , Seeds of maize in2 1 . 2 . 2 Transf ormation method and transferred onto nylon membrane , hybridized with breds were sown in the early May , and plants flowered in ( (DIG labeled probe , detected with CSPD disodium 3- 4- the early and mid-J uly. Maize ears were bagged before ( ) methoxyspiro { 1 , 2- dioxetane- 3 , 2′- 5′- chloro tricyclo 3 ,7 silking. About 0 . 3 g of fresh pollens were collected in the ) ) decan}- 4- y1 phenyl phosphate flores2 3 . 3 . 1 . 1 μmorning , and mixed with about 10g of the plasmid DNA cence staining and exposed to X- ray film. ( in 20 mL of solution with 5 % sucrose The ratio of the 2 Experiment Results () ) plasmid DNA to the pollens W/ Wwas 1?30 000. The 2 . 1 The medium f or pollen mediated transf ormation solution was treated with ultrasonication before and after adding the plasmid DNA. By using J Y92- ?Ultrasonica2 2 . 1 . 1 Sucrose solution If pollens are mixed with tor from Ningbo Xinzi Scientific Instrument Institute the DNA solution directly , they will loss their vitality because parameters for sonication treatment were : sonic intensity they are swollen by water in solution. Therefore , the so2 of 300 W , treatments for 8 times each for 5 s and 10 s in2 lution with a suitable solute has to be selected. We pre2 terval . Then , the treated pollens were pollinated on pared the solution with certain amount of sucrose which clipped maize silks. could maintain normal turgidity of the pollens and facili2 tate them to be germinated. But the optimum sucrose con2 seeds were1 . 2 . 3 D NA dot blot hybridization T 0 centration varies with the pollens from different plant sown in experimental plots next spring. Young leaves were 11 species. collected from plants with 5 - 6 leaves. Total plant DNA 9 A test for selecting suitable sucrose concentration was extracted according to Pich and Schubert, and pu2 was conducted. Seeds from maize inbred lines were sown rified using the instruction of PCR Fragment Recovery in spring. Plants grew normally and silked and bloomed Kit . The plasmid DNA was digested with B am H ? and from middle to late J uly. At full blooming stage the fresh Hind ?, and the chitinase gene fragment of 1 . 1 kb was pollens were taken at around 10 : 00 AM and suspended recovered by electrophoresis on low melting point agarose . () into the solution p H 7 . 0with sucrose of 5 % , 10 % , The recovered DNA fragment was labeled with the DIG 15 % and 20 % , respectively. The pollens in the solution DNA Labeling and Detection Kit from Boehringer were stored at 4 ? for about 5 h and then , observed μMannheim. Teng total plant DNA grom each plant were under an optical microscope . The result was listed in dripped on nylon membrane , which was then baked at 80 ()277 3 期 王景雪等 : 花粉介导法获得玉米转基因植株 英 from 39 . 6 % to 50 . 8 %. We considered that certain de2 Table 1 . It could be seen from Table 1 that the sucrose gree of the sonic intensity was needed to facilitate the solution could , to certain extent , protect the pollens from damage due to water exchange between the pollens and plasmid DNA entering pollens , so 300 W was used in 2 present study. the solution. The solution with 5 % sucrose could best re The physical force produced by ultrasonication not serve the pollens , integrity. With the increase of sucrose concentration percentage of the damaged pollen grains was only can hurt the pollen grains , but also shear the plas2 increased. 68 . 4 % of the pollen grains were damaged mid DNA. We examined the DNA by electrophoresis after when the sucrose concentration was up to 20 %. There2 sonication treatment and found that the parts of the plas2 mid DNA was kept intact . fore , 5 % sucrose solution was used for pollen mediated transformation in the present study. 2 . 2 Identif ication of transf ormed plants Maize ears from inbred lines were pollinated as de2 Ta ble 1 Pollen grain changes in various concentrated sucrose solu2 ()tion var. E28 scribed in 1. 