【作者】 杜照奎；

【导师】 钟章成； 董鸣；

【作者基本信息】 西南大学 ， 生态学， 2014， 博士

【摘要】 大量氯氟烃类(Chlorofluorocarbons,CFCs)气体由于人类的工业活动被释放到空气中,致使大气臭氧层变薄,从而引起太阳辐射中到达地面的中波紫外线(Ultraviolet-B,UV-B,280-320nm)辐射增强,UV-B辐射增强对陆生植物个体、种群及生态系统都产生了较为明显的影响。花生(Arachis hypogaea L.)为豆科落花生属一年生草本植物,富含脂肪和蛋白质,为主要食用植物油来源,目前,有关UV-B辐射增强对花生的生理生态影响报道较少,其影响的分子机制也鲜有探索。本研究以“小京生”花生为试验材料,分析UV-B辐射增强对其幼苗形态、气体交换参数、叶绿体超微结构、活性氧水平、抗氧化酶活性和抗氧化剂含量等的影响；并通过蛋白质组学方法研究UV-B辐射增强对花生蛋白表达的影响；在此基础上,克隆受UV-B辐射增强影响表达下调的果糖-1,6-二磷酸醛缩酶(fructose-1,6-bisphosphate aldolase,FBA)基因AhFBA,明确其生物信息学特征及受UV-B影响的转录情况；将AhFBA基因导入大肠杆菌BL21(DE3)并进行诱导表达,观察宿主菌在UV-B增强处理下的生长情况,旨在回答以下几个问题：(1)UV-B辐射增强如何在生理生态水平对花生造成影响?(2)花生在蛋白质水平如何响应UV-B辐射增强处理?(3)FBA蛋白在抵御UV-B辐射增强中是否有作用?研究结果如下：在补增在强度为54μW/cm2的UV-B辐射处理3d后(每天处理8h),花生叶绿素a和叶绿素(a+b)含量均显著下降(P<0.05),同时,气体交换参数也受到显著影响(P<0.05),表现出净光合速率(Pn)降低了35.42%,气孔导度(Gs)、蒸腾速率(Tr)和气孔限制值(Ls)分别下降了28.12%、26.37%和20.25%,然而,胞间CO2浓度(Ci)上升了16.22%,较对照有显著性差异(P<0.05),表明非气孔限制因素是Pn下降的主要原因。同样,荧光参数Fv/Fm和Fv/Fo均显著降低(P<0.05)。但UV-B处理1d和2d后,以上各指标的变化与对照差异不显著(P>0.05)。透射电子显微镜(TEM)图像显示,处理3d的花生叶片叶绿体膜肿胀、基质类囊体解体,光同化产物淀粉粒数目和大小减少。补增UV-B辐射处理下,花生叶片相对电导率、超氧阴离子(O2·-)、类黄酮和可溶性蛋白质的含量,以及过氧化氢酶(CAT)、过氧化物酶(POD)活性较对照显著升高(P<0.05)；超氧化物歧化酶(SOD)活性先显著升高后降低；过氧化氢(H202)、丙二醛(MDA)、抗坏血酸(AsA)和谷胱甘肽(GSH)含量,以及抗坏血酸过氧化物酶(APX)活性没有显著变化(P>0.05)。可见增强的UV-B能显著提高花生叶片内的活性氧(ROS),而且O2·-是对花生产生氧化胁迫的主要因素,花生幼苗主要通过提高类黄酮含量以及SOD、CAT和POD活性来抵制ROS的胁迫,AsA-GSH循环系统在花生清除ROS中效果不明显。补增UV-B辐射处理下,花生叶片中共检测到丰度变化在2.5倍以上差异表达蛋白点39个(其中22个蛋白质表达上调,17个表达下调),经过MALD1-TOF-TOF分析及数据库检索,成功鉴定出其中的27个蛋白质。被鉴定的27个蛋白质按其功能大致可归为八大类,第Ⅰ类：光合作用相关的蛋白,包括质体蓝素(plastocyanin,PC)、1,5-二磷酸核酮糖羧化酶(Rubisco)小亚基、放氧复合物增强子蛋白1、PsbP结构域蛋白6；第Ⅱ类：糖代谢相关蛋白,包括苹果酸脱氢酶(malate dehydrogenase,MDH)和FBA；第Ⅲ类：能量合成相关蛋白,包括ATP合酶；第Ⅳ类：氨基酸代谢相关蛋白,包括半胱氨酸合成酶；第Ⅴ类：蛋白质加工相关蛋白,包括热激蛋白(heat shock protein, HSP);第Ⅵ类：蛋白质翻译相关蛋白,包括核糖体循环因子(ribosome recycling factor, RRF);第Ⅶ类：防御相关蛋白,包括几丁质酶、过氧化物酶、Cu-Zn超氧化物歧化酶、二羟肉桂酸3-O-转甲基酶(caffeic acid3-O-methyltransferase, COMT),类萌发素蛋白(Germin-like protein, GLPs)和病程相关蛋白(pathogenesis-related protein, PR);第Ⅷ类,未知功能蛋白。表明增强的UV-B辐射可能会通过下调PsbP和RRF的表达破坏花生叶绿体类囊体结构、下调PC表达降低光合电子传递速率、下调Rubisco和光合放氧增强蛋白的表达降低光合速率、下调MDH和FBA的量降低糖代谢速率和下调ATP合酶降低能量产生速率等方式影响花生的生理代谢水平；同时,花生也可能通过上调HSP促进新生蛋白质的折叠,包装及跨膜运输、上调COMT促进木质素的合成、上调GLP表达令细胞壁更为致密,抵抗病毒、细菌感染或采食天敌的侵袭、上调PR表达抵御真菌侵染和上调半胱氨酸合成酶促进GSH的合成,维持细胞内的还原态。以大豆(Glycine max)果糖-1,6-二磷酸醛缩酶基因(GmFBA,GenBank登录号：AY492006.1)作为查询探针,在NCBI网站花生EST数据库中进行BLAST检索,选取与之高同源的花生EST序列,通过EST序列拼接获得一条重叠群(contig),再以拼接后的contig作为新的查询探针,继续搜索花生EST数据库,直至无新的EST序列拼接为止。以最终拼接的contig作为花生的FBA基因,并以该序列为模板设计上下游引物。提取花生总mRNA,后转录得cDNA。以cDNA为模板,加入上下游引物,在55℃退火条件下,PCR扩增得到花生果糖-1,6-二磷酸醛缩酶基囚,长度为1077bp,命名为AhFBA,(?)该序列提交NCBI网站,GenBank登录号为KF470788。AhFBA基因编码蛋白分子式为C1706H2725N469O524S6,由358个氨基酸组成,所含氨基酸组成以Ala最多,酸性氨基酸(Asp和Glu)和碱性氨基酸(Lys和Arg)个数均为40,相对分子质量分别为38.38kD,等电点分别为6.73,不稳定指数分别为30.03,属于稳定蛋白质,无信号肽序列。AhFBA蛋白定位于细胞质中的可能性最大,分值为0.65,定位于线粒体基质、叶绿体内囊体膜的分值均为0.1,而定位于内质网的分值为O。AhFBA蛋白中有2个酰胺化位点(36和285位),1个cAMP依赖的蛋白激酶磷酸化位点(41位),3个蛋白激酶Ⅱ磷酸化位点(114、270和309位),7个蛋白激酶C磷酸化位点(50、201、235、247、304、309和336位),7个N-豆蔻酰化位点(25、97、127、137、232、335和346位),1个醛缩酶活性中心(217-227位,序列为VLLEGTLLKPN),AhFBA属于Ⅰ型细胞质FBA基因。AhFBA蛋白与同属豆科的豌豆、鹰嘴豆、蒺藜苜蓿、菜豆和花生的同源性很高。实时荧光定量PCR结果显示,在自然光照生长条件下,AhFBA基因在根、叶、花和幼果中均有表达,且表达量无显著差异(P>0.05)。但是在受到UV-B辐射增强处理后,该基因的表达发生了变化,照射4h后,AhFBA基因在叶中表达上调了10倍以上,接着表达量开始下调,照射8h、16h和24h后,表达量下调了0.75、0.86和0.29倍。表明AhFBA基因属于UV-B诱导型基因,该基因参与了对UV-B胁迫的应答。根据PCR获得的AhFBA序列设计引物(上下游引物分别引入BamHⅠ和HindⅢ酶位点)扩增其编码区序列,用BamH1和Hind Ⅲ分别双酶切花生AhFBA基因和原核表达载体pET28a,双酶切产物通过胶回收试剂盒进行纯化,连接两种胶回收产物,菌液PCR验证和测序结果表明已经成功构建含有目的基因的重组表达载体pET28a-AhFBA。