【作者】 缪恒彬；

【导师】 陈发棣；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2009， 博士

【摘要】 菊花原产我国,是我国十大传统名花和世界四大切花之一,具有很高的观赏和应用价值,在花卉生产中占有十分重要的地位。但菊花新基因的发掘和分子育种方始起步,本研究在构建菊花花器官EST文库,分析其花序开放过程基因表达谱基础上,对GA 20-氧化酶基因新基因进行了克隆与表达分析。主要结果如下：1.首次以菊花特定发育阶段的花器官为植物材料,利用pBluescript XR载体构建了一个菊花花器官cDNA文库,文库滴度为1.8×107 pfu/ml,扩增后文库滴度达9.0×1011pfu/ml。经过蓝白斑计数,文库的重组率可达90%。平均插入片段大小为1-2kb左右,说明所构建的文库完全符合目的基因分离筛选和表达建库的要求。通过4个花器官优势表达基因的半定量PCR分析,均发现在花器官中的表达量较高,说明该文库具有代表性和有效性,可为进一步开展与菊花发育相关基因的克隆及花器官发育分子机理的探讨等研究奠定重要的基础。2.在构建的cDNA文库中随机挑选了7,500个克隆进行5’端测序,共获得7,307条原始序列。原始序列经去除插入片段小于100bp和污染序列后,共得到6,924条高质量的EST序列,平均GC含量为43.5%,序列递交DDBJ数据库（登录号：DK936567-DK943490）; CAP3程序拼接ESTs得到4,563个Unigenes,其中包括996个contigs和3,567个singlets,冗余度为34.1%。通过序列比对注释,发现有57.2%的Unigenes （2,608）能够通过注释进行功能分类,而42.8%的Unigenes （1,955）不能比对上现有数据库中的任何蛋白质或核酸序列,推测1,955个Unigenes可能是有一些已知基因的非编码区序列,也有可能是一些功能未知的新基因。3.所得到的EST序列中,编码RuBP羧化酶小亚基的EST数量最多,说明rbcs基因在该cDNA文库中的表达量较高。26.7%的Unigenes （1,217/4,563）可以通过COG聚类,其中最高14.79%的Unigenes （180/1,217）编码转录后修饰、蛋白转运相关的酶,而与防卫机制以及细胞运动性相关的Unigenes最少。在所有的6,924条高质量ESTs序列中,35.1%能比对上拟南芥的ESTs,27.2%的序列比对上了水稻的ESTs序列,与菊科植物向日葵、莴苣、非洲菊、百日草的ESTs序列相比较,分别有82.0%、64.6%、48.8%和24.4%的序列能比对上。同时,在6,924条ESTs中,有131个ESTs编码推测的转录因子,占整个Unigenes的2.78%（131／4,563）。其中表达量最高的是C3H转录因子家族,占全部转录因子ESTs的16.0%（21／131）；在4,563个Unigenes中,我们找到了6个已知的花发育相关基因和10个与花色素合成代谢相关的基因。4.在菊花花器官cDNA文库的EST序列中,找到一条与GA 20-氧化酶基因同源的EST（克隆号：rfchcaa0₀07176）.挑选该克隆进行双向测序,并进行3’、5’RACE-PCR得到1,543bp的全长菊花GA 20-氧化酶基因cDNA序列（DgGA20ox,AB360381）,具有一个1,134bp的ORF,编码377个氨基酸及103bp的5’非编码区和306bp包含了多聚A尾的3’非编码区。与其相应的基因组DNA序列（1,468bp,AB360434）比对后发现,该基因基因组DNA序列中包含了90bp和234bp的内含子。通过氨基酸序列比对发现,其具有GA 20-氧化酶所特有的依赖2-酮戊二酸的双加氧酶特性的保守序列,如与2-酮戊二酸结合有关的保守序列NYYPXCQKP,与GA底物结合有关的LPWKET基元以及可能与Fe2+结合有关的His残基（H246和H302）和Asp残基（D248）,并与同科植物莴苣的Ls20ox1氨基酸序列有87%的同源性,确定为菊花的GA20-氧化酶基因。Southern杂交表明,该基因在菊花中存在多个同源拷贝。5.荧光定量PCR结果表明,DgGA20ox基因在菊花的嫩叶中表达量最高,其次是茎和花中,根中的表达量最少,说明嫩叶可能是菊花活性GA生物合成的主要位点；在菊花顶芽中,DgGA20ox基因在一天中的表达呈规律性起伏,在黑暗条件下的转录水平比在光照条件下高,且在光照结束后4h表达量最高。外源激素处理证明了DgGA20ox基因的表达受活性GA的反馈抑制调控,内源GA含量高时,DgGA20ox基因的表达受抑制；内源GA生物合成受阻抑时,DgGA20ox基因的表达量升高,同时内源的IAA含量也升高,说明IAA在GA的生物合成中有重要作用。6.构建DgGA20ox基因正义表达载体,采用农杆菌介导的叶盘法进行遗传转化,通过PCR和Southern杂交鉴定,有3个株系整合进了外源DgGA20ox基因。表型观察发现转基因植株较对照矮化,叶片小,生长势弱；进而进行DgGA20ox基因的表达分析及内源GA含量测定,发现转基因植株中无论是DgGA20ox基因的表达量还是内源GA含量都明显低于对照,推测转基因植株可能出现了共抑制的基因沉默。更多还原

【Abstract】 Chrysanthemum, Chrysanthemum×morifolium （Compositae, Anthemideae）, has historical roots in China, and is one of the ten most famous flowers in China, which is used as the potted flower, cut flower, and applied as the ground-cover plant. Chrysanthemum is valuable for its ornamental character, and is very important for the flower production. Nowadays, discovery the new genes and molecular breeding of chrysanthemum start the first step. In this research, we constructed a cDNA library from inflorescence of chrysanthemum and analysed the expression of genes in the procedure of inflorescence. The main results are as follows:A cDNA library for the floral buds in one special stage of Ch. morifolium was constructed by using pBluescript XR vector. The quality detection indicated that the library had a starting titer of 1.8×107 pfu/ml and a final titer of 9.0×1011 pfu/ml after amplification. It