Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice  

基因枪法和农杆菌介导法对水稻转基因拷贝数和基因重排概率的影响(英文)

作　　者：程在全 黄兴奇 Ray WU 

机构地区：[1]Department of Molecular Biology and Genetics,Cornell University

出　　处：《Acta Botanica Sinica》2001年第8期826-833,共8页Acta Botanica Sinica（植物学报：英文版）

基　　金：SupportedbygrantsfromtheRockefellerFoundation (RF98_0 0 1_60 6)andtheUnitedStatesDepartmentofAgriculture (USDA) ( 95_3730 0_1 5 80 ) . Presentaddress :MolecularBiologyLaboratory ;BiotechnologyResearchInstituteofYunnanProvince ;Kunming 65 0 2 2 3;Chi

摘　　要：Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangemen基因枪法和农杆菌介导法转化的外源DNA整合到植物染色体上是随机进行的 ,因此 ,它们可能会产生不同的转基因拷贝数 ,得到不同的基因表达盒完整率。这反过来会影响基因的表达。为证实这一假说 ,作者首先将同一质粒pAc1PG_CAM分别用基因枪法和农杆菌介导法转化到水稻 (OryzasativaL .cv .TNG6 7)愈伤组织。为了揭示不同质粒是否也出现类似结果 ,也用农杆菌介导法、基因枪法分别将pTOK2 33和pJPM44导入水稻愈伤组织 ,并获得一批转基因植株。R0代植株转基因表达的分析用GUS组织化学染色法。用质粒上的单切点酶酶切基因组DNA后的Southern杂交结果确定转基因拷贝数。总DNA用双切点酶 (分别位于表达盒两侧 )酶切后的Southern杂交结果确定了完整转基因表达盒数目。结果表明 ,农杆菌介导转化法的转基因植株的转基因拷贝数相对少一些 (平均为2 .1和 2 .3) ,而基因枪法转化产生的转基因植株的转基因拷贝数相对多一些 (平均为 4.2和 5.6 )。并且农杆菌介导转化法的转基因植株的基因表达盒DNA重排概率低于由基因枪法转化产生的转基因植株的基因表达盒DNA重排概率———农杆菌介导转化法的DNA重排概率为 0 0 7和 0 1 0 6 ;由基因枪法转化的DNA重排概率为 0 57和 0 6 6。研究还分析了基因表达情况与转基因的拷贝数?

关 键 词：DNA rearrangement gene expression cassette Agrobacterium  _mediated method biolistic method rice (  Oryza sativa  ) 

分 类 号：Q943.2[生物学—植物学] S511.03[农业科学—作物学]