2 . 2. Thirty-four of the maize ears were No . of No . of 2 Percentreated and six ones had seeds. Totally , 32 seeds were pollen Percen2pollen Percen2 tage Sucrose No . obtained and sown in experimental plots. Young leaves grains tage grains tage of concen2 pollen with of with of damaged tration were taken when maize plants had 5 - 6 leaves. Total grains ( )plasmo2 total broken total pollen % DNA was extracted from the leaves and used for molecular lysis wall grains 5 82 2 2 . 4 0 0 2 . 4 assay. 10 59 20 33 . 9 7 11 45. 8 2 . 2 . 1 D NA dot blot hybridization The total DNA 15 47 15 37 . 9 8 17 . 0 54. 9 from 13 transformed Tplants and non- transformed ones as 1 20 57 6 10 . 5 33 57 . 9 68. 4 0 Part More About 95 controls were assayed by dot blot hybridization , 6 out of the 13 plants were positive , and the controls were nega2 ? 2 . 1 . 2 Ultra sonication treatment Xu et al suc2tive . ceeded in introducing foreign fluorescent molecules and 2 . 2 . 2 PCR a mplif ication analysis The primers genes into mammal cells by ultrasonication treatment . 8341/ 8342 was used for PCR amplification of the positive They considered that transient vacuolization induced by ( plants Tai 9101- 3 , Tai 9101- 11 , Tai 9101- 18 , Zong ultrasonication was an important mechanism for foreign ) 31- 4 , Zong 31- 2 and Zong 31- 1identified in DNA dot ( ) molecules including DNAto enter cells. In present blot hybridization in the condition of program 1 . The re2 study , we introduced the plasmid DNA into the pollen sults were shown in Fig. 2 . The primers 49040/ 49041 was grains with transiently released high energy and vacuoliza2 tion by ultrasonication. The pollens could , then , be used for transformation by pollination. Because the intensity of sonication may affect the vitality of pollens , we studied the extent of the damaged maize pollens with various sonic intensities. The results were listed in Table 2 . ( Ta ble 2 Pollen grain changes after ultrasonication treatment var. )Huangzaosi No . of No . of 2 2 SonicNo . of Percenpollen Percen2 pollen Percen2 tage of Sucrose ation treat2 grains tage grains concen2 stre2 ed tage damaged with of with tration ngth pollen of total pollen plasm2 total broken ( ) Wgrains grains olysis wall 5 % 200 101 32 31 . 7 8 7 . 9 39. 6 Fig. 2. PCR analysis of total DNA from transformed Tplants and 1 300 52 20 38 . 5 5 9 . 6 48. 1 () CK 1. 2 % agarose gel electrophoresis. 400 71 30 42 . 3 6 8 . 5 50. 8 ( ) M , molecular marker ; Lane 1 , Zong 31 plant CK; Lanes 2 - 4 , 0 43 13 30 . 2 3 7 . 0 37. 2 transformed plants : 2 , Zong 31- 2 , 3 , Zong 31- 4 , 4 , Zong 31- 1 ; All of the sonication treatments were 8 time treatments with 5 s working time ( ) Lane 5 , Tai 9101 CK; Lanes 6 - 8 , Tai 9101 transformed and 10 s working interval . plants: 6 , Tai 9101- 11 , 7 , Tai 9101- 18 , 8 , Tai 9101- 3 ; 9 , It is indicated from Table 2 that ultrasonication treat2 p GL ?- RC- 1 plasmid. ment could cause some of the pollen grains to be dam2 ) () ( ) ( ) (? Xu N 许宁,Zou X邹翔,Chen B- Q 陈伯权, Zhao N- M 赵南明. aged. The damaged extent was related to sonication inten2 A possible theoritical model for ultrasonication induced transformation cell sity. As the intensity was increased from 200 W to 400 membrane perforation caused by“vacuolation effect ”. Proceedings of the 5th ()national symposium on Bio- Membrane . Haikou ,China ,1993. 178. in Chinese W , the percentage of the damaged pollen grains increased used for PCR of Tplants in the condition of program 2 .seeds could germinate , generate normal roots and shoots 2 under the selection pressure , but the control seeds were Nineteen out of 24 Tplants examined were positive and 2 germinated slowly , and the root development was obvious2 the results were shown in Fig. 3 . ( ) ly inhibited , and the plants died at last Fig. 5 . . Fig. 5. Germination of seeds from transformed and CK maize plants in the solution with 15 mg/ L hygromycin. ( ) Left , Tai 9101 CK; Right , Tai 9101- 3. Fig. 3. PCR analysis of total DNA from transformed Tplants and 2 () CK 1. 