also had a high ratio of recombination （up to 90%） with the inserts at an average size of approximately 1-2 kb. These results showed that this library was highly quality, which is suitable for the isolation and expression analysis of target genes. As four putative flower-preferential genes selected in this cDNA library all expressed most in the floral buds, the cDNA library was proved be effective and could provide an important foundation for the studies on the molecular mechanisms involved in the flower development of chrysanthemum.By large-scale ESTs sequencing of 7,500 randomly selected clones from the constructed library of chrysanthemum inflorescence,7,307 raw sequences were obtained. About 6,924 valid ESTs were generated after cleaning of inserts that is shorter than 100bp, the average GC content of the 6,924 valid ESTs was 43.5%. These EST sequences were submitted to the DDBJ database （Acession No.:DK936567-DK943490）. After clusting by CAP3 procedure, a total of 4,563 unigene including 996 contigs and 3,567 singlets, were ultimately selected corresponding to a redundancy level of 34.10%. About 57.2%of the unigenes （2,608） could be functionally classified after the BLAST search, with the remaining 42.8%unigenes （1,955） showing little of no similarity to any of the proteins and gene sequences in the published databases. Inability to detect matches in nr database for 1,955 chrysanthemum sequences might be due to short or even absent coding regions, consequence of randomness of 5’-reads and low conservation of 5’and 3’UTRs. Thus, many ESTs from genes with homologues in plants exhibit no match. On the other hand, novel genes can’t be excluded from the 1,955 sequences with no match.Among the 6,924 valid ESTs, the most abundant ESTs （101） were coding for ribulose-1,5-bisphosphate carboxylase small subunit （1.46%）. That is to say, the rbcs gene may be the highest expressed gene in this cDNA library. About 26.67%（1,217/4,563） of unigenes from this cDNA library were clustered by COG. The highest percentage of ESTs that were similar to genes in a particular class was 14.79%for those encoding enzymes of Post-translational modification, protein turnover and chaperones. The lowest percentages were for Defense mechanisms （0.16%） and Cell motility （0.16%）.6,924 EST sequences of chrysanthemum were compared with ESTs collections of Arabidopsis, Oryza and four species within Asteraceae using the BLASTn search, respectively.35.1%of chrysanthemum EST sequences were homologous to Arabidopsis ESTs,27.2%to Oryza ESTs. When the comparison is restricted to Helianthus, Lactuca, Gerbera and Zinnia （species within Asteraceae）,82.0%,64.6%,48.8%and 24.4%of chrysanthemum ETSs sequences have a match to ESTs from each of the species, respectively.131 ESTs were encoding putative transcription factors representing 2.78%（131/4,563） of the chrysanthemum unigenes. The most abundantly expressed transcription factor gene family, C3H, accounted for 16% （21/131） of overall transcription factor expression We also found 6 known genes related to the development of flower organ and 10 known genes related to the pigment anabolism in the 4,563 