【作者】 张梦；

【导师】 赵爱春；

【作者基本信息】 西南大学 ， 植物学， 2016， 硕士

【摘要】 植物在生长发育的过程中,不可避免的会遇到各种各样的不利环境,特别是高温、低温、高盐、干旱、氧化损伤等非生物胁迫,它们严重的影响了植物的生长发育及产量,因此研究植物胁迫响应基因具有十分重要的意义。为了适应环境变化,植物自身形成了一套复杂的机制来适应环境的变化。这种机制离不开胁迫信号的识别、转导、传递及相应基因的表达。蛋白激酶是生物内普遍存在的一种响应外界变化的调节激酶,在信号转导、传递中发挥重要的作用。目前研究的参与植物生物胁迫和非生物胁迫的蛋白激酶主要有五类:受体蛋白激酶(receptor-like kinase,RLK)、促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK,MPK)、钙依赖而钙调素不依赖蛋白激酶(calcium-dependent and calmodulin-independent protein kinase,CDPK)、蔗糖不发酵相关蛋白激酶(sucrose non-fermenting-1-related protein kinase,Sn RK)及其它胁迫相关的植物蛋白激酶。MAPK基因作为MAPK级联途径中最下游的蛋白激酶,可通过逐级磷酸化作用,将细胞外的信息级联放大,调控下游基因的表达,与其他基因一起形成庞大的网络,调节生物体内各种生理反应,响应外界环境。目前为止,MAPK基因家族已经在拟南芥、烟草、水稻、玉米、二穗短柄草、棉花、番茄、葡萄、杨树、香蕉和苹果等多种植物中被鉴定。MAPK虽然在多种植物中被鉴定,但其在逆境胁迫中的功能特性及作用机制的研究多集中在草本植物中,在木本植物中还很少。桑树是一种多年生的木本植物,具有耐旱、耐盐碱、耐涝、耐寒、抗逆性强和生长迅速的特性,而桑树抗逆性方面的研究极少。我们以前的研究已从桑树中鉴定了32个Mn MAPKKK,5个Mn MAPKK和10个Mn MAPK。q RT-PCR分析10个桑树MAPK基因在不同胁迫处理下表达情况发现,不同的桑树MAPK在相同的胁迫处理下表达量有所不同,同一桑树MAPK在不同的胁迫处理下表达情况也有所差异,表明桑树MAPK在不同的胁迫处理中可能发挥不同的作用。同时,桑树MAPK5在桑树不同胁迫处理后发现,桑树MAPK5表达量在Na Cl,高温和低温处理后上调,但仅在高温和低温胁迫下存在显著性差异,在脱落酸(abscisic acid,ABA),水杨酸(salicylic acid,SA)和H2O2处理后其表达量显著降低,在干旱和茉莉酸甲酯(Methyl Jasmonate,Me JA)处理后其表达量稍稍降低。桑树MAPK5表达量在不同的胁迫中都表现表达模式不同,暗示桑树MAPK5在逆境胁迫中可能发挥着重要的作用。因而我们选择桑树MAPK5进行功能研究,以期阐明该基因的功能,丰富我们对桑树MAPK基因在桑树胁迫功能的理解。本研究基于已报道的桑树MAPK信息,完成了Mn MAPK5的生物信息学分析,表达谱分析,上游启动子序列分析和亚细胞定位等。通过构建Mn MAPK5过表达载体,以农杆菌为介导转入到拟南芥中,成功获得5个Mn MAPK5过表达转基因拟南芥株系,并对其进行高盐、干旱、低温、高温、氧化等胁迫处理及ABA、SA和Me JA等激素诱导,通过测定种子萌发率,萌发后根长,营养生长阶段胁迫后的生长状况等方法确认其在不同胁迫处理下影响。此外又通过测定H2O2含量,脯氨酸含量,丙二醛(Malondialdehyde,MDA)含量,可溶性糖含量,抗氧化酶活性,胁迫相关基因的表达等指标来探究Mn MAPK5在胁迫响应中的机理。研究结果如下:1.Mn MAPK5基因的生物信息学分析及其启动子分析基于之前的研究,对B族的MAPK基因进行了进化分析,多序列比对分析。多系列比对结果发现,Mn MAPK5蛋白与其他物种B族MAPK蛋白结构保守性很高,含11个保守结构域,在第VII和第VIII结构域之间有TEY的双磷酸化位点,存在P-loop、A-loop、C-loop及docking domain等保守位点。