| Plant in the nature environment have to withstand various stresses caused by environmental changes, such as heat, cold, salt and drought. In order to adapt to these abiotic stresses, plants produce a complex regulatory network. People also improve plants environmental adaptability by germplasm resources screening, transgenic technology and so on. These two methods are associated. Sigificant germplasm resources are firstly selected. Then, available genes can obtained from these selected rescources. Finaly, the available genes can promote plant stresses tolerance by transgenic technology. Therefore, germplasm resources and genes are all important for resistant breeding.Mulberry is a perennial woody plant and has been used as the only natural feed sources for silkworm breeding. Instead of the economic value, the mulberry tree also have the ecological value. For example, the ability for mulberry to resistant drought, salt and water logging can be used to govern ecologically fragile areas, such as land desertification, rocky desertification, salinization and mine ecological recoverment. The traditional mulberry cultivation developed few research for environmental adaptability of mulberry tree. This condition has severely restricted the mulberry application for ecological management and ecological value realization.Thefore, developing abiotic tolerance germplasm resources and hybrid varieties have become the urgent needs to transform and upgrade the mulberry sericulture industry.With the advent of the genomic era, research the mulberry abiotic stress tolerance in genomic level has become necessarily. The completion of genome sequencing opened a new era of molecular research. However, there is little research on mulberry resistant genes and DREB transcription factors. Although, DREB transcription factors have been identified and characterized from various plants, there is little information about genes in woody plants and A-3-A-6 subfamilies. Function analysis about mulberry DREB genes will help to supplement and improve the research on resistant genes and DREB genes in woody plants.Based on these questions, we obtained high tolerance germplasm resources and genes. First of all, better resistant hybrids combination Cesha and Guisangyou series were collected for salt and drought tolerance research. Secondly, the MnDREB4A has been chosen based on previous research to study its function from bioinformation analysis, subcellular localization, promoter activity and gene function respects. The main results obtained in this study are as follows:1. An evaluation on salt and drought tolerance of F1 generations from 13 Mulberry hybrid combinations.The seeds of these hybrid combinations were germinated under the high salt condition (12g/L NaCl). The relative germination rate, germination potential, germination index and salt injury index of these seeds after 6-day treatment were obtained to analyze the salt tolerance based on five level scoring method. As a result, these hybrid combinations were divided into 4 types:Cesah-hybrid 6, Cesah-hybrid 5 and Guisangyou 62 were extremely tolerant to salt, Guisangyou 12, Cesah-hybrid 1, Cesah-hybrid 11, Cesah-hybrid 4 and Cesah-hybrid 7 were highly tolerant to salt, Cesah-hybrid were moderately tolerant to salt, Cesah-hybrid 8, Cesah-hybrid 10, Cesah-hybrid 13 and Cesah-hybrid 12 sensitive to salt. The normal seedlings of these hybrid combination were treated with 400g/L PEG6000. Based on the survival rates after 4-day treatment, these seedlings were divided into 4 types:Cesah-hybrid 6 and Cesah-hybrid 12 were extremely tolerant to drought, Cesah-hybrid 4, Cesah-hybrid 5, Cesah-hybrid 8, Guisangyou 12 and Cesah-hybrid 13 were highly tolerant drought, Cesah-hybrid 1, Cesah-hybrid 11, Cesah-hybrid 10 and Guisangyou 62 were moderately tolerant to drought, Cesah-hybrid 2 and Cesah-hybrid 7 were sensitive to drought. These mulberry were ranked based on their salt and drought tolerance and their ecological function were predicted. Cesha-hybrid 6 are suitable for growing in areas with perennial drought and serious salinity. Cesha-hybrid 12 can be used to restore vegetation in desertification and mining areas. Guisangyou 62 can be chosen to improve ecological environment in salinization areas.2. Cloning and bioinformatics analysis of MnDREB4A.Based on abiotic stresses induced expression profiles, the MnDREB4A were chosen to analyze the function. Bioinformation analysis revealed that MnDREB4A in mulberry showed high sequence similarity especially in the AP2 domain, when aligned with corresponding proteins in other species. In addition, they all contained conserved motifs that were necessary to recognize and bind to DNA, such as YRG, WLG, RAYD, V14 (Valine) and E19 (Glutamic acid). SWISS-MODEL predicted that MnDREB4A can form a classical protein contained 3β-fold and an α-helix. Analysis of the conserved cis-elements in about 1500-bp upstream of the open reading frame (ORF) of MnDREB4A was conducted using PlantCARE. The results revealed that it contained various cis-elements related to abiotic stresses, and suggested that MnDREB4A gene can response to abiotic stresses. By predicting on two websites, MnDREB4A would like to localize in the nucleu. All these results made it possible for MnDREB4A to carry out the function.3. The function of MnDREB4A.To validate the prediction, the MnDREB4A::EGFP or EGFP::MnDREB4A fusion gene under the control of CaMV 35S promoter were transfected into the onion epidermak cells. The green fluorescence signals of the MnDREB4A::EGFP or EGFP::MnDREB4A fusion proteins were observed only in nucleus, implying that the MnDREB4A proteins was a nucleus-localized proteins. The reconstructed plasmid (MnDREB4A pro::GUS) was transformed into Arabidopsis thaliana by the floral dip method. The transgenic seedlings were treated with heat (40 ℃), cold (4℃), salt (150mM NaCl) and drought (20% PEG6000). With the treatment performed, more and more strong GUS expression was observed in transgenic Arabidopsis. The GUS activity was the highest after 12h treatment and then came to decreased. The GUS expression were observed in diverse stages and tissues and predominantly expressed in the vasculature.The recombinant plasmid (CaMV35S::MnDREB4A) was transformed into tobacco (Nicotiana tabacum L.) plants by leaf disc method. Comparing with the WT under normal conditions, the transgenic tobacco plants showed obvious phenotypic differences in terms of leaf and root. For example, the leaves of transgenic tobaccos were smaller and greener than WT, the transgenic tobaccos exhibited lower rates of water loss at each time point than WT after leaf detachment. And the roots of transgenic tobacco were longer compared with WT. After abiotic stresses treatment, the WT showed more obvious leaf wilting and senescence than transgenic line. Compared with the WT plants, the transgenic line showed remarkably higher levels of proline and RWC, but lower levels of MDA under different abiotic stresses. In conclusion, the transgenic plants showed better tolerance to abiotic stresses than WT. NtERD10B, NtERD10C, NtERD10D are dehydrins related genes, NtHSP70, NtHSF2 are heat related genes and NtLEA5 is ignal transduction gene. The expression level of these genes were examine by qRT-PCR. Compared transgenic plants with WT, all these genes exhibited a significantly lower expression level in transgenic lines. This result implied that MnDREB4A can improve plants abiotic stress tolerance by regulating expression of related genes. |
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