Over the past decade, the acreage of GM crops cultivated in commercialization had been rapidly expanded across the world. The global area of transgenic crops had reached 134 million hectares in 2009 (http://www.isaaa.org/). Rice in Asia particular in China is one of the most important cereal crops as well as one of the more serious suffered crops from pests (Han et al., 2006), of which annual economic losses due to pests reached 4-5 million tones. Applying plant genetic engineering techniques to develop insect-resistant transgenic rice would be an important way to solve the problems caused by pests. In 2009, China issued security certificates for the two varieties of insect-resistant transgenic rice, which indicated that China will soon have insect-resistant genetically modified rice going into commercial production (Liang, 2009, private communication; Zhu, 2001). With the development of GM rice in commercialization, the public pays even more attentions about the ecological consequences caused by possible escape of foreign genes after large-scale release of transgenic rice. One of the concerns about biological safety of genetically modified rice is to fear the foreign genes escaping into wild relatives of rice, which might have a direct impact on genetic and ecological adaptations of these wild populations, thereby influencing on the ecological and evolutionary direction of wild rice populations (Lu et al., 2002, Song et al., 2003). Previous studies have been clear that the escaping frequency of transgene should be below 1.0% in the maximum possibility (Rong et al., 2005). China is one of the home of the wild rice, the distributions of wild rice and cultivated rice have a broad overlapping regions as well as matching flowering periods (Song et al., 2003). It might be inevitable that exogenous genes would possible escape into wild rice. Whether could the escaped foreign genes normally inherit in wild rice populations? Could the escaped gene normally expressed? These might be the first step to evaluate the ecological consequences caused by the escaped foreign genes. In this study, we obtained F₁ hybrids and their descendants by artificially making cross between transgenic rice and wild rice to study the behaviors of genetics and expression of foreign genes in the offspring of wild rice, and thus to provide the basis for evaluating the consequences possible caused by transgenic rice released into the environment.

1 Results and Analysis
1.1 Identification of Cry1Ac/CpTI and hpt genes in the offspring of wild rice
In the different generations of wild rice, choosing plants with positive hygromycin resistance to amplify the foreign genes were carried out by using specific primers for Cry1Ac, CpTI and hpt genes, PCR products were separated by electrophoresis, the results showed that all of the foreign genes can be amplified in the sizes of 503 bp, 415 bp and 832 bp, respectively (Figure 1 and Figure 2).

Figure 1 PCR analysis of Cry1Ac and CpTI in hybrids of wild rice crossed with Cry1Ac/CpTI rice and their progenies

Figure 2 PCR analysis of hpt in hybrids of wild rice crossed with Cry1Ac/CpTI rice and their progenies

1.2 The copy numbers of exogenous genes in the offspring of wild rice
Genomic DNA of rice was digested by endonucleases of Xba â…  and Sma â…  that are single restriction sites in the transform vector, Southern blot analysis was performed by using the labeled probe of Cry1Ac gene fragments. The results showed (Figure 3) that copy number of Cry1Ac gene was the same in the parent of kefeng6, and in the generations of F₁, F₂, F₃, F_4, F₅, BC₁F₁ and BC₁F₂, of which occurred in the form of single copy but the size of bands had slightly difference in some generations.

Figure 3 Southern blot analysis of Cry1Ac gene in different generations

1.3 The insertion site of exogenous genes in the offspring of wild rice
Two pairs of primers were designed based on the template of 5' flanking sequence of the T-DNA insertion site of which the upstream primer was located in the rice chromosome and downstream primer was located in the inserted fragment. PCR amplification was performed using DNAs from parental kefeng6 and the offspring derived from different wild hybrid rice as templates, the target fragments were recovered and sequenced, the sequencing results aligned by the CLUSTALW (version 1.83) showed that the sequenced 208 nucleotide bases of rice genomic DNA adjacent to the 5' end of the insertion site were identical to the parental kefeng6 except the 58th site with N instead of G (G→N) in BC₁F₂ generation as well as with A instead of G (G→A) in F₄ generation (Figure 4).

Figure 4 Comparative analysis of the flanking sequences of T-DNA insertion site in different generations

1.4 Cry1Ac protein expressed in the offspring of wild rice at different growth stages and different tissues
The amount of Cry1Ac protein in leaves measured in the stages of tillering, heading, flowering and grain filling (Figure 5) showed that Cry1Ac protein expressing pattern was basically the same in the parental and in the different generations of F₁, F₂, F₃, F₄, F₅, BC₁F₁ and BC₁F₂. Expressing peak occurred in the flowering stage expression being superior to the expressing peak at tillering stage, whereas the expression of Cry1Ac gene was being decreased in the stage of seed ripening. The expression of Cry1Ac protein was no significant difference in statistics in different developmental stages among the various generations.

Figure 5 The content of Cry1Ac protein expressed in leaf at different generations

The expression of Cry1Ac gene in different tissues at the tillering stage (Figure 6) indicated that the amount of expression followed by leaf>stem>root. The overall volume expression protein accounted for a few in ten thousand to a few in a thousand of total soluble protein. SPSS statistical analysis showed that the amount of Cry1Ac gene expressing protein was no any statistical difference in the same developmental stage in the parent, F₁, F₂, F₅, BC₁F₁ and BC₁F₂.