unigenes.An EST （clone No.:rfchcaa0₀07176） homolog to the GA 20-oxidase was found in the ESTs from this cDNA library. A full-length chrysanthemum DgGA20ox cDNA （GenBank accession AB360381） was derived by clone sequencing and RACE-PCR. This sequence contains a single 1134bp ORF, encoding a 377 residue peptide, with a 103bp 5’ untranslated region and a 306bp 3’non-coding region with a poly （A） tail. A comparison of the cDNA and its genomic counterpart （GenBank accession AB360434） demonstrated that the gene is composed of two introns （90bp and 244bp） and three exons. After comparing with other GA 20-oxidase amino acid sequence, the chrysanthemum GA20ox protein shares a high sequence homology with its orthologues. It includes the highly conserved motifs NYYPXCQKP （postulated as being involved in binding the 2-oxoglutarate co-factor） and LPWKET （thought to be involved in binding the GA substrate）, and the conserved H （H246and H302）and D248 residues （involved in Fe2+binding at the active site of isopenicillin N synthase）. The GA20-oxidase sequences of lettuce and chrysanthemum both within the family Compositeae shared the highest 87%similarity. The hybridization pattern of chrysanthemum genomic DNA （Southern blot） with DgGA20ox after restriction digestion suggests the presence of several GA 20-oxidase copies in the chrysanthemum genome.The expression pattern of DgGA20ox was analyzed by using quantitative real-time PCR analyses. A highest transcript level of the DgGA20ox gene was detected in young leaves followed by stems and flowers. The expression of DgGA20ox gene in roots was very low. It can be suggested that the young leaves may be the mainly site for the active GA synthesis. There was a diurnal oscillation in expression of DgGA20ox gene in shoot apex of chrysanthemum, and showed circadian rhythmicity in constant LD conditions. We confirmed that the GA20ox gene was feed-back regulated by active GAs. The transient increase in active GA following the provision of exogenous GA3 decreased DgGA20ox expression. Conversely, decreasing the endogenous content of active GA by the supply of PAC increased the expression of DgGA20ox as well as the endogenous content of IAA. It was also suggested that the IAA might play a role in the GA biosynthesis.A sense plant expression vectors for DgGA20ox were constructed and were transferred into chrysanthemum via Agrobacterium tumefaciens-mediated transformation based on the establishment of a high efficient genetic transformation system. PCR and Southern blot analysis confirmed the integration of foreign genes into the genome of 3 transgenic chrysanthemum. The observation of phenotype revealed the transgenic chrysanthemum with small leaf, semi-dwarf height compared with untransformed plant. Further analysis of the expression pattern of DgGA20ox and the endogenous content of active GA in the transgenic chrysanthemum both decreased compared with untransformed plant. It was suggested that the integration of foreign homologue genes may coordinated and reciprocal inactivation of host genes and transgenes encoding the same sense RNA, and then leaded to a cosuppression phenotypes.更多还原