【作者】 马辉；

【导师】 刘桂丰； 王玉成；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2008， 硕士

【摘要】 二色补血草是一种盐生开花植物,能够在盐碱化土地上正常生长,其优良的耐盐碱特性使之成为研究植物耐盐机理的理想材料。本研究结合表达序列标签（EST）技术和cDNA微阵列技术在转录水平上研究二色补血草耐盐碱机理,并对从文库中获得的液泡膜H⁺-ATPase c亚基基因进行了生物信息学分析、表达模式分析及功能验证。以400 mmol/L NaHCO₃胁迫处理48h的二色补血草叶片为材料构建了cDNA文库,文库的初始滴度为9.6×10⁵ pfu/ml,文库的重组率为95%,PCR检测插入片段的平均长度为0.9 kb。从cDNA文库中随机挑选2880个克隆进行序列测定,共筛选到2358个高质量的EST序列,建立了二色补血草EST数据库,它们GenBank的登录号为EH792855-EH795212;通过BlastX分析,共有1418条序列与已知基因序列高度同源,将这些EST序列根据MIPS分类标准分为12类,其中代谢、光合作用、功能未知、细胞防御及转运相关四类基因的表达丰度最高,分别占已分类EST总数的18.48%、14.6%、11.99%、11%和10.01%。EST分析表明,有245个EST是与早期逆境响应同源的基因,其中最显著的是金属硫蛋白（EH793637）和脂质转运蛋白（EH794695）,它们各自都占总EST的1.4%。选择1330条EST制作cDNA微阵列,获得了NaHCO₃胁迫6h、24h和48h的二色补血草的转录表达谱,筛选到142条差异调节的转录本,6、24和48 h分别有108、43和46个基因出现明显的差异表达。基因芯片研究结果表明,有76%的基因确定是在胁迫早期（胁迫6h）差异表达,因此认为二色补血草在盐碱胁迫初始阶段的响应是重要的。对从二色补血草cDNA文库中获得的液泡膜H⁺-ATPase c亚基（VHA-c）基因进行了生物信息学分析,VHA-c基因cDNA全长729bp,其中开放读码框（ORF）为498bp,编码165个氨基酸,预测蛋白的分子量为16.6kD,理论等电点为8.62。利用实时荧光定量RT-PCR技术对该基因在二色补血草中的表达模式进行了分析,结果表明,NaCl诱导了VHA-c基因在二色补血草叶部的表达,抑制其在根部的表达;NaHCO₃胁迫下,VHA-c基因分别于12h和6h时在叶部及根部表达量最高;Na₂CO₃胁迫6h时VHA-c基因在根部的表达量最大,48h在叶部大量表达。说明VHA-c基因能够对盐胁迫做出应答,确定该基因为耐盐相关基因。将该基因构建到植物表达载体pROKⅡ上,利用农杆菌介导法实现VHA-c基因对模式植物烟草的转化,通过耐盐性试验,对VHA-c基因进行功能验证。试验结果表明,盐胁迫后,转基因烟草的SOD和POD活性较非转基因烟草显著增加,MDA含量低于非转基因烟草,说明VHA-c基因的表达增强了植物清除活性氧的能力,并降低了植物的膜脂氧化程度,一定程度的提高了转基因烟草的耐盐能力,证实了来源于二色补血草的VHA-c基因具有提高植物耐受盐胁迫之能力的功能。植物的耐盐过程是一个复杂的多基因协同作用的体系。我们通过EST技术和cDNA芯片技术等基因组学研究手段,获得了盐胁迫下二色补血草的基因表达信息,为系统阐明二色补血草抗盐碱分子机理奠定坚实的基础,为进一步分析二色补血草的抗逆机理提供了数据;利用基因工程手段使外源VHA-c基因在烟草中组成型表达,不同程度提高转基因烟草对盐胁迫的耐受能力,为将VHA-c基因利用于林木耐性分子育种提供了重要的前提基础。更多还原