采用热激法将重组表达载体转化大肠杆菌表达菌BL21(DE3)感受态细胞,异丙基硫代半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测发现在40kD附近出现一条明显的蛋白条带,表明目的蛋白AhFBA诱导表达成功。IPTG诱导宿主菌2h后开始接受一定时间的UV-B照射,照射后的菌液接种到新的LB培养基中(含卡那霉素),每隔1h测定一次OD600值,绘制12h内的生长曲线。结果显示,转化pET28a-AhFBA和pET28a的宿主菌BL21(DE3)在接受UV-B照射3min后与对照(转化pET28a的宿主菌BL21(DE3)未照射UV-B)的生长并无明显差别,可见短时间的UV-B照射并未对宿主菌的生长产生不利影响,同时,外源蛋白AhFBA的表达也并未影响到BL21(DE3)的生长；而接受9minUV-B照射后,转化pET28a-AhFBA和pET28a的宿主菌BL21(DE3)生长速率均低于对照,但前者却高于后者,表明外源重组蛋白AhFBA可能有助于宿主菌减轻UV-B照射的不利影响。当UV-B照射12mmin后,转化pET28a-AhFBA和pET28a的宿主菌BL21(DE3)生长速率均低于对照,且两者之间没有差别,表明外源重组蛋白AhFBA能在一定照射时间范围内缓解UV-B辐射对BL21(DE3)的伤害。更多还原

【Abstract】 A large number of chlorofluorocarbons (CFCs) discharged into the air due to human industrial activity lead to thinning of the ozone layer. As a result, ultraviolet-B (UV-B,280-320nm) in the solar radiation reach to the ground is enhanced. Many researches have pointed out that elevated UV-B radiation had significant impact on individuals, populations of terrestrial plants and total ecosystems. Peanut (Arachis hypogaea L.) is an annual legume crop, which is rich in fat and protein. At present, the effects of enhanced UV-B radiation on physio-ecological of peanut is seldom reported, as well as the molecular mechanisms. In this study, cultivar "Xiaojingsheng" peanuts was chosen as test materials, the effects of enhanced UV-B radiation on factors including seedling morphology, gas exchange parameters, changes in chloroplast ultrastructure, ROS levels, antioxidant enzyme activity, antioxidant content, and proteome expression profiling have been analyzed. Fructose-1,6-bisphosphate aldolase (FBA) gene AhFBA which was down-regulated by enhanced UV-B radiation, were cloned and analyzed the transcription level of gene AhFBA treaed under enhanced UV-B by real-time quantitative reverse transcription polymerase chain reaction. AhFBA gene in E. coli BL21(DE3) were also expressed and observed the growth curve of the host cell contained recombinant vector pET28a-AhFBA treated with UV-B. The main purpose of this paper is to answer the following questions:(1) What are the effects of enhanced UV-B radiation on peanut in physio-ecology level?(2) How does peanut proteome respond to enhanced UV-B radiation?(3) Whether FBA proteins play a role in resisting enhanced UV-B radiation? The results are as follows:The impact of UV-B radiation on photosynthetic related parameters and chloroplast ultrastructure of A. hypogaea were studied, after an enhanced UV-B irradiation performed8h per day for3days with ultraviolet light (280-320nm) that maintained about54uW/cm2radiations. Contents of chlorophyll a and chlorophyll (a+b) declined significantly after radiation for3days (P<0.05), concomitantly, gas exchange parameters were significantly affected (P<0.05), showing a35.42%reduction in net photosynthesis (Pn),28.12%in stomatal conductance (Gs),26.37%in transpiration rate (Tr) and20.25%in stomata limitation(Ls), however, intercellular CO2concentration (Ci) was increased by16.22%, showing significant difference with that in control (P<0.05), at the end of3days UV-B treatment. Similarly, the fluorescence parameters of Fv/Fm and Fv/Fo were also significantly reduced (P<0.05). However, the differences of above indexes were not significant compareed with the control treated with UV-B after1and2days (P>0.05). Transmission electron microscope (TEM) images revealed that the peanut treated for3d whose chloroplast membranes were swelled and disintegrated, and stromal