[Cotransformation of rice by bar and cecropin B gene expression cassettes lacking vector backbone sequences].
Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). The undesirable vector backbone sequences may not only promote transgene rearrangements and affect transgene or endogenous gene expression negatively, but have disadvantage on the safe assessment of the transformants as "desert DNA". The direct DNA transforming systems can transfer minimal gene expression cassettes (promoter, open reading frame, terminator) into plant genome and generate "safer" transformants, also it can delivery multiple genes of agronomic relevance to economically-important crop plants. But there is seldom researching reports on the topic till now. The present paper studied some factors that affecting the transforming efficiency of liner gene expression cassettes to rice varieties by particle bombardment, and the integration patterns of the gene expression cassettes in rice genome were compared with that of the whole plasmids. The results showed: (1) The transforming frequency of gene expression cassettes to rice via particle bombardment is 0.1%-0.5%, the cotransforming frequency of non-selectable gene is about 50%-60% when two separate gene expression cassettes were used for transformation. Increasing the DNA mole content can increase the transforming frequency and the beside sequences of gene constructs may play an important role on the variation of transforming efficiency between different rice varieties. (2) It's reported that the selectable and non-selectable transgene expression cassettes generated low-copy-number transgenic plants with simple integration patterns. While our results showed that the non-selectable cecropin B gene cassette generated simple integration patterns with 1-3 copies in the rice genome, but the selectable bar gene cassette which got 4-14 copies had much more complex integration patterns than that of the whole plasmids which got 1-3 copies only. As the bar gene is promoted by the CaMV35 promoter, in which there is a 19 bp palindromic sequence could act as recombination hot spot and lead to DNA rearrangement, we presumed that the transgene recombination events happened during the integration course have generated the complex Southern patterns of bar gene expression cassette. The recombination character, the heredity behavior and the expression law of gene expression cassettes in the rice genomes will be reported in our future papers.
Zhao
Yan
Y
College of Life Science, Zhejiang University, Hangzhou 310029, China.
Yu
Yan-Chun
YC
Qian
Qian
Q
Yan
Mei-Xian
MX
Huang
Da-Nian
DN
D000123:Acetyltransferases / Q000235:genetics*; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant; D019476:Insect Proteins / Q000235:genetics*; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
D019476:Insect Proteins; C031162:cecropin B protein, Insecta; D004247:DNA; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase