| Plant transformation technology is one of the most important method applied on both basic science research and modern genetic breeding. Different systems have been developed for the generation of transgenic plants, and have been extensively used in a great deal of plant systems. Among various transformation technologies Agrobacterium-mediated gene transformation is the most popular one. But the genotype-dependency of Agrobacterium-mediated transformation makes it hard to employ this gene transfer method successfully on many elite varieties of particular species.As a model monocotyledon, rice plays a critical role in basic science and world food security. Also it has been the first Monocot species to be successfully transformed with Agrobacterium. However, after many years of extensive efforts to improve the transformation frequency, most indica rice varieties still remain highly recalcitrant to Agrobacterium-mediated transformation, which greatly hamper the function research on indica rice-specific genes and transgenic genetic improvement of indica cultivars. It is believed that it's hard to conquer this recalcitrance just by improving tissue culture condition, and the key to further improve transformation efficiency lies in genetic manipulation of the plant itself. So, it is important for us to know the infection mechanism and the response of plant cell to Agrobacterium infection.In this study, differences in susceptibility of different japonica and indica cultivars to Agrobacterium-mediated transformation were investigated. Using microarray, differences in global gene expression between representative cultivars of japonica and indica variety in the process of Agrobacterium-mediated transformation were monitored, so as to identify differentially expressed genes between the two subspecies. The main results in this study are as follows:1. The differences in transformation efficiency among different japonica rice cultivars result from the differences in T-DNA integration efficiency. Indica rice variety has a lower efficiency in each step of the transformation process compared with japonica rice, resulting in a lower efficiency in both transient and stable infection. But T-DNA integration other than T-DNA transfer and nuclear targeting remains to be the rate-limiting step in Agrobacterium-mediated transformation of indica rice.2. The mature embryo-derived embryogenic calli of two representative cultivars of japonica and indica variety, Nipponbare (Nip) and Zhenshan97(ZS), were infected by Agrobctcterium. In order to analyse the global gene expression patterns across the transformation process, calli of Nip and ZS were sampled just before infection (Oh) and1,6,12and24h after infection, respectively. According to the results of the genomic DNA hybridization, it is likely that the differences in the susceptibility of Nip and ZS to infection by Agrobacterium result mainly from their expressional differences upon Agrobacterium infection rather than the genomic or sequence differences between these two varieties.3. There are more differentially regulated transcripts in ZS calli after Agrobacterium infection, compared to that in Nip, though more transcripts from Nip were detected at RNA level and more transcripts expressed only in Nip. Only3,892(35%of11,105differentially regulated transcripts totally) temporally regulated genes appeared in both Nip and ZS, indicating a variety-type specific effect of Agrobacterium on genome-wide expression patterns.4. In Nip calli, there are more up-regulated genes than down-regulated genes. By contrast, there are more down-regulated genes in ZS calli. Furthermore, more genes that were both up-and down-regulated were found in ZS calli after Agrobacterium infection.5. Most of the differences in gene expression occurred during the early stages of transformation (1-6hour after infection). It seemed that Agrobacterium had both down-regulatory and up-regulatory effects in the first6h after infection (HAI), and had mainly down-regulatory effects in the following18h, while with some genes being up-regulated in the latter12h.6. Defense-related genes play an important role in interaction between Agrobacterium and rice cell, which were both activated and suppressed in Nip and ZS. They also differentially expressed between ZS and Nip calli. Some genes of this class were upregulated early or constitutively activated in ZS calli, whereas most of these genes were repressed in Nip calli, which may may have resulted in the different susceptibility to Agrobacterium infection in these calli.7. Some genes directly related to Agrobacterium-mediated transformation showed differential expression between ZS and Nip. Genes involved in cell division and cell cycle, DNA replication, DNA repairing and recombination, microtubule-based movement, ubiquitin-proteasome system, responding to auxin stimulus and genes coding core histones were suppressed early after transformation in ZS calli but were not apparently suppressed in Nip calli, with some even being activated, which may further decrease the transformation efficiency of ZS. These plant factors which play a crucial role in Agrobacterium-mediated transformation are the focus of our further work and serve as potential candidates for improving transformation efficiency in recalcitrant rice cultivars such as ZS through genetic engineering. |