thylakoids were relaxed and parallel to each other, which might be responsible for worse performance of the photosynthesis. As a result, the size and number of starch grains decreased.The contents of superoxide anion (O2-), flavonoids, and soluble proteins, the activities of catalase (CAT) and peroxidase (POD) in peanut leaves were increased significantly treated with supplementary ultraviolet-B (UV-B), as well as relative electrical conductivity (P<0.05). The activity of superoxide dismutase (SOD) increased at first and then decreased. Nevertheless, there were no significant changes in the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), ascorbic acid (AsA) and Glutathione (GSH), the activities of ascorbate peroxidase (APX) in leaves (P>0.05). These results indicated that elevated UV-B could increase the content of reactive oxygen species (ROS) in peanut leaves, and O2-was the principal factor for oxidative stress. Peanut seedlings resisted the stress of ROS mainly through increasing the content flavonoid and the activities of SOD, CAT and POD. However, As A-GSH circulatory system had no unobvious effect on clearing ROS.A total of39protein spots were differentially expressed by at least2.5fold compared with the controls (22proteins were down-regulated and17were up-regulated) after treatment with supplementary UV-B radiation. Of those protein spots,27were successfully identified by MALDI TOF/TOF MS after a database search. Those27proteins could be classified into eight categories according to their functions:class1, photosynthesis (plastocyanin, ribulose-1,5-bisphosphate carboxylase small subunit, oxygen-evolving enhancer protein1, PsbP domain-containing protein6); class11, carbohydrate metabolism (malate dehydrogenase, and fructose-bisphosphate aldolase); class Ⅲ, energy synthesis (ATP synthase); class IV, amino acid biosynthesis (cysteine synthase); class V, protein biosynthesis (ribosome recycling factor); class VI, protein processing (heat shock proteins); class VII, defense responses (chitinase, peroxidase, Cu-Zn SOD, caffeic acid3-O-methyltransferase, and germin-like protein); class VIII, unknown proteins. In conclusion, we hypothesized that the enhanced UV-B radiation caused a decrease in the photosynthesis rate of peanut leaves mainly via three mechanisms. First, enhanced UV-B may down-regulate the expression of ribosome recycling factor, which caused a decrease in the expression of subunit PsbP in photosystem Ⅱ, thus destroying the thylakoid membrane structure. Second, the reduced plastocyanin expression may have induced a decrease in photosynthetic electron transport efficiency. Third, the down-regulation of ribulose-1,5-bisphosphate carboxylase and fructose-1,6-bisphosphate aldolase resulted in a decrease in carbon assimilation. At the same time, peanut may also enhance its resistance to UV-B stress by increasing the expressions of antioxidant enzymes and non-enzymatic antioxidants, germin-like proteins, pathogenesis-related proteins, and heat shock proteins.Using soybean(Glycine max) fructose-1,6-bisphosphate aldolase gene (GmFBA, GenBank accession number:AY492006.1) as queries, BLAST searches in peanut EST database on the NCBI website were performanced, peanut EST sequences with high homology to soybean FBA gene were selected, a contig named AhFBA was obtained by in silico cloning technology from the selected ESTs. AhFBA was confirmed by RT-PCR, molecular