【作者】 吴敏；

【导师】 陈守才； 唐朝荣；

【作者基本信息】 海南大学 ， 植物分子遗传学， 2011， 博士

【摘要】 天然橡胶是重要的工业原料和战略物资,另外,天然橡胶产业也是我国热带地区农业发展和经济增长的支柱产业。钾是作物生长过程中需要量较大的矿质营养元素,在调节植物细胞渗透势、细胞内多种酶活性以及光合产物运输等方面有非常重要的作用。同样,钾在橡胶树的生长发育过程中也发挥着重要作用,钾素营养缺乏会直接影响橡胶树的生长、产排胶、抗风、抗寒和抗病等。我国属于钾矿资源匮乏的国家之一,在钾肥的生产与需求间存在着极大的失衡。近年来,我国有相当一部分橡胶树的钾营养已处于正常值下限或严重亏缺。实际上,植物在营养性状方面存在基因型差异,植物营养性状的遗传学改良是提高不良土壤营养环境中作物产量的一条有效途径,近年来,有关植物营养缺乏应答分子机理的研究越来越多,但橡胶树钾营养比较生理学及营养遗传研究国内外尚属空白。本研究应用比较生理学阐述了低钾胁迫与钾营养供应正常条件下橡胶幼苗的形态、生理应答特性；应用抑制差减文库(SSH)技术和寡核苷酸基因芯片分离、筛选低钾胁迫橡胶幼苗差异表达基因,并对差异表达基因进行功能注释,同时,利用荧光实时定量PCR (QPCR)对部分差异表达基因的低钾应答特性进行了验证；应用cDNA末端快速扩增法(RACE法)克隆了橡胶幼苗低钾胁迫相关基因HbMPP1和HbCAB1的全长或部分cDNA,应用生物信息学方法对相应基因的编码蛋白做了生物信息学分析,同时,利用QPCR分析了它们在低钾胁迫下的时空表达特性。主要结果如下：1橡胶幼苗对低钾胁迫的形态、生理响应采用水培方法研究了橡胶幼苗在不同低钾胁迫(Omg/L、1mg/L、10mg/L、50mg/L)和钾营养正常供应(250mg/L)条件下的形态和生理特性,结果显示：(1)橡胶幼苗的株高、根长、茎粗等形态学指标,以及根、茎、叶各器官及整株的生物量均随低钾胁迫程度的提高而逐渐降低。(2)低钾胁迫降低了橡胶幼苗各器官中氮和磷积累量,但提高了茎中氮的利用效率。同时,低钾胁迫显著降低了橡胶幼苗各器官的钾含量和钾积累量,但极显著提高了各器官的钾利用效率,表明钾利用效率是反映钾胁迫程度变化的最敏感指标。(3)低钾胁迫降低了橡胶幼苗叶、茎部的皮和根中可溶性磷的含量和比例,而提高了叶、茎部的皮、根中不可溶性磷含量和比例,最终导致低钾胁迫下橡胶幼苗植株磷的利用效率降低。(4)低钾胁迫降低了橡胶幼苗叶部的叶绿素a和叶绿素b含量而提高了二者的比例,同时,10mg/L以下低钾胁迫处理的叶绿素b含量显著低于正常供钾处理。(5)在橡胶幼苗叶部和茎部皮中,不同水平低钾处理对可溶性糖含量的影响不同,Omg/L与1mg/L低钾处理提高了可溶性糖含量,而10mg/L与50mg/L低钾处理却降低了可溶性糖含量；在根中,不同低钾处理均降低了可溶性糖含量。另外,低钾胁迫也降低了橡胶幼苗各器官可溶性蛋白含量。(6)低钾胁迫降低了橡胶幼苗各器官中超氧化物歧化酶(SOD)活性,但各器官中的SOD活性不随低钾胁迫处理浓度的变化而呈规律变化。(7)在根中,1mg/L低钾胁迫处理显著提高了POD酶活性,而其它低钾胁迫处理对PCD酶活性的影响却不明显；在叶和茎部皮中,不同低钾胁迫处理与正常供钾处理POD酶活性的差异均不显著。(8)低钾胁迫显著提高了橡胶幼苗各器官中过氧化氢酶(CAT)活性,但CAT酶活性变化并不随低钾胁迫程度的变化而呈规律变化。(9)低钾胁迫显著降低了橡胶幼苗叶部和茎部皮中的硝酸还原酶活性,但在根中该酶活性降低不显著。(10)低钾胁迫对橡胶幼苗各器官中酸性磷酸酶活性的影响不显著。总之,低钾胁迫抑制了橡胶幼苗的生长,改变了橡胶幼苗体内氮、磷、钾营养代谢特性,降低了叶片叶绿素含量,但低钾胁迫也启动了橡胶幼苗体内细胞的自我防御体系,改变了细胞渗透调节物质如可溶性糖含量和可溶性蛋白含量,调控了橡胶幼苗体内的抗逆酶系统中的超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,表明橡胶幼苗在低钾胁迫下能及时调整其生理代谢状态,从而减轻胁迫伤害、适应胁迫环境。2橡胶幼苗低钾胁迫相关基因的分离(1)首次构建了四个橡胶幼苗低钾胁迫消减文库,即橡胶幼苗低钾叶和低钾根正向消减文库,橡胶幼苗低钾叶和低钾根反向消减文库。四个文库的插入片段长度分布为100-500bp,文库重组率在95%以上,四个文库分别获得432个、1096个、377个和1004个阳性克隆,消减文库的构建为进一步克隆和研究橡胶幼苗耐低钾胁迫相关基因奠定了基础。(2)低钾胁迫下橡胶幼苗叶部、根部的差异表达基因多为低丰度表达基因。其中,在叶正向消减文库和反向消减文库中,低丰度表达的unigenes分别为111个(75.00%)和127个(77.91%)；在根正向消减文库和反向消减文库中,低丰度表达的unigenes的比例要低一些,分别为85个(57.23%)和55个(56.12%)。由此可见,橡胶幼苗低钾胁迫SSH文库的构建有利于低丰度差异表达基因的分离和鉴定。(3)寡核苷酸芯片对橡胶幼苗低钾胁迫SSH文库388个Unigenes进行筛选,获得51个低钾胁迫差异表达基因,分为12个功能类别。包括：未知功能基因(15个,29.41%)、信号传导(7个,13.73%)、转录因子(7个,13.73%)、防御蛋白(5个,9.80%)、次生代谢(4个,7.84%)、蛋白质合成(3个,5.88%)、物质运输(3个,5.88%)、渗透调节物质代谢相关(3个,5.88%)、细胞生长(1个,1.96%)、能量代谢(1个,1.96%)、蛋白质降解(1个,1.96%)、基础代谢(1个,1.96%)。(4)利用QPCR分析了差减文库中13个基因,即soluble starch synthase、14-3-3protein、RNA-binding protein、serine/threonine phosphatase、chlorophyll A-B binding protein、Photosystem Ireaction center subunit N、1rr resistance protein、senescence-associated protein、60S ribosomal protein L5、tubulin-sp ecific chaperone A、zinc transporter3、cation efflux protein/zinc transport er和ATPase等在低钾处理橡胶幼苗叶和根中的表达变化,结果表明这些基因均受低钾胁迫调控,并且它们的表达模式与寡核苷酸芯片筛选的结果一致,证实了SSH文库和寡核苷酸芯片联合应用在橡胶幼苗低钾胁迫响应基因筛选中的可靠性。3两个低钾胁迫相关基因的全长cDNA克隆与分析(1)首次克隆了橡胶幼苗MPP1基因,该基因cDNA全长为801bp,编码一个长度为115aa、分子量为12.7Kda、等电点为5.72的蛋白质,该蛋白最大可能定位于细胞核,在1-65氨基酸间具有MPP超基因家族蛋白的保守结构域,在16氨基酸处具有一个活性位点/金属离子结合位点。系统进化分析表明,HbMPP1蛋白与水稻、拟南芥的丝氨酸/苏氨酸磷酸酯酶蛋白的亲源关系最近,氨基酸序列一致性分别为95%和94%。QPCR结果表明,低钾胁迫处理后1-8d中,HbMPP1基因在根和茎中的表达量均显著提高,但在叶中的表达量却明显降低。(2)首次克隆了橡胶幼苗CAB1基因的549bp cDNA,该基因可能编码一个长度为136aa、分子量为13.9Kda、等电点4.84的蛋白质,该蛋白最大可能定位于叶绿体。系统进化分析表明,HbCAB1蛋白与卷柏的Lhcb1-2蛋白和杨树的CAB亲源关系最近,氨基酸序列一致性分别为95%和98%。QPCR结果表明,低钾胁迫处理4d后,HbCAB1基因在根中的表达量显著提高,但低钾处理1-8d的五个时间点中,该基因在茎和叶中的表达量均显著低于正常供钾处理。本文首次较为系统的阐述了橡胶幼苗低钾胁迫应答的形态、生理特性,构建了低钾胁迫条件下的抑制差减文库并对差异表达基因进行了进一步筛选和验证,同时,克隆了2个低钾胁迫相关基因并研究了其低钾胁迫下的时空表达特性。其研究结果将为天然橡胶生产中钾肥的合理施用提供理论指导,为揭示橡胶低钾胁迫的营养生理和分子机制、以及将来的橡胶营养分子遗传改良奠定基础,具有重要的理论意义和应用前景。更多还原

