| Plants inevitably encounter all kinds of adverse conditions, asespecially the high temperature, low temperature, high salt, drought, oxidative damage, in the growth cycle of plants that seriously threaten the growth and yield of plants. So the research on stress response genes has great practical significance. In order to adapt to environmental changes, plant itself will form a complicated mechanism to adapt to stress, while this mechanism is dependent on the stress signal recognition and transduction, transmission and the corresponding gene expression. Protein kinase is one of biological ubiquitous regulated kinase that response to environmental changes, and play a very important role in signal transduction and transmission. In present study, stress-associated protein kinases mainly contain five categories, they are receptor-like kinase(RLK), mitogen-activated protein kinase(MAPK, MPK), calcium-dependent and calmodulin-independent protein kinase(CDPK), sucrose non-fermenting-1-related protein kinase(SnRK) and other stress-related plant protein kinase. MAPK genes as the downstream protein kinase of MAPK pathway amplify and transfer the intracellular messages through the phosphorylation step by step, regulate downstream gene expression, thus forming a huge network signal pathway with other genes to regulate various physiological responses in the organism and response to the external environment. So far, MAPK gene family has been identified in Arabidopsis, tobacco, rice, corn, Brachypodium distachyon, cotton, tomato, grape, poplar, banana, apple and other plants. Although MAPK have been identified in various plants, but the research on their functional characteristics and mechanism in stress response mainly focused on herbaceous plants, rarely involved in woody plants.Mulberry is a kind of perennial woody plants with the characteristics of dry resistance, salinealkaline resistance, waterlogging tolerance, cold resistance, strong adaptability and rapid growth. While the study on the stress resistance of mulberry is rare. Our previous studies have identified 32 MnMAPKKK, 5 MnMAPKK and 10 MnMAPK genes in mulberry. qRT-PCR analysis of 10 mulberry MAPK genes expression level under different stress treatment showed that the expression level of different mulberry MAPK under the same stress treatment were different, the expression level of the same mulberry MAPK under different stress treatments also were not consistent, suggesting mulberry MAPK genes play different roles in different stress treatments. Meanwhile, expression level of mulberry MAPK5 under different stress treatment in mulberry showed that the expression level of mulberry MAPK5 were up-regulated after NaCl, heat and cold, but only under heat and cold stress exists significant difference; In ABA, SA and H2O2 condition, the expression level of mulberry MAPK5 was significantly decreased; the expression level of mulberry MAPK5 was slightly reduced after drought and MeJA treatment. In other words, the expression level of mulberry MAPK5 in different stress showed great differences, which suggest the mulberry MAPK5 might play an important role in the process of stress. Therefore, we chose mulberry MAPK5 for further functional study to clarify the function of this gene, and enrich our understanding of the function of MAPK gene in mulberry.Based on the previous reported about mulberry MAPK information, this study further completed the comparative bioinformatics analysis of MnMAPK5, expression profile analysis, upstream promoter sequence analysis, subcellular localization and so on. We constructed MnMAPK5 overexpression vector, transferred into Arabidopsis, successfully obtained 5 MnMAPK5 overexpression transgenic Arabidopsis, then treated with high salt, drought, low temperature, high temperature, oxidation stress, ABA, SA, MeJA to measure the seed germination rate, root long after germination, the stress phenotype in vegetative growth stage Arabidopsis to confirm its role. In addition, H2O2 content, proline content, MDA content, soluble sugar content, antioxidant enzyme activity, expression of stress related genes and other physiological index were measured to explore mechanism of MnMAPK5 in stress response. The results of the study are as follows: 1. The bioinformatics analysis and upstream promoter sequence analysis of MnMAPK5Based on previous studies, MnMAPK5 was analysised by genetic evolution analysis, multiple sequence alignment with other group B MAPK were carried out. Multiple sequence alignment found that like other group B MAPK, MnMAPK5 protein structure is highly conserved, containing 11 conserved domains, P-loop, A-loop, C-loop, docking domain and TEY phosphorylation site. Group B MAPK evolution analysis indicate that MnMAPK5 always get together with apple, cotton, tomato, Arabidopsis MAPK. The similarity of MnMAPK5 and MdMPK13-2, GrMPK12, SlMPK4, NtMPK4 reached more than 83%. While NtMPK4, AtMPK4, ZmSIMK1, AtMPK13 which are highly similarity with MnMAPK5 can be induced by a variety of stress, speculating that MnMAPK5 may play a role in the stress response.PlantCARE online prediction software predict that MnMAPK5 promoter sequences contain various cis-acting elements, including defense and stress responsiveness-, hormone responsiveness-, meristem expression and endosperm expression-related cis-acting elements, which provides the possibility for MnMAPK5 responsing to various stress and signal pathways. We connect the GUS gene with the MnMAPK5 promoter sequence, constructed ProMnMAPK5-GUS vector, then transformed