【作者】 马乐园；

【导师】 于喜艳；

【作者基本信息】 山东农业大学 ， 蔬菜学， 2011， 硕士

【摘要】 甜瓜(Cucumis melo L.)是世界上重要的园艺作物之一,甜瓜果实含有大量的类胡萝卜素,其中β-胡萝卜素含量最为丰富。β-胡萝卜素被人体消化吸收后转变为维生素A,所以β-胡萝卜素也被称为维生素A原。医学研究表明,类胡萝卜素在增强人体免疫力、防止心血管疾病和癌症等方面具有重要的作用。八氢番茄红素合成酶是甜瓜类胡萝卜素合成途径中的关键酶,对甜瓜果实类胡萝卜素的合成起着关键作用,因此,利用现代分子生物学手段研究甜瓜八氢番茄红素合成酶的活性及其调节机制对改善甜瓜品质有重要意义。本研究通过HPLC测定了不同基因型甜瓜果实发育时期β-胡萝卜素含量,克隆到了甜瓜果实Psy基因全长cDNA,并对该基因进行了表达特性分析和遗传转化研究。主要研究结果如下:1.利用HPLC测定了不同基因型甜瓜果实发育时期β-胡萝卜素含量,结果表明,三个基因型(M01-3、Homoken和J-01)甜瓜在果实发育中,β-胡萝卜素含量的变化趋势是一致的,即果实发育前期,β-胡萝卜素含量很低。在果实接近成熟的时候,β-胡萝卜素含量迅速升高,成熟后,β-胡萝卜素含量又有所下降。橙色果肉的Homoken临近成熟的果实中β-胡萝卜素含量最高,为白色果肉的M01-3和浅绿色果肉的J-01的十几倍到几十倍。2.根据GeneBank中登记的甜瓜果实的Psy基因的序列设计引物,利用RT-PCR技术从M01-3甜瓜果实中克隆到了Psy基因,该基因全长1443 bp,编码422个氨基酸。在GenBank中登记号为GU361622。利用Real Time RT-PCR进行了该基因的表达特性分析。结果表明,Psy基因在根、茎、叶、花和果实中的表达水平不同,在果实发育前期,Psy基因的相对表达量很低,在接近成熟时达到最大值,成熟期又趋于下降。Psy基因在甜瓜果实发育中的表达趋势与β-胡萝卜素的变化趋势一致。3. Psy基因编码区与原核表达载体pET-30a(+)连接,并转化大肠杆菌BL21感受态细胞,构建重组质粒pET-Psy,并用8%的SDS-PAGE电泳分析。经IPTG诱导的目的蛋白分子量为46.7 kDa,说明该基因经诱导后可正确表达PSY蛋白4.甜瓜Psy基因的编码区连接在pMD18-T克隆载体上并测序。正、反向重组质粒pMD18-T-Psy分别用BamHⅠ和SalⅠ双酶切并与经BamHⅠ和SalⅠ双酶切的表达载体pBI121连接,转化大肠杆菌感受态细胞DH5α,获得正、反义表达载体。利用农杆菌介导法将正、反义表达载体分别对甜瓜进行遗传转化,已成功获得了Kan抗性的转化苗。更多还原

【Abstract】 Melon (Cucumis melo L.) is one of the most important horticultural crops in the world, it contains a lot of carotenoids, especiallyβ-carotene.β-carotene is transformed into the vitamin A after digested and absorbed in the body, so theβ-carotene also known as the original vitamin A. Medical studies have shown that carotenoids have important roles in enhancing human immunity, preventing cardiovascular disease and cancer. Phytoene synthase (PSY) is the key enzyme in the melon carotenoid biosynthesis pathway, and plays a key role in the synthesis of carotenoids in melon. To improve the quality of melon, it has important significance to study melon phytoene synthase activity and its’regulatory mechanism by the use of modern molecular biology methods. In this study, we measuredβ-carotene content at different developmental stages of melon fruit by HPLC. We cloned the full length of melon PSY cDNA by RT-PCR. We also analyzed the Psy expression characteristic and its genetic transformation to melon.The main results are as follows:1.β-carotene content in different genotypes’muskmelon fruit at different development stages were measured by HPLC. The results showed that in the fruit development of three genotypes (M01-3, Homoken and J-01), there are very littleβ-carotene content in early melon fruit development andβ-carotene content rapidly increased in nearly mature fruit and followed by declined after mature. Orange flash Homoken had the highestβ-carotene content in nearly mature fruit and significantly higher than white flash M01-3 and light green flash J-01.2. Primers were designed according to Psy gene conservative sequence in GenBank, A 1443bp cDNA was cloned from M01-3 melon fruit by RT-PCR, it encoded 422 amino acids and the registration number in GenBank is GU361622. Real-time RT-PCR was used to analyze Psy expression characteristic. The results showed that Psy expression level were different among root, stem, leaf, flower and fruit tissues. The Psy expression level was very low in the early development stage of fruit and reached the maximum at nearly mature fallowed decreased after mature. The expression trend of Psy was consist with the trend ofβ-carotene content in the development of fruit. 3. The ORF of Psy was ligated to pET-30a(+), and transformed into E. coli BL21 (DE3) competent cells to construct recombinant plasmid pET-Psy and analyzed by 8% SDS-PAGE. The specific expressed proteins induced by IPTG were about 46.7kDa in molecular weight. The results indicated that the protein was induced in vivo protein expression in the melon.4. Whole ORF of Psy was cloned in to clone vector PMD18-T followed by sequencing determination. The recombinant plasmids PMD18-T-Psy were isolated, double digested with BamHI and Sal1, ligated into BamHI - SalI digested pBI121 and transformed into E.coli competent cells DH5αand obtained sense and antisense expression vector, respectively. Agrobacterium-mediated method was used to genetic transformed sense and antisense expression vector into the melon,respectively. Melon seedlings with Kan resistance have been successfully obtained and they will be identified in a few weeks.更多还原