【Abstract】 Natural rubber is an important raw material for both industry and national defense. The natural rubber industry is the pillar industry of the agricultural development and economic growth in tropical regions. Potassium (K) is a kind of Mineral nutrition element which is greatly needed in the process of plant growth and plays a role in a range of physiological functions in plants:photosynthesis, enzyme activation, protein synthesis, osmotic potential. In Hevea brasiliensis (Hevea hereafter), the availability of K affects the characteristics of latex production, latex flow, wind resistance and cold hardiness, etc.China is short of K fertilizer resources. The situation that the supply of K fertilizer does not meet the demand becomes increasingly serious. The acreages of K-deficient soil are increasing rapidly in rubber plantations.In fact, the genetic improvement of plant nutrition traits which exist genotype differences in different plants is one of useful ways improving crop yields under poor soil nutrient environments.Recently, the researches of molecular response mechanisms of plant nutrient deficiencies becomes more and more common.However, in Hevea,similar research reports are almost blank, and the physiological researches of K nutrition are also scarce.The response mechanisms of Hevea seedlings to low K stress were investigated in the aspects of morphological and physiological characteristics. Furthermore, the differentially expressed Hevea seedlings genes to low K stress were firstly separated and screened by constructing the suppression subtractive hybridization (SSH) libraries and hybridizing the oligonucleotides chip. On the basis of these, the functions of the differentially expressed genes were preliminarily annotated by BlastX and BlastN, and the response characteristics of part differentially expressed genes to low K stress were verified by the real-time quantitative PCR(QPCR,hereafter). What’s more, the full-length or part-length cDNA of HbMPPl and HbCABl were cloned by the rapid amplication method of cDNA ends (RACE), the functional predictions and spatio-temporally expressed characteristics of two genes were done by Bioinformatics methods and QPCR, respectively.The major results were as follows.1.Morphological and physiological responses of Hevea seedlings to low k stressThe hydroponic culture was carried out to investigate the differences of morphological and physiological characteristics between the treatment of normal K supply and four treatments of low K supplies. The five K levels settled were OmgL-1,1mgL-1,10mgL-50mgL-1and150mgL-1(normal), respectively. The major results obtained were summarized as follows.(1)The low K supplies gradually decreased the plant height, root length, stem girth and biomass of the root, stem, leaf and whole plant.(2)Under low K stress, the accumulations of nitrogen and phosphorus in all Hevea seedling organs were decreased, but the N utilization efficiencies of the stem being increased.Moreover,the K contents and accumulations in all Hevea seedling organs were dramatically decreased, but the K utilization efficiencies, the most sensitive low K-responsive indicators, being significantly increased.