| Both bar and ceropinB gene expression cassettes (GEC, including promoter, opening reading frame and terminator)without plasmid vector backbone sequences and the whole plasmids pCBl and pCB4 carrying bar and ceropinB genes were introduced to the two Japonica rice varieties Xiushui04 and Jia59 using bar as selectable marker gene. The factors affecting transformation frequency were studied, the integration character, the hereditary behavior and the expressive law of exotic genes were studied and compared between the GECs and whole plasmids. Moreover, the hereditary and expressive behavior of exotic genes in crossbreeding transfer were analyzed. The esults showed:1 The transforming frequency of GEC to rice via particle bombardment is 0.1 ~ 0.5% and the co-transforming frequency of non-selectable gene is about 50~60% when two separate GECs used for transformation. The non-selectable cecropinB GEC generated simple integration patterns with 1 ~3 copies in rice genome, but the selectable bar GEC which got 4~14 copies had much more complex integration patterns than that of the whole plasmids which got 1~3 copies only.2 The liner DNA of bar GEC is prone to be recombined and truncated when integrate into rice genome delivered by particle bombardment, bar GECs and its fragments were linked with each other head-to-head, head-to-tail or tail to tail in rice genome. Multiple copies of bar GEC generated 1-3 "transgene array" and integrated in a larger near area of rice chromosome. The CaMV35S promoter in the bar GEC which carrying similar sequence to DNA recombination hot spots may play a important role on the recombination of bar GECs.3 The segregation and mutual expressive interference of multiple bar GEC copies in rice genome made the segregation ratios of Basta resistance in GEC DNA transformed plant lines more complex than that of the whole plasmid DNAtransformed plant lines, but about 30% transgenic plant lines carrying GECs can get homozygotes in TI generation.4 The selectable marker bar gene and the non-selectable cecropinB gene are link-inherited at low frequency in TI generation of more than fifty percent (4/7) co-transformed rice plant lines when two separated GECs used to co-transform. It's easy to remove the marker bar gene in transgenic plants because the Basta resistance is very easy to test. According to the co-transformation and segregation strategy, two marker free transgenic plants carrying cecropinB GEC were gained through PCR selection on the Basta sensitive plants in TI generation. These two marker-free plants are really safe transgenic plants without any dirty DNA but cecropinB GEC. Therefore, it provide a effective new way to generate safe transgenic plant by removing marker genes through hereditary segregation after co-transformation of separate GECs via particle bombardment. This is the first originating point of the present study.5 The expression level of cecropinB is mainly influenced by position effect and has no direct corresponding relationship with mat GEC or plasmid DNA used for transformatin, Actin or rbcS as promoter and diploid or tetraploid the transgenic plant be. There's no remarkable corresponding relationship between the resistance to rice blight leaf disease and the mRNA transcript level in transgenic plants too.6 The hereditary behavior of exotic single-copy bar gene in the transgenic tetrapoid rice mutant XIP-1 is complex, but mainly segregated according to 15:1 ratio. New Southern hybrid bands of bar and cecropinB gene were produced in the progeny of tetraploid plant line, which completely different to the TO generation and maybe caused by the chromosome breakage and recombination in the course of meiosis that involved in the transgene loci. This is the first time to report the transgene hereditary behavior in the rice tetraploid mutant plant and make the second originating point of the present study.7 There are low seed germinating frequency and albino variation characters among the transgenic plants carrying bar GECs, which m... |
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