cloning and sequencing, the complete conding sequence (CDS) of AhFBA was1077bp in length, and the sequnence was submitted to NCBI database with an accession number KF470788.AhFBA gene encoding a protein AhFBA composed of358amino acids with the formula C1706H2725N469O524S6, in which Ala was the most abundant amino acid, the number of acidic amino acids (Asp and Glu) and basic amino acids (Lys and Arg) were all40, the relative molecular mass was38.38kD, isoelectric point was6.73, instability index was30.03, and it had no signal peptide sequence. Bio informatics forecast that AhFBA protein was in most cases located in the cytoplasm with the score of0.65, scores that located in the mitochondrial matrix and chloroplast thylakoid membrane were all0.1and localized in the endoplasmic reticulum was0. AhFBA protein has2amide sites (36and285), a cAMP-dependent protein kinase phosphorylation sites (41),3protein kinase II phosphorylation sites (114,270and309),7protein kinase C phosphorylation sites (50,201,235,247,304,309and336),7N-myristoylation sites (25,97,127,137,232,335and346), an aldolase activity center (217-227bit sequence VLLEGTLLKPN), AhFBA belongs to type I cytoplasmic fructose-bisphosphate aldolase gene.Real-time PCR results showed that:when the A. hypogaea was grew in natural lighting conditions, AhFBA gene was constitutively expressed in roots, leaves, flowers and young fruit, and there is no significant differences in expression levels among them (P>0.05). However, AhFBA gene’s expression up-regulated in leaves more than10times when the plant treated with enhanced UV-B for4h, and then down-regulated after treated8h,16h and24h, reduced the expression of0.75,0.86and0.29times. It indicated AhFBA belongs to UV-B inducible gene, which participated in the UV-B stress response.Primers (forward and reverse primer was introduced BamH I and Hind III restriction enzyme site, respectively) were designed according to AhFBA gene, and the sequence was amplified by PCR. The fragment of vector pET28a and AhFBA was connected after digested with BamH1and Hind III enzymes. PCR and DN A sequencing results verified that the recombinant expression vector pET28a-AhFBA has been successfully constructed containing the target gene AhFBA. Heat shock method was employed to transforme pET28a-AhFBA into E. coli expression strain BL21(DE3), isopropyl thiogalactoside (1PTG) induced the expression of gene AhFBA and about40kD protein band was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The host bacteria were irradiated by UV-B for certain minutes after induced by IPTG, then, they were inoculated into new LB medium that contains Kanamycin, OD600value was measured every one hour. The results showed that:there are no significant differences in the growth anmong bacterial BL21(DE3) contain pET28a-AhFBA and pET28a with control (host strain BL21(DE3) contains pET28a unirradiated UV-B) after receiving UV-B irradiation for3min. Accepted9min UV-B irradiation, host strain BL21(DE3) contain pET28a-AhFBA and pET28a growth rates were lower than control, but the former is higher than the latter. The growth rates of BL21(DE3) contain pET28a and pET28a-AhFBA were both lower than control after12min UV-B irradiation, and there is no difference between them, indicating that exogenous recombinant protein AhFBA may help the host bacteria mitigate the adverse effects of UV-B radiation to some extent.更多还原