Figure 6 The content of Cry1Ac protein expressed in different tissues

2 Discussion
With the development of GM crops in commercialization, it is the consensus of scientists to assess the ecological risk caused by the release of genetically modified crops, especially for genetically resistant enhanced crops, people worried that these exogenous genes might gradually colonize in wild populations through gene flowing introgression, leading to the wild relatives acquiring a selective advantages to form super-weeds (Dale, 1992; 1994; Dale and Irwin, 1995), alternatively, leading to lose of genetic diversity caused by wild alleles loss due to transgene fixed in wild populations (Lu et al., 2009), which is an important aspects of ecological risk that may arise after the release of genetically modified crops to the environment.

The escape of foreign genes through the spread of transgenic pollen is one of the main ways. Studies showed that the outcrossing rate between cultivated rice and wild rice (O. rufipogon) was from 6% to 10%, the hybrids usually have high fertility and the gene flow might be more likely to cause genetic drift. Wild rice is an ancestor of common cultivated rice and most closely related to cultivated rice, widely distributed in South Asia, Southeast Asia and northern Australia, which have broad overlapping distributions as well as matching flowering seasons with cultivated rice. The frequency of gene introgression between the both is very high. Natural hybrid of the both often happens in these areas (Song et al., 2003), which might be the relatives at the risk of the transgene introgression.

In this study, the exogenous genes were introduced into common wild rice through artificial hybridization, in order to study the genetic behaviors of foreign genes in common wild rice, then to predict whether transgene escaping to wild rice will colonize in the wild populations as well as whether the foreign genes make an influence on inheritance and genetic diversity of wild rice. Through the studies on foreign gene introgressing in wild rice by consecutive selfing for five generations and backcrossing for 2 generations, the results showed that foreign genes in common wild rice can be stably inherited. The exogenous genes in wild rice genome with different size of blotting bands had slightly different in offspring, suggesting that this might be due to the changes of the restriction sites near the insertion sites caused by pairing and recombination of chromosome in the period of meiosis, or possible caused by structural rearrangement of the chromosome near insertion site (Guo et al., 2000, Science Bulletin, 45(19): 2086-2090). Sequencing results of the flanking sequence of insertion sites in different generations, insertion sites of foreign genes also have stable inheritance.

Exogenous insect-resistant proteins have been expressed in various tissues of the wild hybrid rice. There were a few differences in the expressing levels in different tissues and in different growth stages, the overall trends is that the amount of expression varied with the change of growth periods, the higher in the tillering stage, followed by a decrease and then reach a high expressing level in the flowering stage, decreased in seed ripening stage. The overall volume expression protein accounted for a few in ten thousand to a few in a thousand of total soluble protein. The expression of Cry1Ac protein was no significant difference in statistics in different developmental stages among the various generations. However, Gene silencing at protein expressing level happened in some offspring, a similar phenomenon in transgenic cultivated rice have also occurred (Duan et al., 2002, Biological Bulletin, 37(3): 15-16). Regarding the relationship of the expressing level of Cry1Ac protein to the ecological fitness of wild rice as well as whether exogenous gene would affect the biological diversity of wild rice, we are ongoing the follow-up monitoring surveys in long-term.

In summary, the studies on the inheritance and expression of exogenous gens by conducting selfing five generations and backcrossing two generations showed that the expression and inheritance of exogenous genes in the genetic background of wild rice were the same as in transgenic cultivated rice. Exogenous genes escaping into wild rice can also be inherited from generation to generation. However, the previous studies indicated the gene flow between rice crops (crop-to-crop) in the case of zero distance the frequency of gene flow is very low, usually below 1.0%, in case of 6 meters the frequency of gene flow will be rapidly attenuation (Rong et al., 2005). As known as genetic isolation exists in the cultivated rice and wild rice, the frequency of genetic drift would be far much smaller. Nevertheless, the possibility of gene flow drafting into common wild rice might be still in existence. Therefore, large-scale production of transgenic rice in future should attach importance to isolate from the geographical distributions of wild rice.

3 Materials and Methods
3.1 Materials
Common wild rice (Oryza rufipogon Griff) line F45 from Pingxiang county of Jiangxi province as the maternal parent, kefeng6 with Cry1Ac and CpTI that harboring in the genome of MingHui 86 as paternal parent that is a T5 generation line having genetic stability. kefeng6 is stable transgenic insect-resistant line obtained by Agrobacterium-mediated transformation in this laboratory in 2000 (Su et al., 2003), small scale trials approved in 2000 (Approval license No. 2000B-01-0400), now production trials has been passed.