into Arabidopsis, successfully obtained 3 transgenic Arabidopsis. Under high salt, high temperature, low temperature and drought stress, the GUS signal increased in the beginning, and then became lower in transgenic Arabidopsis, which showed that MnMAPK5 promoter could be involved in the stress response. Otherwise, the activity of MnMAPK5 promoter was observed in different stages of Arabidopsis. Results found that GUS signal can be clearly detected in 3 days after seedling germination. With the growth of plants, the GUS signal is getting stronger and stronger, indicating that MnMAPK5 promoter may participate in the growth of seedlings after germination. The activity of MnMAPK5 promoter in different Arabidopsis tissues showed that GUS signal can be detected root, stem, leaf, calyx, filament, anther, stigma and long pods, but its expression levels in different tissues were also different, the expression level of MnMAPK5 in calyx, filament and anther were lowest, only in the microtubule organization exits less expression. In conclusion, GUS genes drove by the MnMAPK5 promote rcan response to various stress conditions and GUS sinal were expressed in various growth stages and tissues, which shown tissue-specific expression patterns. This provides a basis for understanding the characteristics of MnMAPK5 gene and how MnMAPK5 gene response to various stress and signaling pathways. 2. The subcellular localization and expression profile analysis of mulberry MAPK5subcellular localization prediction software predict that the MnMAPK5 can located in both nucleus and cytoplasm. Using cNLS Mapper and NucPred nuclear localization signal prediction software predict that the nuclear localization signal of MnMAPK5 are weak. In order to further confirm the localization of MnMAPK5 in cells, the 35S-MnMPK5:EGFP and 35S-EGFP overexpression vectors were constructed and transiently transformed into onion epidermal cells. The results showed that MnMAPK5 is located in both the cytoplasm and nucleus of onion epidermal cells, suggesting that MnMAPK5 may not only can act as a part of the signal transduction remaining in the cytoplasm to phosphorylate downstream specific target proteins, but also could enters the nucleus to activates the transcription factor, regulate of gene expression. These further illustrates the MnMAPK5 may respond to a variety of stress, participate in multiple signal paths. All of these further illustrateed that MnMAPK5 might be involved in many signal pathways and response to multiple stresses.Expression profile analysis discovered that MnMAPK5 is expressed in all parts of the mulberry, especially in root, stem, leaf, bark, flower and fruit, but the expression levels in different tissues were also different, its expression in stem and fruit were highest and in flowers was lowest. Stress inducible expression analysis in Yuesang No.69851 showed that the expression level of mulberry MAPK5 gene was upregulated in leaves treated with 40℃ and in stem treated with 30%PEG; the expression level of mulberry MAPK5 genes appeared to incease first and then decline in leaves after 4℃, 30%PEG and MeJA treatment, in stems after 4℃ and ABA treatment, in roots after 4℃ and 40℃ treatment; while the expression level of mulberry MAPK5 genes was significantly inhibited in leaves after SA treatment, in stems after NaCl, H2O2 and SA treatment, in roots after 30%PEG, NaCl and SA treatment; the expression level of mulberry MAPK5 genes in leaves and roots after ABA and H2O2 treatment, in stems after MeJA treatment appeared to decline first and then incease; In addition, mulberry MAPK5 gene expression pattern is more complicated in leaves after NaCl and H2O2 treatment, in roots after MeJA treatment. Obviously, the expression pattern of mulberry MAPK5 gene in different stress is different, which may be related to the different functions of the gene expression in different stress. 3. Function study of MnMAPK5 transgenic Arabidopsis in different stressesWe transformed MnMAPK5 genes into Arabidopsis through floral dip method, successfully obtained 5 positive MnMAPK5 overexpressed transgenic Arabidopsis. Among them, the T3 generation transgenic Arabidopsis whose expression level of MnMAPK5 were high(OE3), medium(OE5) and low(OE1) were selected as materials to further research its function under different stress.Under salt stress, the germination rate and the root length in OE lines was significantly lower than that of WT, Growth condition in OE lines was worse than that of WT. Thus indicated that MnMAPK5 overpression transgenic Arabidopsis reduced the resistance to salt stress. In addition, physiological indexes and stress related genes were tested under 300 mM NaCl treatment, the results showed that the content of MDA in OE lines was significantly higher than that of WT. While the the soluble sugar content, the CAT activity, the expression level of CAT and RD22 were significantly lower than that of WT, these results above indicated that MnMAPK5 under salt stress conditions produce excessive H2O2 by reducing intracellular CAT expression level, CAT enzyme activity, soluble sugar content, generate lipid peroxidation, destroy the structure of cell membrane; On the another hand, the stress-related genes, such as the RD22 and CAT gene were declined to reduced the resistance to salt stress. It also suggested that MnMAPK5 may plays a negative regulatory role in responsing to NaCl stress.Under drought stress, the germination rate and the root length in OE lines were significantly lower than that of WT, the phenotype of