盐碱地星星草根cDNA文库的构建及初步分析

以盐碱地原位取材的星星草根为材料,利用SMART技术构建了cDNA文库。扩增后文库的滴度为1.747×109CFU/mL,插入片段分布在0.5-2 kb之间,重组率为92%。文库ESTs序列的初步分析表明,从盐碱地星星草根的cDNA文库中筛选到耐盐相关基因S-腺苷甲硫氨酸合成酶2基因和钙牵蛋白基因的EST片段。该文库可用于进一步从中筛选星星草耐盐基因。盐碱地星星草根cDNA文库的构建为揭示星星草耐盐分子机制、挖掘星星草耐盐基因、培养耐盐植物奠定了重要基础。     

盐生植物海马齿盐胁迫全长cDNA文库的构建与分析

为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下:采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN+TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。     

紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。     

基于Linux的cDNA文库序列分析平台的构建与应用

本研究构建了基于Linux的cDNA文库序列分析平台,该分析平台可大批量自动处理测序后的序列,包括载体序列的去除、序列格式的转换、序列的自动拼接、序列对数据库的相似性搜索及全长ORF的预测等,可加速对大规模测序数据的分析和利用。用该平台对构建的野生大豆盐胁迫全长cDNA文库部分测序结果进行分析和利用。用该平台对构建的野生大豆盐胁迫全长cDNA文库部分测序结果进行分析,获得了较好的结果,已得到多个具有潜在价值的新基因序列。     

盐芥（Arabidopsi salsuginea）cDNA文库的随机测序

实验以盐生植物盐芥为材料,提取经盐处理的盐芥总RNA,分离mRNA后,构建cDNA文库。从cDNA文库中随机挑取克隆进行测序。结果共测得53个表达序列标记（EST）,有37个EST（69．8％）,与拟南芥的基因在多肽水平上有较高同源性;21个EST（39．6％）的功能未知。     

盐芥(Arabidopsis salsuginea)cDNA文库的随机测序

实验以盐生植物盐芥为材料 ,提取经盐处理的盐芥总RNA ,分离mRNA后 ,构建cDNA文库。从cDNA文库中随机挑取克隆进行测序。结果共测得 5 3个表达序列标记 (EST) ,有 37个EST( 6 9 8% )与拟南芥的基因在多肽水平上有较高同源性 ;2 1个EST( 39.6 % )的功能未知     

用限制性cDNA文库制作K562细胞基因表达谱芯片探针

以人红白血病K562细胞为材料,应用限制性显示PCR（RD-PCR）技术构建cDNA文库,该文库通过PCR引物3′端延伸两个不同碱基形成136对引物对cDNA进行限制性扩增,得到136组不同的PCR扩增产物,纯化后与载体连接并转化细菌,即为限制性cDNA文库,根据不同的分组进行克隆的鉴定和分离。并进行大量扩增制备cDNA芯片探针,该方法构建的文库因经过了限制性分组扩增,每组均含有特定的cDNA,因而大大加快了随后克隆的分离 和鉴定的速度,为基因芯片探针制备提供了一个新方法。     

通过cDNA微阵列鉴定水稻(Oryza sativa L.) 盐胁迫应答基因

利用水稻耐盐品种兰胜构建了一个高质量的cDNA文库以鉴定水稻盐胁迫应答基因, 从cDNA文库中提取约15000个质粒, 并用Biomek 2000 高密度点阵系统或手工操作, 将这些质粒点于尼龙膜上. 通过这种方法鉴定了30个盐应答基因, 对其中的12个基因通过Northern杂交进行表达分析, 确证了cDNA微阵列的杂交结果. 30个基因中, 18个基因受盐诱导, 另外12个基因受盐抑制, 其中27个在GenBank数据库中有同源序列. 根据功能, 这些基因大致可以分为5类：光合作用相关基因、物质运输相关基因、代谢相关基因、耐逆相关基因以及其他未分类的基因. 研究结果表明, 盐胁迫影响了植物生长发育的多个方面, 其中有些基因可能在植物体的耐盐过程中具有重要作用.     

