| RSG (repression of shoot growth) in tobacco containing a basic leucine Zip,is a GA synthetic regulatory transcription factor, which control endogenous GA content through regulate the expression of GA synthesis key enzyme KO (Kaurene oxidase) and GA20ox1 (GA18 oxidase). The combine of RSG and GA20oxl promoter regulated feedback by GA content in tobacco.14-3-3 protein is a important regulatory protein in tobacco, which regulate the respond of plant to external environmental stimulate by interact with multiple signal transduction proteins.14-3-3 protein in cytoplasm reduce the ability to combined with RSG and then promote RSG into nucleus, what’s more, RSG combine with KO and GA20ox1 promoter then enhancing the expression of KO and GA20ox1 to stimulate GA content, When decrease GA content in tobacco or inhibit its synthesis by PAC (synthesis inhibitors of endogenous GA). Opposite with it, add exogenous GA will promote the combination of 14-3-3 protein and RSG then RSG can not into nucleus, further more, it’s inhibit the combination of RSG to KO and GA20ox1 then decreasing the expression of the two promoters, which lead to GA content decrease compared with no add exogenous. Studies in our lab have shown that stimulating tobacco with methanol and ethanol influenced the expression of GA biosynthesis-related genes and 14-3-3 proteins; applicated exogenous GA to plant enhanced the resistance to drought stress. Besides, some studies indicated that GA also relate to the response of Al stress and it’s tolerance to Al stress. In this study we treated wild type tobacco with exogenous GA and PAC to change GA content,and obtained transgenic tobacco through overexpression or RNAi interference the expression of RSG/14-3-3.Analysis the expression and interaction of RSG and 14-3-3 as well as the mode of RSG binding to GA20oxl promoter response to the stiumlation of methanol/ethanol and drought and Al stress in WT and transgenic tobacco.Study the bmolecular mechanisms of RSG and 14-3-3 proteints in tobacco respond to methanol or ethanol. We obtained the results as follows:In order to investigated wether methanol and ethanol promote plant growth related to GA synthesis,we analyzed the affection of exogenous GA and PAC to the stimulation of 2mM methanol and ethanol in WT tobacco on MS solid medium. The results showed that the addition of exogenous GA enhanced stimulation of methanol and ethanol on tobacco growing, while adding PAC inhibited stimulation of methanol and ethanol on tobacco growing. Analysising GA content confirmed the stimulation of methanol and ethanol was increased when add exogenous GA. RT-PCR analysis showed that methanol and ethanol stimulation induced ranscription and expression of 14-3-3 genes in tobacco,and also induced the expression of RSG protein. Immunoprecipitation analysis methanol and ethanol stimulation enhanced the interactions of 14-3-3 protein and RSG, as well as the combination of RSG and GA20oxl promoter. These indicated that methanol and ethanol stimulation promote GA synthesis by increasing the expression of 14-3-3 proteins and RSG in tobacco to enhance the binding of RSG and GA20ox1 promoter and GA20oxl promoters so that to increase GA content and increase the interaction of 14-3-3 proteins and RSG.In order to verify the effect of RSG and 14-3-3 protein in tobacco response of methanol and ethanol stimulation, We construct over expression RSG vector pK2-35S-RSG and interference vector pK7-35S-RSG-I-RSG with CaMV 35S promoter, Transform WT tobacco and obtained 4 RSG overexpression lines (RO1-4) and 3 RSG inhibits expression of lines (RI7,8,10), four RO strain of GA was higher than that of WT, while the three RI strain of GA was less than WT. The laboratory master’s thesis of Guo Chuanlong prepared 14-3-3 protein respectively with 2 mM methanol and ethanol (overexpression lines S23, inhibit the expression of strain RE5) and RSG transgenic (RO3 and RIIO) plants, the amount of root elongation of S23 and RO3 and increased the fresh weight were significantly greater than WT, in RI10 and RE6 in contrast to the results of. Compared with WT, increase the content of RO3 and RE5 in GA, and the content of RIIO and S23 reduced GA. Analysis confirmed that the combination of RSG and GA20oxl promoter RO3 and RE5 in plant was larger than WT immunoprecipitation, and the combination of RSG and GA20oxl promoter RI10 and S23 in the plants is less than WT. The expression level of RSG in RO3 and is responsible for the phosphorylation of S23 in plant protein kinase CDPK is higher than WT, so the interaction between 14-3-3 protein and RSG is greater than WT; and the expression level of CDPK in RI10 and RE5 is less than WT, so the interaction between 14-3-3 protein and RSG is less than WT.Treatment of WT and RO3, RI10 transgenic tobacco with 2%PEG, results shows RO3 loss rate is less than WT under PEG stress, but the transpiration rate, net transduction rate and intracellular photosynthetic rate, stomatal CO2 concentration was greater than WT; while RI10 water loss rate is greater than WT, but the transpiration rate, net transduction rate and cell photosynthetic rate, stomatal intercellular CO2 concentration was less than WT. These results suggest that the expression of RSG increase tobacco drought tolerance and net photosynthesis under drought stress on the photosynthetic rate, inhibit the expression of RSG decreased the ability of drought resistance and net photosynthetic rate. In the 2%PEG stress treatment 12h, the binding capacity of RSG and GA20ox1 promoter is greater than WT, and the GA content of RO3 is higher than that of WT, RSG and 14-3-3 protein interaction level is also higher than the WT; the ability to combine RSG and GA20oxl promoter in RI10 is less than WT, which makes the GA content is lower than WT, RSG and 14-3-3 protein interaction level is less than WT.The most obvious symptom of AI toxicity is the inhibition of root growth. In 50μM under Al stress, RRG (relative growth rate) of WT tobacco roots decreased, content in the root GA decreased gradually, the synthesis of aluminum stress inhibited the root of tobacco GA. Addition of exogenous GA under Al stress, elongation of WT tobacco roots recovered obviously, and the addition of PAC, aggravate the inhibition of root elongation of aluminum stress. Immunoprecipitation analysis showed that the combination of aluminum stress also reduced RSG and GA20oxl promoter. RT-PCR analysis showed that the transcription of WT 14-3-3 gene in tobacco induced by aluminum stress in a, b, d, e, h and i subtypes and the expression of RSG, while there had no significant effect on transcription of CDPK. Western blot analysis showed that the expression of aluminum stress enhanced the expression of WT tobacco 14-3-3 protein and RSG protein, decrease the interaction of 14-3-3 protein with RSG. Overexpression of RSG enhanced GA content in RO3 that is extremely resistant to aluminum stress, enhance the combination of RSG and GA20ox1 promoters, inhibiting the expression of RSG and reduce the content of RI10 in roots of GA extremely tolerance to aluminum, weakened the combination of RSG and GA20ox1 promoter... |
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