Study On Five Zinc-finger Proteins (ZFPs) Related To Stresses In Cotton(Gossypium Hirsutum L.)

Cotton is an important economic crop in the world, and provides most natural raw material for textile industry. Upland cotton is the major cultivar of cotton, and products more than90%of the total cotton fiber of the world. However, most cotton cultivars are cultivated in arid and semi-arid areas. Natural disasters, such as drought, salinity, low temperature, Fusarium and Verticillium wilt, are seriously hindering of cotton yield and quality improvement. Breeding cotton varieties with high resistance is one of the main tasks of cotton breeding.Zinc finger proteins are a type of proteins which can combine with zinc ions and folds into a finger-like structure, is the largest members of the transcription factors in number. Zinc finger domain binding zinc ion mainly relies on cysteine and histidine. The arrangement of systeine and histidine determines the mode of zinc finger protein binding zinc ion. Zinc finger domain, can combine with DNA, RNA, protein and lipid and involved in all aspects of life process. In plants, some zinc finger proteins, including some plant-specific zinc finger proteins, involved in responses to biotic and abiotic stresses.In our previous sutdy,5genes, GhSAP1, GhBBX1, GhRCHY1, GhZFP2, and GhZFP3, encoding Znic-finger proteins, were cloned from cotton. Their expression levels under different abiotic stresses were detected by real-time quantitative PCR. The results showed that the expressions of the five Znic-finger genes were significantly induced by drought, NaCl, and ABA. In this research, we constructed the plant over-expression vectors, and created the transgenic tobaccos with these exogenous genes, further, investigated the phenotypes and functions of the transgenic plants under drought and salt stress. The main results as follows:1. The five Znic-finger genes were integrated into a plant over-expression vector pBI121with a35S promoter to promote the genes. These genes were transferred into tobacco and10clones of T0plants for each gene were obtained, respectively. The seeds in T1 transfomation tobacco were screened with different concentration of kan, and the optimization screened concentration of kan resistance150mg/ml was confirmed. Further screening was finished to get T1resistant plants.9single lines from3different clones were sampled from each transformation material to extract DNA and RNA from the leaves. The insertion and expression of exogenous target genes was verificated by PCR and RT-PCR analysis. T2pure lines were obtained by selfing the T2seeds, combining with PCR verification.2. Three clone lines of the transgenic tobacco for each gene were treated with salt stress. Seven indexs involving to salt tolerance, including chlorophyll content, carotenoid content, contrast of SOD, MDA content, sucrose and total sugar content, and K+/Na+, were measured respectively. We determined the modest concentration of NaCl was200mM according to the treatments of transgenic materials under different concentration salt stress. The further researches were done using this salt stress level, and the results of the correlated indexs showed:the chlorophyll content the GhSAP1, GhZFP2, GhZFP3, GhBBX1,GhRCHY1transgenic plants under salt stress was significantly higher than wild type. The carotenoid content of the GhBBX1transgenic plants was significantly higher than wild type, and the level of GhSAPl, GhZFP2, and GhRCHY1was significant. Meanwhile, the carotenoid content of GhBBXl is significantly higher than the level of GhSAPl, GhZFP2, and GhRCHY1. The SOD contrast of all transgenic plants was significantly higher than wild type, and the activity level of GhBBX1transgenic plants was significantly higher than the others. The MDA content of all transgenic plants was significantly lower than the wild type, and the level of GhBBX1and GhSAP1transgenic plants was significantly lower than the others. The sucrose and total sugar content of all transgenic plants was significantly higher than wild type, and the sucrose content level of GhBBXl transgenic plants was significantly higher than the others, while the total sugar content level of GhBBXl transgenic plants was significantly higher than the others. The K+/Na+of aerial parts of all transgenic plants was significantly higher than that in wild type, and the K+/Na+of underground parts of GhBBX1and GhZFP2transgenic plants was significantly higher than that in wild type. The results indicated that under salt stress the photosynthesis pigment synthesis ability of the transgenic plants is higher than the wild type; the accumulation of the osmosis regulatory substance is more than the wild type; the enzyme activity of clearing reactive oxygen species is higher than the wild type; the plasmalemma oxidation products is less than the wild type; the outside Na+and K+can be tansfered from roots to leaves to keep the nutritive equilibrium.The research proclaimed the salt tolerance ability of the transgenic tobacco of the five genes from high to low is GhBBXl, GhSAP1, GhRCHYl, GhZFP2, and GhZFP3.3. Three clone lines of the transgenic tobacco for each gene were treated with drought stress. Six indexs involving to drought tolerance, including chlorophyll content, carotenoid content, contrast of SOD, MDA content, sucrose and total sugar content, were measured respectively. The results showed:under the drought treatment, chlorophyll content of GhSAPl, GhZFP2, GhZFP3, GhRCHY1transgenic plants was significantly lower than that in the wild type. The carotenoid content of the GhZFP2and GhZFP3transgenic plants was significantly higher than wild type. The SOD contrast of all transgenic plants was significantly higher than wild type, and the activity level of GhZFP2and GhZFP3transgenic plants was significantly higher than the others. The MDA content of all transgenic plants was significantly lower than the wild type. The sucrose and total sugar content of all transgenic plants was significantly higher than wild type, and the level of GhZFP2and GhZFP3transgenic plants was significantly higher than the others. The results indicated that the transgenic plants under drought stress synthesize more chlorophyll and carotenoid to ensure the photosynthesis; accumulate more sucrose and other soluable sugar to keep the osmoregulation and stability of osmotic potential; the SOD activity is much higher than the nontrangenic control, which can clear the reactive oxygen species preferably; the low MDA content and less plasmalemma oxidation guatantee the integrality and stability of the plasmalemma.The research proved the drought tolerance ability of the transgenic tobacco of the five genes from high to low is GhZFP3, GhZFP2, GhSAP1, GhRCHYl and GhBBX1.