In October 2002, the wild rice line F45 that artificial cut stigmas were delivered with pollens from kefeng and then was labeled and bagged. Harvesting seeds about a month after bagging generated F1 sown in April of 2003 , Selfing F₂ seed harvested in November of the same year. While BC₁F₁ was obtained by F₁ as female parent backcrossing with wild rice line F45 as male parent in September of the same year. BC₁F₁ was sown in next year to harvest BC₁F₂. F₂ was sown in April of 2004 to harvest selfing F₃ seed in November of 2004. F₃ was sown in April of 2005 to harvest selfing F₄ seeds in November. F₄ was sown in April of 2005 to harvest selfing F₅ seeds in November. All generated seeds except F₁ and BC₁F₁ for preservation by ratooning were dried to be stored at -5℃ cold storage.

All seeds of the generations mentioned above sown on April 2008 prior to pre-soaking at 37℃ 2 days. Until in three-leaf stage, Picking up leaves to rapidly extract DNA, simultaneous to amplify hpt and Cry1Ac gene. The hpt and Cry1Ac gene-positive and negative individuals were transplanted separately.

Rice grown in the GMO experimental station of Fujian Academy of Agricultural Sciences at Wufeng of Fuzhou, where surrounded by 2 meter high fence isolated from the surrounding. 21 individuals of each line planted in a plat with three replicates with total of 63 plants and randomly arrangement. Conventional measurement was applied to field management and on any pesticides used in growing season.

3.2 Sampling
Taking leaves at five sites of each plat to mix all samples in the stages of tillering, heading, flowering, grain filling. The mixed samples divided into two parts, one part for extraction of DNA, another part for detecting Cry1Ac insecticidal protein. While taking roots and stems at five sites of each plat at the tillering stage was carried out, then quickly frozen in liquid nitrogen, stored in -80℃ refrigerator until all samples were completely collected ready for use.

3.3 PCR detection of foreign gene
genomic DNA was extracted by using CTAB method in the samples of different generations as templates. The primers for Cry1Ac gene amplification are F: 5'-CAAGGATTCTCCCACAGG-3' and R:5'-TTTCTAACACCCACGATG-3' with expected 503 bp fragment in length; for hpt gene amplification are F: 5'-ACACAGCCATCGGTCCAGA3' and R: 5'-TAGGAGGGCGTGGATATGTC-3', the expected fragment in length of 832 bp; For CpTI gene amplification are F: AAAATGTGAAGAGCACCATCTTC-3' and R: TCTAGAGTTCATCTTTCTCATC-3', the expected fragment in length of 415 bp. PCR reaction procedures are following as 94℃ pre-denature for 7 min and then 30 cycles with 94℃ denature for 1 min, 58℃ annealing for 1 min and 72℃ extending for1 min, finally 72℃ extending for 8 min. Taking 5 μL of PCR products for electrophoresis with using 1% agarose gel.

3.4 Copy number of foreign gene detected by Southern blotting
According to the plasmid structure of transformation vector (Figure 7), choosing two single restriction sites, Xba â…  and Sma â… to digest the 20~30 μg of genomic DNA overnight, then separated by electrophoresis, following the steps of transferring to the membrane, blotting and pre-hybridizing. Cry1Ac probes were prepared by the alkaline phosphatase probe labeling kit produced by ROCHE and completely following up the method with the kit. Plasmid pCUBAC-hpt used as a positive control and non-transgenic receptor of rice as a negative control. Hybridization signal were recorded using FluorChem SP gel imager with 50 mm f11.4 lens.

Figure 7 Diagram of single restriction enzymetic sites in pCUBAC vector

3.5 Detection of T-DNA insertion sites
In order to detect genetic stability of insertion site of the foreign gene, we designed a pair of primers based on known T-DNA insertion site in parental kefeng6 located in the site of 33049063 on chromosome 4 (Genebank accession number: AP008210) with 1 346 bp of flanking sequences., one primer, F: 5'-CATGTCACGGTGTCTGTC-3', located on the site near the foreign gene insertion site in chromosome 4, another primer, R: 5'-TCTCATCATCTTCATCCCT-3', located on the insertion fragment, to amplify the transgenes in genetically modified parent and in different wild rice generations. The amplified fragment expected in length of 376 bp. Reaction procedure as the same as above. PCR products were recovered to be sequenced in TaKaRa Company.

3.6 Determination of transgenic Cry1Ac insecticidal protein
The content of Cry1Ac insecticidal protein content was determined by using ELISA approach (Xie and Shu, 2001). ELISA kits for Cry1Ac protein purchased from the Agdia Company of United States. The protocols are completely following up the instruction provided with the kit. Total protein content determined by Brandford Protein Assay Kit. The relative content of Cry1Ac protein to total proteins was calculated and analyzed with SPSS17.0 software.

Author’s contributions
JS completed the experimental design, result analysis and manuscript preparation and revision; GYZ is the main executor of the experimental work, completed data analysis and involved in writing. WJY participated in parts of the experimental work to build population and gene expression analysis. HS took part in the experimental work to build the population and to manage materials in the field. All authors have read and agreed the final content.

Acknowledgements
This research was supported by the National Transgenic Key Projects titled Technology on Environmental Safety Assessment of Genetically Modified Rice (2008ZX08011-001). The line F45 of common wild rice (Oryza rufipogon Griff) kind presented by Professor Lu from Fudan University.

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