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| Enhanced Cold Tolerance of Tobacco (Nicotiana tabacum) Transformed with Carrot (Daucus carota) AFP Gene Driven by Prd29A and CaMV 35S Promoter |
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Abstract Abstract Antifreeze proteins (AFPs) is a kind of special small molecule protein produced by cold-resistant organisms for adapting low temperature environment, and it can decrease ice point and inhibit ice recrystalization. In this research, the expression level of DcAFP from carrot (Daucus carota) driven by environment inducible promoter Prd29A (responsive to desiccation 29A) and constitutive promoter Cauliflower mosaic virus 35S (CaMV35S) in transgenic tobacco (Nicotiana tabacum) were detected. The results showed that the DcAFP gene has been integrated into tobacco genome and could express stably in progeny. The phenotype of inflorescence in T1 transgenic tobacco was dysplasia, and its capsule was easily to detach from the carpopodium, and few fruits could develop normally. After 4 ℃ acclimation for 10 d, -1 ℃ low temperature treatment for 24 h and 25 ℃ recover for 7 d, the survival rate of T1 tobacco plants transformed with Prd29A:AFP was 34.6% higher than that transformed with CaMV35S:AFP, and was 61.1% higher than wild type tobacco plants. T1 tobacco plants transformed with Prd29A:AFP grew well, while wild type tobacco was most dead. The chlorophyll fluorescence parameters (Fv/Fm) of all kinds of tobacco plants were decreased under cold stress, but after 25 ℃ recover, the Fv/Fm in Prd29A:AFP transgenic tobacco restored to the normal level, but the Fv/Fm in CaMV35S:AFP transgenic tobacco was just a little higher than that in wild type tobacco. Compared with wide type tobacco, proline content, soluble sugar content and soluble protein content in the two DcAFP transgenic tobacco under cold acclimation at 4 ℃ were increased, and the activity of superoxide dismutase (SOD) and peroxidase (POD) enhanced, and isozyme bands were increased. At the same condition, proline content, soluble sugar content, soluble protein content as well as SOD and POD activity in Prd29A:AFP transgenic tobacco were 45.1%, 12.1%, 20%, 19.7% and 8.6% higher than that in CaMV35S:AFP transgenic tobacco, respectively. The results demonstrated that the expression of DcAFP driven by Prd29A promoter could significantly enhance the cold tolerance of transgenic tobacco and had fewer effects on plant growth. These results have great reference value for breeding new cold tolerant transgenic plant varieties.
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Received: 23 February 2017
Published: 20 July 2017
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