Development Of The Elictor-coding Gene PeaT1 Transformation System Of Cotton And Tobacco And Its Functional Analysis

PeaT1 is a protein elicitor derived from fungus, Alternaria tenuissima. The preliminary research studies indicate that it promotes crop growth, improves quality, and induces disease resistance. The elictor-coding gene, peaT1 transformation system in cotton and tobacco was developed to study its function. The integration, transcription and expression of transgenic plants were identified by molecular biological methods. The biological functions of elicitor such as resistance to insect pest and diseases, and effect on certain agronomic traits were also analyzed. The main results are as summarized as follows:1. Construction of plant expression vectorThe plant expression vector pCAMBIA2300-G4AS-peaT1 was constructed. It was found to improve the expression level which contains enhancer and poly-terminator. The DNA constructs were verified by restriction enzyme digestion and sequencing, then the plant expression vector was transformed into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method.2. Development of transformation system in cotton and tobacco(a) The elictor-coding gene, peaT1 was transferred into tobacco (Nicotiana tabacum) by Agrobacterium-mediated method. The medium composition and culturing conditions were optimized to obtain primary tobacco transformants.(b) The regeneration ability of different cotton genotypes was analyzed. It was found that the induction and regeneration of the embryonic calli in H05, H08 and H15 was difficult as compared with Coker312 and CCRI24.(3) The elictor-coding gene, peaT1 was transferred into cotton hypocotyls as the receptor by Agrobacterium-mediated method. The sets of culturing conditions such as concentrations of Km Agrobaterium tumeficiens, hormones, optimum temperature, time of co-cultivation, and prevention of browning rate were also studied. The set of medium was confirmed which was suitable for Coker312 and CCRI24, and a lot of regenerated cotton seedlings were obtained successfully.3. Transgenic plants were confirmed by polymerase chain reaction (PCR). The integration of peaT1 gene was further confirmed by Southern blot analysis, transcription was verified by reverse transcription polymerase chain reaction (RT-PCR) and protein expression in tobacco was detected by Western blot.4. The growth parameters in cotton and tobacco transforments were analyzed. It was found that the transgenic tobacco was matured earlier as compared to control. The stem diameter of the transgenic cotton obtained by pollen-tube pathway method was 2.42cm, and it was higher than the average value of the control which was 1.41cm. The transgenic cotton had yielded 33 bolls, while it was 8.1 in non-transformed control. These results indicated that the expression of PeaT1 in cotton and tobacco could promote plant growth.5. The transgenic tobacco lines K1 and K2 at T1 generation were challenged with TMV (Tobacco Mosaic Virus). It was found that there was reduction in TMV lesions by 40.86 and 46.22 % respectively in K1 and K2 transgenic lines as compared to wild-type tobacco infected with TMV. The feeding of transgenic cotton leaves to Sylepta derogata Fabricius and Helicoverpa armigera Hubner revealed that the transgenic cotton also showed the insect resistance.