B族MAPK系统进化分析表明,Mn MAPK5与苹果,棉花的B族MAPK聚在一起,且与Md MPK13-2、Gr MPK12、Sl MPK4、Nt MPK4的相似性很高,达到83%以上。而与Mn MAPK5相似性较高的Nt MPK4、At MPK4、Zm SIMK1和At MPK13能够被多种胁迫所诱导,暗示Mn MAPK5可能和它们有相似的功能。Plant CARE在线预测软件预测Mn MAPK5启动子序列中含多种与植物胁迫响应、激素信号和生长发育相关的顺式作用元件,暗示Mn MAPK5可能参与多种胁迫及信号路径。用Mn MAPK5启动子驱动GUS基因,构建Pro Mn MAPK5-GUS载体并转化到拟南芥中,成功获得3株转基因拟南芥。Mn MAPK5启动子实验中发现,转基因拟南芥在受到高盐、高温、低温和干旱胁迫时,GUS信号表现出先增加后降低的趋势,说明Mn MAPK5启动子能够参与这些胁迫响应。观察Mn MAPK5启动子驱动的GUS基因在拟南芥不同时期表达发现,GUS信号在幼苗萌发后3天就可以被清晰的观察到,随着植物的生长,GUS信号越来越强,Mn MAPK5启动子驱动的GUS基因在拟南芥的根、茎、叶、花萼、花丝、花药、柱头及长荚果中都有表达,暗示,桑树Mn MAPK5启动子是一个组成型启动子。2.Mn MAPK5的亚细胞定位及表达谱分析亚细胞定位预测软件预测发现,Mn MAPK5在细胞核和细胞质中都有分布,核定位信号预测软件预测发现Mn MAPK5的核定位信号很弱。为确认Mn MAPK5蛋白在细胞中的分布,我们构建35S-Mn MAPK5:EGFP和35S-EGFP过表达载体,瞬时转化到洋葱表皮细胞中。结果发现,与EGFP在细胞中的分布一致,35S-Mn MAPK5:EGFP的融合蛋白在细胞的细胞核和细胞质中都有分布,说明Mn MAPK5在细胞质和细胞核处都有分布,暗示Mn MAPK5可能在细胞质和细胞核中都发挥作用。为确认Mn MAPK5在桑树中的表达情况,定量检测发现Mn MAPK5在川桑的根、茎、叶、皮、花、果各个组织都有表达,不同的组织中它们的表达量不同,在茎和果中的表达量最高,在花中的表达量最低,暗示Mn MAPK5是一个组成型表达的基因。桑树胁迫诱导表达分析发现,40℃处理的叶中和30%PEG处理后的茎中,桑树MAPK5基因的表达量显著性上调;在40℃处理的根中,4℃处理后的根、茎、叶中,30%PEG处理后的叶中,ABA处理后的茎中及Me JA处理后的叶中,桑树MAPK5基因的表达呈先增加后降低的趋势;在30%PEG处理后的根中,Na Cl处理的根和茎中,H2O2处理的茎中及SA处理后的根茎叶中,桑树MAPK5基因的表达显著受到抑制;在ABA处理后的根叶中,H2O2处理的根叶中及Me JA处理后的茎中,桑树MAPK5基因的表达则呈先降低后增加的趋势;而在Na Cl和H2O2处理的叶中及Me JA处理后的根中,桑树MAPK5基因的表达模式更为复杂。由此可见,桑树MAPK5基因的表达响应模式在不同胁迫中差异显著,暗示,桑树MAPK5可能在不同胁迫中有不同的功能。3.Mn MAPK5转基因拟南芥在不同胁迫下的功能研究通过浸花法转化拟南芥,获得了5株Mn MAPK5阳性的转基因拟南芥,选择Mn MAPK5表达量高(OE3),中(OE5),低(OE1)的转基因拟南芥作后续材料来进一步研究该基因在不同胁迫下的功能。在盐胁迫条件下,OE拟南芥显著的比WT的萌发率低,根长短,长势差。由此可见,Mn MAPK5在转基因拟南芥中降低了对盐胁迫的抗性。此外,测定300m M Na Cl胁迫后的一些生理指标和胁迫相关基因发现,OE拟南芥中的MDA含量显著的比WT的高,而可溶性糖的含量,CAT表达量及酶活性和RD22表达量都比WT低。表明Mn MAPK5在盐胁迫条件下一方面通过降低细胞内CAT的表达量及酶活和可溶性糖含量,使细胞内产生过量的H2O2,质膜发生膜脂过氧化物作用,破坏细胞膜的结构;另一方面通过降低胁迫相关基因,如RD22基因来降低对盐胁迫的抗性,暗示Mn MAPK5可能在Na Cl胁迫响应中起到了负调控的作用。