(3)Under low K stress, the contents and the percentages of soluble phosphorus were decreased in different organs, but the insoluble phosphorus reversely being changed, and the ultimate P utilization efficiencies being decreased.(4)The low K supplies decreased the contents of chlorophyll a and chlorophyll b, but significantly increasing the ratio of chla/chlb. The contents of chlorophyll b were significantly different between the treatments of low K (below10mg L-1) supplies and the treatment of the normal K supply.(5) The soluble sugar contents (SSC) in the leaves and in the barks of the stem were increased in low K supplies of0and1mg L-1, but being decreased in the low K supplies of10and50mg L-1. However, all treatments of low K supplies decreased the SSC in roots, decreasing the soluble protein contents (SPC) in all organs.(6)The low K supplies irregularly decreased the superoxide dismutase (SOD) activities in different organs of Hevea seedlings.(7) In root,the low K supply of1mg L-1significantly increased the activities of peroxidase (POD),but other low K supplies having not significantly effects on the activities of POD.In leaves and barks of the stem,the activity diffirences of POD were little between the low K supplies and the normal K. supply.(8) The low K supplies significantly increased the activities of catalase (CAT), but the activities of CAT varied irregularly in different levels of low K supplies.(9) The low K supplies significantly decreased the activities of nitrate reductase(NR)in the leaves and the barks of the stem,but insignificantly decreasing the activities of NR in the roots.(10) The low K supplies affected little on the activities of the acid phosphatase.In general, low K stress suppressed the growth of Hevea seedlings, changing the characteristics of nutrient metabolism and decreasing the contents of chlorophyll, but also launching the systems of cell defenses,changing the contents of Osmotic regulation substances(such as SOC and SPC),regulating the activities of SOD,POD and CAT in anti-enzyme system.Thus, Hevea seedling can timely regulated the statuses of physiological metabolism to relieve the stress harm and to adapt the stress environment. 2. Seperation of low k-induced Hevea seedling genes(1)Four suppression subtractive hybridization (SSH) libraries firstly were constructed, including two leaf libraries (forward and reverse) and two root libraries (forward and reverse). The forward libraries of the leaf and root had432and1096cDNA clones, respectively. The reverse libraries of the leaf and root had377and1004cDNA clones, respectively. The clones revealed a recombination efficiency of95%, and containing cDNA inserts of200-500bp.(2)Most of the seperated unigenes of Hevea seedling under low K stress were low-abundantly expressed. The numbers and percentages of the low-abundantly expressed genes were111(75.00%),127(77.91%),85(57.23%) and55(56.12) in leaf forward, leaf reverse, root forward and root reverse library. So construction of Hevea SSH libraries under low K stress was useful for the seperation and identification of low-abundant differentially expressed genes.