wilting and death were more serious than of WT. Thus indicated that MnMAPK5 overpression transgenic Arabidopsis reduced the resistance to drought stress. Moreover, physiological indexes and stress related genes under drought treatment were tested, the results showed that the content of H2O2 and MDA in OE lines were significantly higher than that of WT. While the content of proline and soluble sugar, the activity of POD and CAT, the expression level of POD, CAT and MYB44 were significantly lower than that of WT. As stated above, MnMAPK5 under drought stress conditions produce excessive H2O2 by reducing expression level of POD and CAT, activity of POD and CAT, content of proline and soluble sugar, generate lipid peroxidation, destroy the structure of cell membrane; On the another hand, the stress related genes, such as the POD, CAT and MYB44 gene to reduced the resistance to salt stress.Under cold stress, the germination rate in OE lines were slightly lower than that of WT, the chlorophyll content was in OE lines significantly lower than that of WT, the phenotype of wilting and death were more serious than of WT, indicating that MnMAPK5 transgenic Arabidopsis reduced the resistance to cold. Moreover, physiological indexes and stress related genes under cold treatment were tested, the results showed the content of proline and soluble sugar, the activity of POD and CAT, the expression level of POD, CAT, RAB18, RD29 A and RD22 were significantly lower than that of WT. As stated above, MnMAPK5 under cold conditions can not increase the resistance to cold by increasing content of proline and soluble sugar, the activity of POD and CAT, the expression level of POD, CAT, RAB18, RD29 A and RD22. At 35℃, the germination rate of WT plants was significantly higher than that of OE, but its root length exhibits no significant difference, which suggested that MnMAPK5 may have an influence on the germination of plants under heat conditions.Under oxidative stress, the germination rate in OE lines were significantly lower than that of WT and the growth condition in WT were better than that in OE transgenic Arabidopsis. It was showed that MnMAPK5 in Arabidopsis decreased the resistance to oxidative stress. Moreover, physiological indexes and stress-related genes under oxidative treatment were tested, the results showed that content of MDA in OE lines were significantly higher than that of WT. While the content of proline and soluble sugar, the expression level of POD, CAT and ABF4 were significantly lower than that of WT. As stated above, MnMAPK5 under oxidative stress conditions on the one hand produce excessive H2O2 and produce peroxidation damage by inhibiting the activity of antioxidant enzymes to reduce oxidative stress resistance, on the other hand inhibit the content of osmoregulation substance such as proline, soluble sugar to increase the sensitivity to oxidative stress.The expression of OE transgenic Arabidopsis under hormone stress found that the seed germination rate and root long in OE lines under ABA treatment were reduced; The growth condition in OE transgenic Arabidopsis under MeJA treatment at the vegetative growth stage were better than that in WT; The seed germination rate in OE transgenic Arabidopsis under SA treatment at 5 days after seed germination were lower than that in WT, and the chlorosis phenomenon were more serious in OE transgenic Arabidopsis. Taken together, MnMAPK5 may be involved in ABA, SA, MeJA signal transduction processIn conclusion, MnMAPK5 protein contained 11 conserved domains, P-loop, A-loop, C-loop, docking domain and TEY phosphorylation site. The similarity of MnMAPK5 with MdMPK13-2, GrMPK12, SlMPK4, NtMPK4 reached more than 83%. MnMAPK5 promoter sequences contain various cis-acting elements, including defense and stress responsiveness-, hormone responsiveness-, meristem expression and endosperm expression-related cis-acting elements, which provides the possibility for MnMAPK5 responsing to various stress and signal pathways. The transgenic Arabidopsis harbouring GUS gene controlled by MnMAPK5 promoter can respond to high salt, high temperature, low temperature and drought stress at different time points, indicating that the MnMAPK5 promoter could be involved in the stress response. Subcellular localization experiment showed that MnMAPK5 is located in both the cytoplasm and the nucleus of onion epidermal cells, suggesting that MnMAPK5 may not only can act as a part of the signal transduction remaining in the cytoplasm to phosphorylate downstream specific target proteins, but also could enters the nucleus to activates the transcription factor, regulate of gene expression. Expression profile analysis discovered that MnMAPK5 could expressed in all parts of the mulberry, especially in root, stem, leaf, bark, flower and fruit, but the expression levels in different tissues were also different, its expression in stem and fruit were highest and in flowers was lowest. Stress-inducible expression analysis discovered that mulberry MAPK5 gene can be induced by various stress and plant hormones, but stress response mode difference were great in different adversity stresses, which may be related to the different roles it played in different stresses. MnMAPK5 transgenic Arabidopsis under different stress conditions found that MnMAPK5 may reduced resistance to NaCl, PEG, heat, cold, H2O2 stress by reducing the antioxidant enzymes activity and expression level of the corresponding stress-related gene expression, increasingthe accumulation of ROS. |
PDF Full Text Request