【Abstract】 Limonium bicolor,a halophytic species of flowering plant,thrives in saline and saline-alkali soil.It’s with high capacity of salt resistance,is an excellent target for plant salt tolerance research.We employed expressed sequence tags（EST） analysis combined with cDNA microarray in order to have a better view of transcript expression in L.bicolor during saline-alkali induced stress.And we cloned a Vacuolar H⁺-ATPase Subunit c gene from the library,the analyses of biological information,the expression pattern,and function identification of H⁺-ATPase Subunit c（VHA-c） were conducted.A cDNA library was constructed from the leaf of Limonium bicolor exposed to 0.4 mol/L NaHCO₃ for 48 h.Primary titer of the library was 9.6×10⁵ pfu/mL,and the rate of recombination was 95%,with an average inserts size of 0.9kb,which were determined by both PCR and sequencing analysis.A Total of 2358 high quality sequences were generated from 2880 sequenced clones which were selected randomly from the library.The GenBank accession numbers of these ESTs are EH792855-EH795212.BlastX search results of 2358 ESTs showed that 1148 sequences had high homologous to known sequenees.The database-matched ESTs（1418） were grouped into 12 function categories.The most recurrent categories were metabolism（18.48%）,photosynthesis（14.6%）,unclassified（11.99%）,cell rescue, defense（11%） and transport facilitation（10.01%）.EST analysis showed that 245 ESTs were homologous to genes earlier implicated in stress responses,most prominently metallothionein （EH793637） and lipid transfer protein（EH794695）,each of which accounted for 1.4%of total ESTs.1330 ESTs were selected for construction a cDNA microarray.We obtained transcript profiles of L.bicolor response to NaHCO₃ for 6,24 and 48 h.A total of 142 transcripts were differentially regulated under the conditions studied,and 108,43,and 46 transcripts were differentially regulated by NaHCO₃ stress for 6,24 and 48 h,respectively.The microarray results showed that 76%of differentially regulated transcripts were identified at the early stress period（stress for 6 h）,suggesting that the response at an early stage is important for saline-alkali stress tolerance in L.bicolor.This study provides informative preliminary data and a starting point for more in-depth analyses of extreme stress tolerance in L.bicolor.The biological information analysis of the Vacuolar H⁺-ATPase gene（VHA-c） was analyzed.Sequence analysis showed that the VHA-c gene is 729 bp in length,including 498 bp of open reading frame（ORF）,and encodes a protein of 165 amino acids with a predicted molecular mass of 16.6kD and pI 8.62.In addition,the expression patterns of VHA-c gene in L. bicolor exposed to NaCl,NaHCO₃ and Na₂CO₃ were performed by real time RT-PCR.The result showed that the expression of VHA-c gene was induced by NaCl at leaves of L.bicolor and inhibited in root;reached to the highest level at leaves and root after NaHCO₃ stress for 12 hand 6h;and reached to the highest level after Na₂CO₃ stress for 6 h,48 h at the leaves of the highest expression.Above all results indicated that the VHA-c gene can be responsive to abiotic stress;therefore,it may be a salt tolerance gene.The VHA-c gene was inserted into the plant expression vector pROKⅡ,and transferred into model plant,Nicotiana tabacum cv.Petit Havana SR-1,using Agrobacterium-mediated transformation method.The function of the exogenous VHA-c gene was examined by the salt tolerance test of transgenic lines.The result indicated that compared with nontrangenic line,the expression of exogenous VHA-c gene increased the activities of SOD and the POD,and reduced the content of MDA in transgenic lines,enhancing the salt tolerance of the transgenic plant.our results indicated that the expression of VHA-c gene may increase the salt tolerance of plant by protection of the plant active oxygen elimination enzyme class,such as POD,SOD and so on,and reduction of the membrane oxidation in plants.This study confirmed the VHA-c gene from L.bicolor can confer salt tolerance to plant.The process of plant bears salty is a complex system associated with multi-genetic coordinated operating.Our research obtained that the genes expression profiles through the EST analysis and cDNA microarray,and will make a solid foundation for revealing the molecular mechanism of salt-alkali tolerace in L.bicolor.This study provides informative preliminary data and a starting point for more in-depth analyses of extreme stress tolerance in L. bicolor.The experiments results indicated that the constitutively expressed VHA-c gene from L. bicolor can improve the tolerance of plant to adverse environments,and made a foundation for employing the VHA-c gene in plant genetic.更多还原