正常人肝细胞cDNA文库的构建及应用分析

目的利用Gubler-Hoffman法构建了正常人肝细胞的cDNA文库以筛选肝细胞内部与乙肝病毒感染相关的基因。方法首先采用TRIzol法提取正常人肝细胞总RNA,纯化mRNA。逆转录合成单链cDNA,然后合成双链cDNA。用Spin Column回收0.4kb以上片段,然后与Vector pAP3neo进行连接,利用电刺激转化法导入E.coliDH10B,利用PCR法检测文库的重组效率。结果扩增后的文库重组率为93.3%。结论已经成功地构建了正常人肝组织的cDNA文库,该文库可用于筛选与乙肝相关的基因及用于基因芯片的制作。     

厚藤cDNA文库的构建和重金属镉耐受相关基因的筛选

厚藤（Ipomoea pes-caprae（L.） Sweet）是一种具有重要生态、观赏及药用价值的沙滩植物,对重金属镉（Cd）具有一定的富集能力,可作为Cd污染滨海地区的修复植物进行引种栽植和利用。本研究通过Gateway技术构建厚藤的cDNA文库,将该文库质粒转化酵母对Cd敏感的突变株ycf1△,采用全长cDNA过表达基因捕获系统（FOX）筛选厚藤重金属Cd胁迫耐受相关基因,并采用酵母互补实验进行基因的功能验证。本研究获得了2个能够恢复ycf1△对Cd敏感表型的重组质粒,经测序分析,该重组质粒包含的cDNA全长序列分别对应厚藤植物螯合肽合成酶基因（phytochelatin synthase）和金属硫蛋白基因（metallothionein）,分别将其命名为IpPCS和IpMT,通过功能分析,初步认定该基因为编码Cd耐受和解毒相关蛋白的候选基因。     

The analysis of differential gene expression in early somatic embryogenesis on Lycium barbarum

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can induce somatic embryogenesis. Somatic embryogenesis of Lycium barbarum L. is controlled artificially by regulating 2,4-D concentration. The total RNA that was isolated from calluses, embryonic calluses and early somatic embryos was used for analyzing differential genes expression. We obtained three cDNAs from early somatic embryogenesis which were not found in calluses. The results indicate that these cDNAs were early embryogenesis-specific cDNAs and this gene expression was induced in cultured calluses after a transfer to an auxin- free medium. A cDNA library was constructed using poly(A)⁺-RNA derived from early somatic embryos of Lycium barbarism L. Two full-length cDNAs were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of the full-length cDNA only existed in embryogenic calluses and early somatic embryos of Lycium barbarum L. This revised version was published online in June 2006 with corrections to the Cover Date.     

硫腺苷甲硫氨酸合成酶基因的克隆及其在盐胁迫条件下的不同表达

硫腺苷甲硫氨酸作为甲基供体在转甲基反应中起到重要作用.为了解硫腺苷甲硫氨酸在盐地碱蓬(Suaedasalsa (L.)Pall)耐盐中的作用,我们对可能编码硫腺苷甲硫氨酸合成酶的基因(SsSAMS2)进行了分析.该基因在经400 mmol/L NaCl处理的盐地碱蓬地上部分的λ-Zap cDNA文库中克隆到,其插入片段全长1 531 bp,包含一个395个氨基酸的开放阅读框架,该基因推断的分子量约为43 kD.SsSAMS2与长春花(Catharanthus roseus)的SAMS2在氨基酸水平上的一致性为93%.Southern杂交显示,SsSAMS2在盐地碱蓬基因组中可能是两个拷贝.Northern分析显示硫腺苷甲硫氨酸合成酶基因受NaCl等胁迫的正调控.酶活性检测表明,NaCl胁迫条件下该酶活性增强.     

蝶兰胚珠的cDNA文库构建及其特异基因表达的研究

以蝶兰(Phalaenopsis“Mt.Kaala”cv SM9108)为材料,分别提取大孢子母细胞时期胚珠和成熟胚珠的PolyA RNA,反转录成cDNA,构建起两个cDNA文库。克隆筛选采用差异杂交法。从上述两个cDNA文库中,各选择一个筛选出的cDNA,对其在植物体不同器官和不同发育时期的胚珠内的表达进行了分析。结果表明该两个cDNA均为胚珠特异,并且分别在胚珠发育的特定时期表达。推测该两个cDNA的表达受胚珠内部的不同因子调控。     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.     