(3)The388Hevea seedling Unigenes in SSH libraries under low K stress were screened by oligonucleotidechip.The58gained genes were divided into12categories,including of15unkown functional genes(29.41%),7signal transduction (13.73%),7transcription factors(13.73%),5defence and stress(9.80),4secondary metablism(7.84),3protein synthesis(5.88%),3substance transportation(5.88%),3metablism of osmoregulation substance,l cell gowth(1.96%),1energy metablism(1.96%),1protein degradation(1.96%)andⅠ primary metabolism(1.96%).(4)The relative expressional characteristics of13genes in roots and leaves were studied by QPCR in the treatments of low K supplies,the genes including of soluble s tarch synthase,14-3-3protein,RNA-binding protein,serine/threonine phosphatase,chlorophy Ⅱ A-B binding protein,Photosystem I reaction center subunit N,lrr resistance protein,sen escence-associated protein、60S ribosomal protein L5,tubulin-specific chaperone A,zinc t ransporter3,cation efflux protein/zinc transporter)and ATPase. The results of QPCR in dicated the expressions of13genes were regulated by the low K stress, their expresse d patterns were consistent between QPCR and oligonucleotides, so the united applicati on of QPCR and oligonucleotides chip increased the reliability of screening low k-ind uced genes in Hevea seedling.3. Clone the Hevea seedling full-length cDNA sequences induced by low K stress(1)The full-length801bp cDNA sequence of HbMPPl firstly was cloned by meth od of rapid-amplification of cDNA ends(RACE,hereafter). The HbMPPl encoded a115amino acids(aa) protein whose molecular weight and isoelectric point were12.7Kda a nd5.72,respectively. The HbMPP1which most probably locates on nucleus has95%a nd94%amino acid sequence identities to the serine/threonine phosphoesterase family P rotein of Arabidopsis_thaliana and Oryza_sativa_Japonica_Group.Moreover, the HbMPP1has a conserved domain of MPP-superfamily superfamily between the first to66th a mino acid, having a active site/metal-binding site in the16th amino acid. The results o f QPCR indicated the low K stress from the first to the eighth day significantly incre ased the expressed levels of HbMPPl in root and stem,but decreasing the expressed levels in leaf.(2)The549bp cDNA sequence of HbCAB1firstly was cloned by RACE.The Hb CAB1probably encoded a protein composed of133amino acids(aa).The molecular we ight and isoelectric point of HbCAB1were13.9Kda and4.84,respectively.Moreover,the HbCAB1which most probably locates on chloroplast has95%and98%amino acid se quence identities to the Lhcbl-2of Selaginella tamariscina and the CAB of poplar.The results of QPCR indicated the expressed levels of HbCAB1significantly were increas ed in root after the fourth day of low K stress,but siginificantly being decreased in st em and leaf in five time points of the low k stress.This dissertation made a comprehensive study of the responses mechanisms of Hevea seedling to K-deficiency from the aspects of physiology and molecular biology, constructing4SSH libraries, further screening and validating the differentially expressed genes,cloning two low k-induced Hevea seedling genes.The results presented here would lay a basis for revealing the response mechanism in aspects of morphology,physiology and molecular biology,facilitate the rational application of K-fertilizer in rubber plantations, and provide a guidances for genetic modification of Hevea for improved K-nutrition.更多还原