【作者】 陈银华；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2005， 博士

【摘要】 乙烯作为一种植物激素,对植物种子萌发、生长发育、衰老、果实成熟、性别分化等有广泛的影响,同时与逆境胁迫如机械伤害、冷害、渍水、病原菌入侵等方面有关;并参与植物细胞的程序性死亡。植物体内乙烯的生成量主要由ACC合成酶(ACS)和ACC氧化酶(ACO)的活性大小决定,而这两个关键酶活性的提高是其mRNA表达的结果。利用基因工程克隆植物乙烯ACS和ACO基因,通过反馈调节、反义表达等调控乙烯合成,进而调控乙烯对植物的生理效应,是当前对乙烯研究的主要途径。国内有关实验已在番茄等一些跃变型果实中获得成功。研究乙烯合成酶基因和氧化酶基因的克隆、表达及调控机理,对全面了解乙烯对植物的生理效应及化控机理具有重要的指导意义。 本研究的目的:克隆番茄、黄瓜、花椰菜等3种蔬菜作物的ACC氧化酶基因,对其结构进行分析;克隆ACC氧化酶基因的启动子,研究ACO基因的表达规律,为从分子水平上调控该基因的表达打下基础。最后,构建正义、反义、RNAi植物表达载体,调控ACC氧化酶基因的表达。结果如下: 1.克隆了番茄ACO基因LeACO11基因组序列(gDNA)及相应的cDNA序列。测序结果分析表明:该基因gDNA序列长1697bp,由4个外显子、3个内含子组成。相应的cDNA序列长1018bp。生物信息学工具软件分析,预计该基因CDS全长951bp,编码316个氨基酸。RT-PCR结果与预测的CDS一致。gDNA序列与cDNA序列比对发现,内含子与外显子交替的位置总是以AG碱基出现,并被剪切。对该基因cDNA推断的蛋白质进行同源性分析,与番茄ACO3(CAA90904)的同源性为85%,ACO1(CAA41212)的同源性为82%。 2.利用载体介导的PCR方法克隆了LeACO基因的上游调控序列2000bp。测序结果分析表明,2000bp的片段含有起始密码子上游1946bp,+97～+99位为翻译起始密码子,(-29～-22)之间为TATA盒(TATAAATA),(-193～-189)为CAAT盒(CCAAT),-1724～-1300与-1037～-592高度重复,存在100多个转录因子结合位点。 3.对番茄植株不同组织及不同发育时期的器官(茎、叶片、花、果实的未成熟期、绿熟期、破色期、红熟期)ACO基因的表达分析发现,LeACO11基因在叶片中有微量表达,在未成熟期果实中没有表达,在绿熟期、破色期、红熟期果实中均有高水平的表达,且随成熟度的增加其表达量增强。推断该基因的表达具有时间和空间特异性。更多还原

【Abstract】 Ethylene, a phytohormone, plays an extensive role in plant growth and development, such as seed germination, floral differentiation, sex determination and fruit setting as well as in various stresses: wounding, freezing, drought, plant disease. Ethylene biosynthesis in plants has been discovered. ACC oxidase is the key enzyme in the course. The antisense transgenic plants have been acquired in tomato. The research in cloning, expression of ACO and ACS gene is important to learn the role of ethylene and the mechanism of manipulation.The objective of this research was to clone the ACO genes of tomato, cucumber and cauliflower, and analyze the gene structures and expression patterns. Then, plant expression vectors with sense and antisense orientations and RNA interfering have been constructed and the manipulation of endogenous gene expression was done with the Agrobacterium-mediated transformation.1. The gDNA and cDNA of tomato ACO gene (LeACO11) was cloned using PCR. The full length of LeACO11 gDNA is 1697 bp with four exons and three introns. The length of LeACO11 cDNA is 1018 bp, including the coding sequence 951 bp and poly--A. It encoded 316 amino acids. The alignment results of the gDNA and cDNA showed that AG was always on the splicing site.2. 2000 bp upstream of LeACO11 gene was obtained using the method of vector-mediated PCR. Sequence analysis showed that it contains the regulatory elements: TATA box (-29 ~ -22), CAAT box (-193 ~ -189), wound and drought response elements. -1724 ~ -1300 and -1037 ~ -592 were repeated. 100 transcription factor binding sites exist in the upstream.3. Northern blotting of different tissues was conducted. The expression of LeACO11 is week in both leaves and the mature green fruits, however it expressed strongly in breaker and red ripen fruits.4. Five plant expression vectors were constructed, which consist of the sense, antisense, RNAi of LeACO11 cDNA sequence and two fragments of upstream sequences. Transgenic plants were acquired using the Agrobacterium-mediated transformation. Expression analysis showed the inhibition of the endogenous gene.5. The promoter regulatory elements were chereterized with histochemical analysis of GUS activity in various tissues. The results showed that the two fragments of the promoter were able to direct fruit-specific gene expressions. The expression driven by 2000 bp fragment of regulatory element increased with the maturation of fruits. The fragment of 1200 bp could direct uidA gene expression in fruits with the induction of drought and wound. The regulatory region for fruit-specificity was probably located in the 1200 bp of 5’-flanking sequence and some positive regulatory elements or enhancers may exist in the region from 1200 to 2000 bp.6. The cucumber ACO gene of CsACO4 was cloned using the method of homologous sequence with the GenBank accession number: AY450356. The length of the genome sequence is 2200 bp. The spliced length of mRNA was 933 nucleotides (nts) and it encoded 311 amino acids. The length of 5’ untranslated region of CsACO4 is 795 bp. The core region of the deduced promoter was located at -51- -11, TATA box at -47--39, and no CAAT box. Nine primary response elements concerning signal transduction were found in the upstream of TATA box, they are wound response elements, heavy metal response elements, drought response elements and auxin response elements.7. Northern blotting showed that the gene expressed among female flowers of gynoecious and monoecious genotypes, it could not express in other tissues or organs. This implied that the gene might be correlated with the female behavior positively.8. Plant expression vector of RNAi was constructed and genetic transformation was done using the Agrobacterium-mediated method. Five transgenic plants were identified by southern blotting. The morphological exploration showed that the inserted target fragment inhibited the endogenous gene expression and it contributed to the male differentiation in the lower nodes.9. The cauliflower ACO gene of BoACO was cloned using the method of homologous sequence with the GenBank accession number: AY676466. The size of BoACO is 1202 bp of nucliotides. Three exons and two introns were identified in this sequence. The spliced length of mRNA is 756 nts and encodes 252 amino acids. This gene expressed in the flowers and leaves, the levels in flower was higher than in the leaves.10. Plant expression vector of RNAi was constructed and genetic transformation was performed using the Agrobacterium-mediaied method. Three transgenic plants wereidentified by southern blotting. The aboundence of the endogenous mRNA in the transgenic plants was lower than in the wild type based on Northern blotting. As well the activity of ACO enzyme was inhibited significantly.更多还原