【作者】 于喜艳；

【导师】 何启伟； 赵双宜；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2003， 博士

【摘要】 甜瓜(Cucumis melon L.)是世界性的重要园艺作物。甜瓜营养品质主要取决于果实中可溶性糖的含量和种类。因此，在甜瓜果实发育过程中可溶性糖的积累一直得到了人们的关注，国内外许多学者进行了这方面的研究，得出了一致的结论：甜瓜果实中总糖，尤其是蔗糖含量在距果实完熟期15天左右开始迅速提高，而糖的积累是与蔗糖代谢酶的变化息息相关的，在果实发育早期，葡萄糖和果糖含量相对较高，而此时酸性转化酶活性也很高；而在果实发育后期，蔗糖和总糖含量急剧上升，伴随着的是酸性转化酶活性的下降和蔗糖磷酸合成酶活性的提高。由此说明，甜瓜果实中糖的积累和组成是严格受糖代谢酶的调控。植物生物技术和分子生物学的发展为研究甜瓜代谢酶基因和开展甜瓜品质育种开辟了一条新途径。 本研究首先构建了甜瓜幼果的cDNA文库，并通过RT-PCR方法克隆到了甜瓜果实酸性转化酶基因cDNA片段，构建了反义表达载体。通过农杆菌介导分别转入烟草和甜瓜，获得了转基因烟草植株和甜瓜抗性愈伤组织，具体研究结果如下： 1．甜瓜幼果cDNA文库的构建 在20世纪90年代初，美国的CLONTECH公司发明了SMART(Switching Mechanism At5’end of RNA Transcript)技术，使用一个改造了的Oligo(dT)引物，引导cDNA第一链的合成。当反转录进行到mRNA的5’端时，逆转录酶的末端转移酶活性在cDNA的3’末端加上几个C，恰好与溶液中的引物SMARTⅣ Oligonucleotide的3’端的几个G配对，此时反转录酶转换模板，继续复制SMARTⅣ Oligonucleotide的5’端，从而生成了一条含SIdARTⅣ Oligonucleotide的全cDNA。自SMART技术问世以来，在短暂的十几年内就构建了大量的cDNA文库。象其它的许多分子生物技术一样，SMART技术最初也是应用于动物，当他们在动物方面的研究比较成熟后，才被应用到植物中。目前SMART技术在植物方面的应用已有报道，在甜瓜的应用未见报道。本实验首先把SMART技术应用于甜瓜的分子生物学研究，成功地构建了一个质量高，代表性较好的甜瓜幼果cDNA文库。 以‘伊利莎白’甜瓜授粉后5天的果实为材料，采用一步法提取总RNA，利用SMART技术合成cDNA第一链，再经23个循环的长距离PCR(LD-PCR)合成双链cDNA。双链cDNA经蛋白酶K消化、纯化cDNA、SfiⅠ(A和B)酶切、CHROMA SPIN-400柱——层析除去 400hp以下的小分子后，与经过 5’端脱磷的入 TiP匝XZ侣fi…和 B）／CIP载体进行连接，经体外包装和感染 XL；8。宿主菌，得到了 8．54X”个独立的噬菌体克隆。从中随机挑选 100个克隆进行 PCR插入片段的鉴定，电泳结果显示 PCR产物大于500hp的片段占81％，插入片段平均长度为720hp；蓝白斑筛选鉴定表明文库的重组率为99．53％；以甜瓜果实酸性转化酶基因CDNA片段为探针，初步筛选文库，得到了阳性克隆。由此认为甜瓜幼果CDNA文库己经建立，为今后研究甜瓜果实糖代谢酶基因与其发育相关基因奠定了基础。2．甜瓜果实酸性转化酶基因CDNA片段的克隆及反义表达载体的构建 反义技术能抑制目的基因而不会改变其序列，由反义DNA介导后整合到宿主细胞基因组中能产生稳定的目的基因的关闭，这是常规育种无法达到的。反义技术能用于产生作物新种质，并且可用于验证一些未知功能的基因的生物学功能。反义技术在农业作物品种改良中具有广阔前景。改善甜瓜品质，提高含糖量尤其是适当提早蔗糖积累的重要突破口就是适时降低果实发育中酸性转化酶的活性。因此，研究甜瓜果实酸性转化酶基因具有重要意义，可以从分子水平上研究甜瓜果实蔗糖积累与分解的分子机制，并通过基因工程手段改良和调控甜瓜品质。本研究率先克隆了甜瓜果实酸性转化酶基因的。DNA片段并构建了反义表达载体，为从分子水平改善甜瓜品质奠定了基础。 根据GenBank中己登记的番茄、马铃薯、胡萝卜等的酸性转化酶基因保守序列设计了一对引物，以‘伊利莎白’甜瓜总RNA为模板，经RT。PCR反应获得了约比 的特异性产物，与预期扩增产物的分子量相符。将该 F CR产物插入到 pMD丁载体中得到pMAI。双向测序结果表明克隆的CDNA片段长1038hp，推导的氨基酸序列长为346个氨基酸。与GenBank中的同源序列比较，本研究所分离。DNA片段的氨基酸序列与其它物种酸性转化酶基因的氨基酸序列同源性多介于70％～99％，其中与茄科作物的酸性转化酶基因氨基酸的同源性最高，与番茄同源性为99％，与马铃薯为96％，与辣椒为93％；其次是胡萝卜、甘薯，氨基酸同源性都在80％以上。据此认为我们己率先成功克隆到了甜瓜果实酸性转化酶基因的cDNA片段，在GenBank中登记号为AF490425。 用 Hindlll和 EcoR消化植物表达载体 pBllZI，将其 Gus基因、355启动子和多克隆位点切除，将pSPROK的多克隆位点和355启动子插入，从而获得了pROKZ植物双元表达载体。用 BalnHI和 Hinc 11消化 pMAI，获得 MAI基因，回收纯化后定向插入pROKZ和 pROK3（将 pROKZ的 npt 11基因切除，由于占东完成）的 BamH侣 位 2更多还原

