【作者】 哈斯阿古拉；

【导师】 扈廷茂；

【作者基本信息】 内蒙古大学 ， 动物学， 2004， 博士

【摘要】 以甜瓜品种河套蜜瓜（Cucumis melo L. cv Hetao）成熟果实RNA为模板,分别经反转录合成和PCR扩增得到编码ACC合成酶（1-aminocyclopropane-1-carboxylic acid synthase, ACS）、ACC氧化酶（1-aminocyclopropane-1-carboxylic acid oxidase, ACO）和多聚半乳糖醛酸酶（polygalacturonase, PG）基因cDNA,将其分别克隆于pUC19质粒中获得重组质粒。三个基因cDNA长分别为627bp、545bp和1183 bp。与已报道的甜瓜ACS基因、ACO基因和PG基因cDNA相应序列比较同源性很高。 Cucumisin是甜瓜类枯草杆菌丝氨酸蛋白酶,其表达具有果实特异性。本文应用PCR方法从甜瓜基因组DNA中扩增出该基因自转录起始位点至上游一段长310bp片段,并将其克隆到pUC19载体中,序列分析表明该序列与已报道的相应序列完全相同,且具有TATA-box、CAAT-box、G-box和I-box-like等功能域,具有典型的果实特异性启动子特征。成功获得甜瓜果实特异性表达基因的启动子,为下一步实现外源基因在甜瓜果实中特异表达奠定基础。 为建立甜瓜简便易行转化频率高的转基因技术,以甜瓜（Cucumis melo L.）品种河套蜜瓜为受体材料,以含gus基因的植物双元表达载体pPZP221为外源基因供体,进行了花粉管通道法转基因研究。自交授粉后分别于1、2、3、4、5、6、7、8、9、10小时,切去柱头上半部分,并立即滴加供体DNA溶液,果实成熟后收获转化种子。对T₀代植株进行PCR检测,结果表明不同时间处理所得到的T₀代植株均具有较高的转化频率,其中授粉后7h滴加DNA更多还原

【Abstract】 Total RNA was extracted from mature fruits of Hetao melons (Cucumis melo L.cv Hetao). The cDNA fragments encoding 1-aminocyclopropane-l-carboxylic acid synthase (ACS), 1-aminocyclopropane-l-carboxylic acid oxidase (ACO), and polygalacturonase (PG) were amplified by reverse transcription polymerase chain reaction (RT-PCR). These cDNA fragments were cloned into pUC19 and transformed into the E.coli DH5 a strain. The recombinant plasmids were designated as pCMACS, pCMACO, and pCMPG, respectively. The analysis of the nucleotide sequence showed that the length of the cDNA clones are 627 bp (ACS), 545 bp (ACO), and 1183 bp (PG), respectively. Their nucleotide and deduced amino acid sequences shared high similarity to those of the melon ACS, ACO and PG reported previously.Cucumisin, an extracellular subtilisin-like serine protease, is highly expressed in the fruit of melon (Cucumis melo L.cv Hetao) and accumulates in its juice. Thepromoter region of the cucumisin gene from the nucleotide at the position of-310 to -1 relative to the transcriptional initiation site was amplified from the melon genomic DNA by PCR and cloned into the pUC19 vector. The sequence analysis showed that the nucleotide sequence is completely identical to the published sequence. The genomic fragment contains some signature motifs typically found in the fruit-specific promoters, such as TATA-box, CAAT-box, G-box, and I-box-like box. This cloned promoter region may be useful for genetic engineering of the melon fruit.In order to establish a simple and practical transformation technique with a high transformation rate in melon (Cucumis melo L.cv Hetao), the pollen-tube pathway transformation method has been developed. The melon cultivar, hetao melon, was used as recipient and the binary vector pPZP221 containing a GUS (B glucuronidase) gene was used as the donor DNA of transgene. The donor DNA solution was applied to the cut surface of stigma at 1, 2, 3, 4, 5, 6, 7, 8, 9, and10 h after hand-pollination, respectively. PCR results show that the transformation rate in the different transformation events was very high with the highest rate of 30% at the 7 h time point. Southern blot analysis confirmed that all of the T1 plants indeed contained the transgene.The cDNA clones of the ACC synthase and ACC oxidase genes from melon were constructed reversely into the plant binery vector pROKII. The melon (Cucumis melo L.cv Hetao) cultivar, Hetao was transformed with these constructs using the pollen-tube pathway transformation method. A large number oftransformed seeds were obtained. About 10 T6 strains with improved storage capacity and other desired traits have been selected from two thousands of T0 seeds. The PCR and Southern blot analyses showed that the transgene was inserted into the recipient’s genome and inherited into the progeny of the primary transgenic plants. Northern analysis showed that the transgene was expressed in the fruits. The production of ethylene in the fruits of the transgenic plants was reduced approximately to 1% of that of the non-transgenic controls. In the field, non-transgenic melon fruits turned yellow upon ripening and yielded an aromatic flavor. The transgenic fruits remain green even at the late stages of ripening. The fruits from transgenic melon were stored for 2 months at room temperature without over-ripening or fungal attack. However, non-transgenic fruits have completely rotten and liquefied within two weeks of storage. The firmness of the fruits from transgenic melon was not changed during the storage period. The initial difference observed between the transgenic and non-transgenic fruits was the color of the rind. There was no noticeable difference between the transgenic and non-transgenic fruits in other physiological and biochemical parameters. The transgenic fruits turned yellow by the treatment of exogenous ethylene, and subsequently produced the aromatic flavor.更多还原