2 % agarose gel electrophoresis. 3 Discussion M , molecular marker ; Lanes 1 - 3 , descendant of Tai 9101 trans2 formed plants , Tai 9101- 3- 5 , Tai 9101- 18- 6 , Tai 9101- 3- 4 , re2 It was reported that generative cells were used as 12 ] ( ) spectively ; Lane 4 , Tai 9101 CK; Lane 5 , p GL ?- RC- 1 plas2 vectors for genetic transformation in animals. There mid. has not been any similar successful results done in plants till now , though some attempts were made by Sanford et 2 . 2 . 3 PCR- Southern blot hybridization The above 13 ] 14 ] 15 ] 16 ] al , Ohta , Booy et al , and Li et al . We detected positive Tplants were assayed again with PCR- 2 have here reported an easy- applicating genetic transforma2 Southern blot hybridization. Eleven of the 15 tested plants tion approach using plant pollen as a vector . ( ) were positive Fig. 4. The results implied that the chiti2 15 ] Booy et al reported that the digestion of plasmid nase gene was introduced into maize inbred lines. DNA by nuclease was a major hurdle for using pollen as a genetic transformation vector . They attempted to wash off nuclease or to inhibit the activity of the enzyme with EDTA. They found that the former could only eliminate partial enzyme and the latter inhibited the germination of pollens completely. We conceived that the ultrasonication treatment at certain intensity before adding plasmid DNA might destroy the activity of nuclease , meanwhile , not severely harm the germination activity of the pollens. We adopted the ultrasonication treatment at the intensity of 300 W before adding plasmid DNA to destroy the nuclease on the pollen surface . 17 ] Dong et al obtained transformants of maize and Fig. 4. PCR- Southern blot hybridization of total DNA from trans2 millet with the frequencies of 0 . 05 % - 0 . 21 % and formed Tplants and CK. 2 0 . 18 % , respectively by pollinating with bombarded pol2 ( ) 1 , plasmid ; 2 , Zong 31 CK; 3 , Zong 31- 1- 3 ; 4 , Zong 31- 3- 8 ; 5 , Zong 31- 1- 6 ; 6 , Zong 31- 4- 2 ; 7 , Zong 31- 1- 2 ; 8 , Zong 31- 4- lens. We assumed that it was difficult for the bombarded ( ) 2 ; 9 , Tai 9101 CK; 10 , Tai 9101- 3- 5. pollens to compete with the normal pollens due to the pol2 lens wounded by hitting of microparticles. Therefore , the 2 . 2 . 4 Seedlings resistant to hygromycin The seeds transformation frequency would be low even though some from transgenic and non- transgenic maize plants were ger2 transformants could be got . Further more , the pollen () (minated in 25 mg/ L for Zong 31or 15 mg/ L for Tai might be splashed up by bombardment . In our approach , ) 9101hygromycin solution in dark. The transgenic plant ()279 3 期 王景雪等 : 花粉介导法获得玉米转基因植株 英 plants expressing an insecticidal protein derived from B acil2 the transformed and untransformed pollens have the vitali2 lus thuringiensis . Bio/ Technology , 1993 , 11 :194 - 200. ty at the same level , thus , the transformation frequency Ishide Y , Saito H , Ohta S , Hiei Y , Kmari T , Kumashiro 5 ] would be relatively higher . ( ) T. High efficiency transformation of maize Zea mays L . Using pollen- mediated transformation approach , we mediated by Agrobacterium tumef aciens . N at Biotechn , could avoid tedious tissue culture procedures adopted in 1996 , 14 :745 - 749. A grobacterium and bombardment approaches. Thus , it is D’ Halluin K , Bonne E , Bossut M , Beuckeleer D M , Lee2 6 ] easy to apply this approch in practice and ready to inte2 mans J . Transgenic maize plants by tissue electroporation. grate it into conventional breeding program. Facilities Plant Cell , 1992 , 4 :1495 - 1505. Lorito M , Woo S L , Fernandez I G , Colucci G , Harman G 7 ] used for this approach is relatively simple and cost less. E , Pintor- Toro J A , Filippone E , Muccifora S , Lawrence C The study on enhancing seed setting of the transfor2 B , Zoina A , Tuzun S , Scala F. Genes from mycoparasitic mants and their progenies resistant to maize head smut fungi as a source for improving plant resistance to fungi ( ) ( Sphacelotheca reilianaand leaf spot Cochliobolus het2 pathogens. Proc N atl Acad Sci USA , 1998 , 95 : 7860 - ) erostrophus are undergoing. 