【Abstract】 The content and composition of soluble sugar in melon fruit play key roles in melon quality. Sugar accumulation and composition in melon fruit are controlled by sugar metabolism enzymes. The developments of plant biotechnology and molecular biology create a new path for studying on genes of melon metabolism enzymes and melon quality breeding.In this research, we constructed melon fruit cDNA library and cloned a cDNA fragment of melon fruit acid invertase gene. Melon fruit acid invertase (MAI) gene antisense expression vector was constructed and transferred into tobacco and melon especially mediated by agrobacterium tumefaciens strain LBA4404. Transgenic tobacco plants and kanamycin- resistance melon callus were obtained. The details were as follows:1. Construction of melon fruit cDNA libirayAt the beginning of 1990s, Clonetech in America invented SMART( the Switch Mechanism At the 5’ end of RNA Transcript) technique. A modified oligo(dT) primer primes the first-strand synthesis reaction, and the SMARTIV Oligo serves as a short, extended template at the 5’ end of the mRNA. When the RT reaches the 5’ end, the enzyme’ s terminal transferase activity adds a few additional nucleotides, primarily deoxycytidine, to the 3’ end of the cDNA. The SMARTIV Oligo, which has an oligo(G) sequence at its 3’ end base-pairs with the deoxycytidine stretch, creating an extended template. RT then switches templates and continues replicating to the end of the oligonucleotide. The resulting full-length ss cDNA contains the complete 5’ end of the mRNA which then serves as a universal priming site in the subsequent amplification by LD PCR. However, at present this technique is seldomly applied to plants and the applicatin for melon hasn’ t been reported. We firstly constructed a high quality cDNA library of yong melonfruit using SMART technique.Total RNA extracted from five days after pollination melon fruit was reverse transcripted into first strand cDNA using SMART technique. The double strand cDNA digested by sfi I (A and B) was ligated to SfiA and SfiB sites in the vector # TriplEx2. 8.54X105recombinants had been obtained after the mixture of ligation was packed in vitro and the host strain E. coli XLl-Blue was infected.100 clones was chosen randomly from the library and were tested cDNA inserts using PCR. The result indicated 81 percent of PCR products were over 500bp and the average length of cDNA inserts was 720bp. The recombinant rate of the library was 99. 53 percent by Blue/White Screening. These results showed that the cDNA library of melon fruit had been constructed successfully. This will provide theoretical basis for studying on the metabolism enzymes of melon fruit and other genes in the future.2. Cloning of a cDNA fragment of acid invertase gene from melon fruit and constructing of antisense expression vectorAt present, antisense RNA is used to inhibite some enzymes activities successfully. Antisense RNA can integrate into the genomic DNA of host and express stably. Antisense technology has been used in plant inprovement successfully for many species. The main measure for improving melon quality and increasing sugar content of melon fruit is to decrease the acid invertase activity in suitable period during the fruit development. Therefore, in this study we cloned a cDNA fragment of melon fruit acid invertase, constructed antisense expression vector and transformed it into tobacco and melon. These provides theoretical basis for transferring antisense acid invertase gene into melon and improving melon fruit quality at the level of molecular biology.PCR primers were designed based on the consensus domain of some acid invertase genes in GenBand. The aimed product with molecular weight of about 1kb was amplified from the total RNA isolated from ’Elizerbirth’ melon fruit via RT-PCR.Then it was inserted into pMD18-T vector and sequenced. The result showed this cDNA fragment contained 1038bp and 346 amino acids determined by this fragment. The amino acid sequence shows 70-99 percent homo更多还原