Reconstruction Of Split-recombinase FLP And Its Recombination Activation In Transgenic Tobacco

Genetically modified (GM) plants are being grown on more than 67 million hectares (ha) in 18 countries worldwide, and the amount is increasing by 10% or more annually. The commercial, economic, and social benefits resulting from the use of herbicide and insect resistance traits is now widely established in North and South America as well as Australia and Asia. Seven GM crops (cotton, canola, maize, papaya, potato, soybean, and squash) are currently being grown, but most of the world’s bioengineered hectarage is in cotton (7 million ha), maize (10 million ha), and soybean (33 million ha). Two agronomic traits—resistance to herbicides and resistance to insect pests—account for virtually all planted hectares.However, biosafety concerns have been raised regarding the deployment and release of genetically engineered plants. This debate has divided the farming and consumer communities over acceptability of genetically modified foods. GM plants could cause environmental harm. Potential adverse effects include the genetic contamination of related wild species and impacts on ecosystems if the GM crop itself becomes invasive. Critical debate of environmental liability has been lacking and the Government has used this to avoid addressing the issue. The concerns about the biosafety of GM plants have limited the development of transgenic technology. Hence, in order to comfort people without apprehensions, a new system used for foreign gene deleted in hybrid crops must be necessitated.Our studies showed that the "Gene-deletor" could excise efficiently all the foreign genes in transgenic plants, but the current vision can not be inevitably applied to hybridization breeding. To accommodate this system to field production and improve to solve the security problem of genetically modified, we refined the "Gene-deletor" by splitting the FLP recombinase into two independent parts, namely N-domain and C-domain, and obtained a new "Gene-deletor" system. The transformation of tobacco verified that the system had an efficient activity. The results are as follows:1. Construction of plant expression vectorThe target DNA sequences of FLP recombinase gene, including N terminal fragment, C terminal fragment and the full length were amplified with high-fidelity enzyme and cloned into pCXSN cloning vector. Then the recombinant vectors were digested with BglⅡand Nco I to gain the target fragments with sticky ends and were retrieved and cloned into two direction FRT sites of FRT-NOS-FRT-pUC19 vector. Finally, the cassette flanking by FRTs were cloned into the plant expression vector pCambia1305.1 and the 12 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 and A. rhizogenes C58C1.2. GUS staining of transgenic hairy rootsSpliting proteins of FLP recombinase were introduced into tobacco via leaf disc transformation with A. rhizogenes C58C1 and transgenic hairy roots of tobacco were obtained. Our results showed that GUS activity was detected in transgenic hairy roots containing full-length FLP recombinase gene, indicated that FLP/FRT system had a deletion function in the hairy roots. When either the N terminal or C terminal of FLP recombinase existed, there was no GUS activity detected, suggestion that the protein was lack of recombinase function. By contrast, when the system included both N and C terminal domains, GUS activity dramatically emerged in the hairy roots. This result demonstrated the activity of split FLP could partially recover when reconstruction.3. Molecular analysis for transgenic plantsBased on the plant expression vector T-DNA interval sequence, two pairs of specific primers were designed to detect the integration of the exogenous genes into transgenic plant genome. PCR results showed that all of T-DNA vector had been successfully integrated into trangenic tobacco genome.Genomic DNA of transgenic hairy roots containing the different FLP recombinase gene fragments was extracted as the PCR templates. The primers coincided with the both ends of recognition sites FRT were designed for PCR amplification. A 377-bp amplified band was detected in all transgenic hair hairy roots which GUS staining was positive, indicating that the cassettes flanked by the FRT recognition sites were excised successfully. These PCR products were sequenced further and the results showed that all exogenous genes except one recognition sequence FRT site were accuratually deleted in transgenic plants.4. Analysis of deletion efficiency of spit-protein system The deletion efficiency of the recombinant enzyme system was analyzed in transgenic hairy roots. A total of 30-50 independent transgenic hairy roots for each construct were selected from transformed tobacco leaf discs and GUS-positive hairy roots were accounted. The statistics of each hairy root transformation vector were repeated three times. The results showed that the deletion efficiency was up to 37.8% in transgenic hair roots containing natural FLP recombinase, consistent to previous reports. Higher deletion efficiency (43.5%) was observd in transgenic hair roots carring FLP recombinase plus the nuclear localization signal (NLS). There was no recombination activity when the recombinase FLP was split into N-terminal domain and C-terminal domain. While these split fragments (N-and C-terminal domains) were constructed into the same binary vector and integrated into the tobacco genome, the recombination activity of FLP recombinase was recovered and the deletion efficiency was 29.4%. Further, combination of NLS into the FLP enzyme N- and/or C-terminal could significantly improve the the recovery of restructuring activity and the deleted efficiency was 57.1%,63.6% and 75%, respectively.