7865. Sambrook J , Fritsch E F , Maniatis T. Molecular Cloning : 8 ] Ackno wledgements : We deeply appreciate Prof . S. A Laboratory Manual . 2nd ed. New York : Cold Spring Muthukrishnan working in Biochemistry Department , Harbor Laboratory Press , 1989. ( ) ( ) ( Fu R- Z 傅 荣 昭 , Sun Y- R 孙 勇 如 , Jia S- R 贾 士9 ] Kansas State University , and Prof . SU Shu- Wen in the ) 荣. Technical Manual for Plant Genetic Transformation. Crop s Genetics Institute , Shanxi Academy of Agricultural ( Beijing: China Science and Technology Press , 1994. in Sciences , for providing the chitinase gene construct , )Chinese p GL ?- RC- 1 ; and maize inbred lines , respectively. ( ) ( ) Wang G-L 王关林, Fang H- Y方宏筠. Principle and 10 ] Technology for Plant Gene Engineering. Beijing : Science References : ()and Technology Press , 1998. in Chinese () Hu S- Y 胡适宜. Experimental methods in plant embryol2 11 ] ( ) ( ) ( 1 ] Ding Q- X丁 群 星, Xie Y-J 谢 友 菊, Dai J- R 戴 景 ) ( ogy ?determination of pollen viability. Chin B ull B ot ) ( ) ( ) ( 瑞, Mi J-J 米景九, Li T- Y李太元, Tian Y- C 田 () () ()植物学通报, 1993 , 10 2:60 - 62. in Chinese ) () () 颖川, Qiao L- Y乔利亚, Mang K- Q 莽克强, Liu B- ( ) ( ) Liu H-L 刘 红 林 , Chen Y- F 陈 宜 峰 . Research 12 ] () () () L 刘宝兰, Wang Y王音, Feng P- Z冯平章. Study progress on foreign DNA transferring mediated by sperm. on introducing Bt toxin gene into maize with ovary injection. () () Bio- Engineering Progress 生物进展, 1997 , 17 2: ( ) () Sci China Ser B中国科学 〃B 辑, 1993 , 23 : 707 - ()27 - 37. in Chinese ()713. in Chinese 13 ] Sanford J C , Skubik K A , Reisch B I. Attempted pollen- Zhang H , Xie Y-J , Dai J- R , Xu N , Zhao N- M. Gene 2 ] mediated plant transformation employing genomic donor transfer into maize by ultrasonication. Agricultural biotech2 Theor Appl Genet , 1985 , 69 :571 - 574. DNA. nology. YOU C B , CHEN Z L . Proceedings of Asia- Pacific Ohta Y. High- efficiency genetic transformation of maize by a 14 ] Conference on Agricultural Biotechnology. Beijing : China mixture of pollen and exogenons DNA. Proc N atl Acad Sci Science and Technology Press , 1992. 311 - 312. USA , 1986 , 83 :715 - 719. ( ) ( ) ( 3 ] Wang G- Y王 国 英 , Du T-B 杜 天 兵 , Zhang H 张 15 ] Booy G , Krens F A , Huizing H J . Attempted pollen- medi2 ) () () (宏, Xie Y-J 谢友菊, Dai J- R 戴景瑞, Mi J-J 米景 ated transformation of maize . J Plant Physiol , 1989 , 135 : ) ( ) ( ) 九, Li T- Y李 太 源 , Tian Y- C 田 颖 川 , Qiao L- Y 319 - 324. () ( ) 乔利 亚 , Mang K- Q 莽 克 强 . Transferring Bt toxin Li Y H , Tremblay F M , Seguin A. Transient transformation 16 ] gene into maize with bombardment and plant regeneration of ( ) ( ) transformations. Sci China Ser B中 国 科 学 〃B 辑 , ( of pollen and embryogenic tissues of white spruce Picea ()1995 , 25 :71 - 76. in Chinese ( ) ) glauca Moench. Voss resulting from microprojectile Koziel M G , Beland G L , Bowman C , Carozzi N B , Cren2 bombardment . Plant Cell Rep , 1994 , 13 :661 - 665. 4 ] shaw R , Crossland L , Dawson J , Desai N , Hill M , Dad2 () () (Dong Y- Z董云洲, Duan S-J 段胜军, Zhao L- Y赵连 17 ] well S , Launis K , Lewis K , Maddox D , Mcpherson K , ) () () 元, Yang X- H杨秀海, Jia S- R 贾士荣. Production Meghji M R , Merlin E , Rhodes R , Warren G W , Wright of transgenic millet and maize plants by particle bombard2 M , Evola S V. Field performance of elite transgenic maize () () ment . Sci Agric Sin 中国农业科学, 1999 , 32 2: 9 - ()13. in Chinese () 责任编辑 : 谢 巍