| Cotton belongs to the important economic crop, while affected increasingly by drought and salinity soil, the yield and quality of cotton decreased obviously, bringing to big losses in production and economic value. With the constant development of biotechnology, genetic engineering is found to be one of the most effective method to create new drought and salt resistant cotton germplasm. Transcription factors of ERF (Ethylene responsive factor), special type of genes in plants, and generally involved in regulation of plant response to the all kinds of stress, are crucial to adapt adjustment factor. In order to improve drought and salinity tolerance ability of cotton, two transcription factor genes, PeDREB2a and KcERF, cloned from Euphrates poplar (P. euphralica) and bell sting (H. halodendron), respectively, with the resistant marker gene CP4,are transferred into the R15 cotton lines using agrobacterium-mediated method in the study. The transgenic cotton plants thus obtained were analysed and identified for the intergration and expression of the foreign genes using molecular biological methods. The drought and salt tolerance transgenic plants were also evaluated in field. The main results as follows:(1) The most effective bivalent expression vector, pCAMBIA1302-KcERF PeDREB2a, harboring rd29A as the promoter, was used to the transformation of cotton cultivar R15, and 32 transgenic cotton plants were obtained.(2) The T3 generation of the transgenic cotton plants were obtained after cultivation in Hainan and Hebei provinces for two years.(3) R15 cotton plants were preliminary screened out by spraying the plants with kanamycin solution of 1500 mg/L. Selected the kanamycin-resistance cotton lines using 1500mg/L kan.(4) The prliminary screened the T3 plants were further analysed using PCR, reverse transcription PCR and Reverse Transcription PCR for identification of the intergration and expression of the foreign genes. A total of 6 transgenic cotton strains were obtained finally. Southern blot analysis showed that the exogenous genes had been inserted into the receptor cells in the form of single copy. The genetic transgenic plants followed Mendel's law, and gene silencing phenomena is none in the progeny.(5) The T3 cotton of transgenic plants showed significant resistance to herbicide, while the the controls R15 died after the spraying of the herbicide, proving that the CP4-epsps had been expressed in the transgenic plants normally.(6) The transgenic plants were treated with with 15% PEG6000 or 200 mmol/LNaCl, and their tolerance to drought and salt were analysed by detecting the related physiological indicators under drought and salt stress. The results showed that the transgenic cotton grew significantly superior to the controls under the stress conditions, and the content of MDA in transgenic cotton was lower than the control, CAT,SOD activities and free proline content were higher than those of the control. As compared with the control, roots of the transgenic cotton are also more developed. The qPCR analysis of the transgenic cotton plants showed that over-expression of KcERF and PeDREB2a genes can be induced by drought and salt stress treatment. In conclusion, PeDREB, KcERF could improve the tolerance of cotton to drought salt tolerance stress. |
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