Use of an epitope-tagged cDNA library to isolate cDNAs encoding proteins with nuclear localization potential

We have combined epitope tagging with an expression cDNA library in order to isolate cDNAs encoding nuclear proteins. This system allows us to detect proteins expressed from the cDNA library by using antibodies against the epitope tag. As a tag, we used the 85-aa N-terminal peptide of the SV40 T antigen which lacks the nuclear localization signal (NLS). A strong expression vector, pEF204 [Kim et al., Gene 91 (1990) 217–223], was modified into an epitope-tagging vector, pTkim, by putting the tag-coding region and a cDNA cloning site immediately after its promoter. From cDNA libraries constructed using pTkim, we isolated eight cDNA clones whose tagged proteins were localized within the nuclei. From partial sequence analysis, two cDNAs were shown to code for the ribosomal (r-) proteins, simian L44 and human L21, and the others were shown to be new. Furthermore, six cDNAs including those encoding the r-proteins could direct a non-karyophilic T antigen [Fischer-Fantuzzi et al. Virology 153 (1986) 87–95] into nuclei, showing that they have NLSs. These results indicate that this system is useful for isolating new cDNAs which code for nuclear proteins.     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理（submergence-treated,ST)的玉米（Zea maysL.)幼苗根部cDNA为目标群体,未处理（untreated,UT)的玉米幼苗cDNA为对照群体,进行抑制差减杂交。用经过UT差减的STcDNA构建了一个含有大约2000个独立克隆的差减文库。对随机挑取的408个克隆进行差异筛选。获得了184个在ST中特异表达或表达增强的候选克隆。对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段。GenBank中BLAST查询结果表明;6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%-90%;21个克隆在GenBank中无法查到对应的同源序列。可能代表了新基因。或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性。     

The timing of maize leaf senescence and characterisation of senescence-related cDNAs

Leaf senescence was characterised in two Zea mays lines, earlier senescence (ES) and later senescence (LS). Loss of chlorophyll was delayed in LS compared with ES, but the decline in photosynthesis occurred simultaneously in the two lines. Western analysis detected transition points during senescence of both lines when major quantitative and qualitative changes occurred in a number of leaf proteins. Differences in the pattern of translatable mRNAs were apparent earlier than alterations in pigment or protein levels. A cDNA library was constructed using mRNA from ES leaves early in senescence and differential screening was employed to isolate senescence-related clones. Two senescence-enhanced cDNAs showed sequence homology with cDNAs for seed proteins - a cysteine protease and a protein-processing enzyme. These findings suggest that there are similarities between gene expression during seed maturation, germination and leaf senescence. Other senescence-enhanced cDNAs were related to genes implicated in gluconeogenesis and chlorophyll breakdown.     

A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues

CgA is a 72Kd protein of unknown function that is present in many neuroendocrine tissues and co-secreted with their resident hormones. We prepared a cDNA library to the mRNA from CgA-producing human medullary thyroid carcinoma (MTC) cells in the expression vector lambda gt11. The library was screened with a panel of one polyclonal and two monoclonal antibodies to CgA. The specificity of the antibodies for CgA was demonstrated by immunoassay, immunohistology, and immunoprecipitation of the in vitro translation products of mRNA from CgA-producing tissues. A chromogenic second antibody identified five immunoreactive clones. Their cDNA inserts were isolated after EcoRI digestion and agarose gel electrophoresis. These cDNAs were 32P-labelled and used as probes in Northern hybridization studies. An mRNA of approximately 2.3Kb was detected with the cDNA probes in human cell lines from MTC and lung cancers that were shown to produce CgA and in human pheochromocytoma and bovine adrenal medulla tissue. To confirm its identity, one of the putative CgA cDNAs was subcloned into a plasmid and was used to hybridization-arrest the in vitro translation of CgA mRNA. These studies demonstrate the cloning of cDNAs which hybridize with CgA mRNA from diverse neuroendocrine tissues.