干旱胁迫条件下,OE拟南芥的萌发率,根长比WT的要低,植株萎蔫、干枯死亡的现象比WT严重,由此可见,Mn MAPK5过表达的转基因拟南芥提高了对干旱胁迫的敏感性。此外,干旱胁迫后OE拟南芥中的H2O2、MDA含量显著的比WT要高,而脯氨酸含量、可溶性糖含量、POD活力及表达量、CAT活力及表达量和MYB44表达量都比WT低。综上所述,在干旱胁迫条件下,Mn MAPK5过表达的转基因拟南芥中的POD、CAT的表达量和活力都降低,产生大量H2O2,诱发细胞发生膜脂过氧化物作用,破坏细胞膜的结构,而细胞内的渗透调节物质脯氨酸和可溶性糖含量不能弥补干旱胁迫对其造成的伤害;另一方面,干旱胁迫下,Mn MAPK5还降低了MYB44基因的表达,从而降低了其对干旱胁迫的抗性。冷胁迫条件下,OE拟南芥萌发率稍比WT的低,叶绿素含量显著的比WT低,OE拟南芥出现萎蔫,干枯死亡的现象比WT严重,由此可见Mn MAPK5过表达的转基因拟南芥提高了冷胁迫敏感性。此外,冷胁迫条件下OE拟南芥脯氨酸含量,可溶性糖含量,POD活力及表达量,CAT活力及表达量,ABF4,RAB18,RD29A和RD22基因表达量都比WT中的低。以上结果表明,Mn MAPK5过表达的转基因拟南芥在冷胁迫条件下,并不能通过增加渗透调节物质脯氨酸,可溶性糖含量,POD和CAT含量及酶活和胁迫相关基因的表达来提高对低温的抗性。在35℃条件下,WT种子的萌发率显著的比是OE拟南芥高,但是其根长却没有显著的差异,暗示了Mn MAPK5在高温条件下可能对植物的萌发有影响更大。氧化胁迫下,OE拟南芥的萌发率、长势都不如WT,说明Mn MAPK5在拟南芥中降低了对氧化胁迫的抗性。此外,OE拟南芥的MDA含量显著的比WT高,而脯氨酸含量,可溶性糖含量,POD、CAT和ABF4表达量比WT低,暗示Mn MAPK5过表达的转基因拟南芥提高对氧化胁迫的敏感性很可能是Mn MAPK5过表达的转基因拟南芥在氧化胁迫条件下,H2O2积累,Mn MAPK5基因调控的抗氧化酶的表达量降低,破坏了细胞膜结构,而细胞本身并不能通过增加渗透调节物质脯氨酸,可溶性糖来抵抗氧化胁迫并通过过氧化作用破坏细胞结构降低对氧化胁迫的抗性。在研究激素诱导下对Mn MAPK5转基因拟南芥的影响发现,ABA诱导下,Mn MAPK5过表达的转基因拟南芥的种子萌发率及根长都降低;Me JA诱导下,营养生长阶段OE拟南芥的长势要比WT拟南芥长势好;SA诱导下,拟南芥种子萌发后5d时,WT种子萌发比OE拟南芥萌发率稍高,且幼苗萌发后WT黄化现象比OE拟南芥严重,暗示Mn MAPK5可能与ABA、SA、Me JA信号转导过程有关。综上所述,Mn MAPK5与其他物种B族MAPK一样,其蛋白结构保守性很强,都含有11个保守结构域,在第VII和第VIII结构域之间有TEY的双磷酸化位点,含P-loop、A-loop、C-loop及docking domain;Mn MAPK5与Md MPK13-2、Gr MPK12、Sl MPK4、Nt MPK4的相似性很高,达到83%以上。Mn MAPK5启动子序列中含有与胁迫相关的,植物激素相关的及植物生长发育相关的顺式作用元件,暗示Mn MAPK5可能参与多种胁迫和信号路径。含Mn MAPK5启动子驱动的GUS基因的转基因拟南芥能够在不同的时间点响应高盐、高温、低温、干旱胁迫,说明Mn MAPK5启动子能够参与这些胁迫响应。亚细胞定位实验发现,Mn MAPK5在细胞质和细胞核中都有定位,暗示可能在细胞质和细胞核中都有作用。表达谱分析发现Mn MAPK5在川桑的根、茎、叶、皮、花、果各个组织都有表达,在不同的组织中它们的表达量不同,在茎和果中的表达量最高,在花中的表达量最低;在受到不同的胁迫时,其表达模式差异较大,这可能与它在不同胁迫中所起的作用不同有关。而Mn MAPK5转基因拟南芥在不同胁迫处理后发现,Mn MAPK5转基因拟南芥可能主要是通过降低抗氧化酶的活性和表达量及相应的胁迫相关基因的表达,增加ROS的积累,降低了对Na Cl、PEG、低温、高温、H2O2胁迫的抗性。更多还原

【Abstract】 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.更多还原