【作者】 徐颖；

【导师】 戴锡玲； 王全喜；

【作者基本信息】 上海师范大学 ， 植物学， 2013， 硕士

【摘要】 1987年，秦仁昌系统将中华水韭归属于蕨类植物小型叶植物门(Microphyllphyta)，水韭亚门(Is?ephytina)，水韭目(Is?etales)，水韭科(Is?etaceae)。2008年，刘红梅等人认为中华水韭属于石松类植物，石松亚门(Lycophytina)，水韭目(Is?etales)，水韭科(Is?etaceae)，水韭属(Is?etes)。国内外对水韭的研究几乎都集中在孢粉学领域和孢子体方面，对其配子体和胚胎等研究较少。在分子水平上仅有一些学者采用分子标记技术分析了中华水韭的遗传结构。到目前为止，还没有对中华水韭的功能基因和分子机理进行研究。本实验以蕨类中的模式植物水蕨(Ceratopteris thalictroides (L.) Brongn.)为参考，利用RACE技术成功克隆出中华水韭Fe-SOD基因，并命名为IsFeSOD基因。本研究首先提取中华水韭孢子体(包括大、小孢子)的总RNA后反转录，获取cDNA。利用植物的同源性，设计简并引物，从中华水韭中得到一个418bp的核心片段。再利用RACE的方法，分别进行5’RACE和3’RACE，得到5’和3’两个亚克隆，分别获取了464bp的5’端片段和1182bp的3’端片段。最后根据已知的序列设计特异性引物，利用PCR技术，得到IsFeSOD1469bp的全长序列，ORF区为744bp，编码247个氨基酸。利用生物信息学分析，在中华水韭中获得IsFeSOD基因的序列与Gene bank中公布的其他植物的Fe-SOD基因完整序列进行比对，发现中华水韭中也含有TGGGA保守序列。通过比对莱茵衣藻、地钱、扭口藓、中华水韭、水蕨、海岸松、水稻、拟南芥等8种植物的Fe-SOD完整的mRNA序列可以看出，这些核酸序列从100到750之间的同源性较高。通过氨基酸序列比对还发现，中华水韭中该酶的氨基酸序列中含3个Fe-SOD的保守序列，即FNNA、FGSGW和WEHAYY，同时，所有的植物除了含有这3个保守序列外，还含有WNHHFF和HWGKH这样的序列。将IsFeSOD的ORF区片段与pET32a载体连接后转化入大肠杆菌BL21，之后用1mM的IPTG诱导6h，表达形成一个大小43KD的蛋白，与预计大小符合。得到的重组蛋白为不可溶蛋白。采用Western-blotting的方法，利用实验已制备水蕨Fe-SOD抗体和从公司购买的拟南芥Fe-SOD抗体检测了中华水韭中Fe-SOD的真实性。虽然DNA分子标记、基因克隆、基因芯片和RNAi等技术已经在蕨类植物的各项研究中得以应用，但与这些技术在种子植物中的应用相比，无论是技术应用的成熟程度，还是所能够解释的科学问题都是刚刚起步。目前，蕨类植物中还没有成熟的转化体系，给研究蕨类植物的分子机制和功能基因带来了一定的困难。同时，蕨类中已知的序列信息相对比较少，也是分子生物学研究进展相对较慢的原因之一。本研究成果为蕨类的功能基因研究提供了一定的数据和参考。更多还原

【Abstract】 According to Ching’s system, Isoetes sinensis belong to Isoephytina. However,some scholar considered that/sinensis should be classified to Lycophytina accordingto molecular data. The palynology and sporophyte on I. sinensis have been wellstudied, though，the plant biology of gametophyte and embryo of that remain lessunderstood. Furthermore, there are few studies on the genetic structure of L sinensisbased on selected molecular marker. So far, the function of genes and the molecularmechanism of I. sinensis were not stuidied. The present investigation successfullycloned Isoetes sinensis Fe-SOD gene, termed IsFeSOD,The total RNA was extracted using I. sinensis containing macrospore andmicrospore as experimental material. Then the cDNA sequence of Fe-superoxidedismutase was cloned by RACE. A pair of degenerate pirmers designed based on thehighly conserved sequences to clone the418bp mid-fragment of SOD gene fromIsoetes sinensis-The full-length cDNA was obtained by the rapid amplification ofcDNA ends (RACE) technique. It consisted of1469nucleotides and an open-readingframe of744bp encoding247amino acids.The bioinformatics analysis was used to compare IsFeSOD with other plants，gene sequences in Gene Bank, which indicating that IsFeSOD also contains TGGGAconservative sequence. We also found that Fe-SOD mRNA sequence are highlyhomologous from100to750with several DNA sequences from Chlamydomonasreinhardtii, Marchantia polymorpha, Barbitla unguiculaia, I. sinensis,Ceraiopteris thalictroides, Pinus pinaster, Oryza saliva, Arahidopsis lhaliana.By the amino acid sequences also found3conserved sequence: FNNA、FGSGW、WEHAYY, and other two: WNHHFF and HWGKH.Furthermore，the recombinant pET32-FeSOD plasmids were transformedinto host E. coli BL21for expression. The recombinant protein about43kDa was induced by IPTG, in line with the expected size. The recombinant proteinwas used for western boltting detection.Ferns as important transition groups in the plant kingdom，is an important andindispensable part to study biological evolution and phylogeny. In addition, there aremany very important plant groups, plenty of valuable medicinal plants, some specialstress-resistant plants. Research of these ferns functional genes and metabolicpathways have great value in medicine, industry, agriculture, horticulture, food and tomaintain ecological balance.DNA molecular markers, gene cloning, microarray and RNAi technology hasjust been applied in studing ferns. But these techniques has become maturity in seedplant molecular operation. The results of this study provide some data and referencefor ferns.更多还原