【作者】 岳海林；

【导师】 彭抒昂；

【作者基本信息】 华中农业大学 ， 果树学， 2007， 博士

【摘要】 钙是决定果实品质形成的重要矿质元素，本研究通过对钙吸收和钙离子信号转导相关基因——钙调素(CaM)和Aux／IAA进行克隆及其在子房(幼果)和体细胞胚胎发生中的表达研究，探讨其在果实钙吸收和体细胞胚胎发生中的作用机理。主要研究结果如下：1、CaM和Aux／IAA的克隆及序列分析(1)以温州蜜柑国庆4号(Citrus unshiu Marc．cv．Guoqing 4)基因组DNA和盛花前后子房和幼果的总cDNA为模板进行特异性扩增，分别得到CaM基因的两条DNA和4条cDNA片段以及一条Aux／IAA的全长cDNA。CaM基因的碱基序列和氨基酸序列之间高度保守，在柑橘基因组上均有1-2个拷贝，推测可能是两种不同的钙调蛋白亚型或一个基因的两个不同拷贝，命名为CuCaM。Aux／IAA的全长cDNA在柑橘基因组中为单拷贝，命名为CuAux(2)从红肉脐橙(C．sinensis Osbeck‘Cara Cara’)果肉cDNA文库中，筛选得到了一条Aux／IAA基因的全长cDNA，命名为CsAux。Southern杂交表明：CsAux在柑橘基因组中为多拷贝基因，在柑橘上较为保守。2、钙调素(CuCaM)和CsAux在子房(幼果)和体细胞胚胎发生过程中的表达研究(1) CuCaM在黄花梨(Pyrus pyrifolia NaKai cv．Huanghua)盛花前后子房和幼果中的表达研究。Northern杂交和mRNA原位杂交结果表明，钙调素基因在黄花梨子房和幼果中的表达水平，花前较高，至盛花时最高，花后又下降；钙调素mRNA在梨果中的表达多集中于果皮、果肉、胚珠和果肉微管组织，而在果心的表达较少，且表达水平为果皮＞果肉＞果心，珠心＞胚囊＞珠被。在细胞水平上，CuCaM的表达主要集中于细胞的胞间隙和胞间层及细胞核。钙调素在盛花期子房／幼果中的大量表达，可能与果实钙的增加有一定关系。(2) CuCaM和CsAux在柑橘盛花前后子房和幼果中的表达研究。结果表明：CuCaM和CsAux在国庆4号盛花前和盛花期子房中均有较高表达，花后趋向下降；而CuCaM和CsAux在华农本地早橘子房和幼果中的表达盛花前后一直保持上升趋势。华农本地早橘花后受精不良的幼果及胚珠中CuCaM和CsAux表达水平明显低于同期正常发育的幼果。CuCaM和CsAux在胚囊中的表达，盛花前呈上升趋势，直到花后3天。CuCaM和CsAux在胚珠中的表达存在极性分布，在合点端的表达高于珠孔端。CuCaM和CsAux在两品种子房和幼果中的分布基本一致，主要分布在外果皮、胚珠等分裂旺盛的部位。细胞水平上，CuCaM主要分布于细胞壁、胞间层及胞间隙。由此表明，受精作用促进了CuCaM和CsAux的表达，钙调素和CsAux可能参与调控细胞分裂和胚胎发生。3、CuCaM和CsAux在体细胞胚胎发生中的表达研究(1) CuCaM在默科特橘橙(C．reticulata×C．sinensis)体细胞胚胎发生中的表达和CsAux在纽荷尔脐橙(C．sinensis Osbeck cv．‘Newhall’)体细胞胚胎发生中的表达基本一致。CuCaM和CsAux在胚性愈伤组织和非胚性愈伤组织中均有表达，在胚性愈伤组织中的表达水平远高于非胚性愈伤组织；随着体细胞胚的发育，在多细胞原胚、初生小球胚和球形胚中的表达水平高于胚性愈伤组织，在心形胚、鱼雷胚和子叶胚中的表达水平相当，但低于球形胚时期；在已分化的胚状体中，在原表皮和原形成层等细胞分裂旺盛的部位表达水平明显较高。CuCaM和CsAux在胚状体中的表达存在极性分布，倾向于集中在胚芽端大量表达，在胚根中的表达也较高。细胞水平上，CuCaM和CsAux集中在细胞的细胞核、细胞质、胞间层和细胞间隙中表达。由此表明，CuCaM和CsAux参与调控体细胞胚胎的发生和形态建成。(2) 2，4-D处理对伏令夏橙(C．sinensis Osb．cv．Valencia)二倍体胚性愈伤组织体细胞胚胎发生和CsAux表达的影响。组织切片观察发现，2，4-D处理一周时，胚性愈伤组织在早期胚胎发生能力增强，非胚性愈伤相对减少，但随后胚性能力开始下降，胚性细胞开始退化，随着处理时间延长，胚性能力逐渐丧失，在胚性细胞团的表面开始产生大量非胚性的薄壁细胞。随着2，4-D的处理后体细胞胚胎发生能力的改变，CsAux表达水平也呈现先升后降。说明高浓度的2，4-D抑制柑橘体细胞胚胎发生和形态建成，对CsAux表达也有一定的调控作用。更多还原

【Abstract】 Calcium is one of ion elements for fruit quality formation. In this study, genesrelated to calcium absorption and Ca²⁺ signal, such as calmodulin and Aux/IAA, werecloned and their expression patterns during ovary and fruitlet development and somaticembryogenesis were profiled. In addition, the mechanism on calmodulin and Aux/IAAplaying their roles in these processes was discussed. The main results were as follows:1. calmodulin gene and Aux/IAA cloning and sequence analysis（1） DNA and cDNA fragments of calmodulin and Aux/IAA from Citrus unshiu Marc.cv. Guoqing No. 4 were obtained using the genomic DNA and total cDNA of the ovariesand fruitlets before and after blossom as template. The nucleotide and amino acidsequences of the two CaM genes were highly conserved. Southern blotting revealed thatthere were 1-2 copies existed in Citrus, and it suggested that they were two differentsubforms of calmodulin or two copies of a CaM, named CuCaM. The Aux/IAA gene wasa single copy in Citrus, named as CuAux.（2） Complete cDNA fragment of Aux/IAA, termed as CsAux, was selected from thecDNA library of C. sinensis Osbeck ’Cara Cara’ sarcocarp. Southern blot revealed thatmultiple copies of CsAux existed in Citrus, and conserved in different species andcultivars.2. Expression patterns of CuCaM and CsAux in the ovary （fruitlet） development andsomatic embryogenesis.（1） Temporal and spatial expression patterns of CuCaM in pear’s （Pyrus pyrifoliaNakai cv. Huanghua） ovaries and fruitlets were investigated. Northern hybridizationdemonstrated that the expression level of calmodulin gene increased gradually beforeflowering, reached the peak at full blossom, and then decreased after flowering, mRNA insitu hybridization showed that CuCaM was located on pericarp, flesh, ovule and fleshvascular bundles, while expression signal could not be detected at core. The expressionlevels of calmodulin mRNA were as follows: pericarp＞flesh＞core; nucellus＞embryonic sac＞integument. At cellular level, CuCaM was located at nucleolus, interstitial space andintercellular layer. The high expression of calmodulin in ovaries and fruitlets atblossoming was probably related to the calcium uptake.（2） Temporal and spatial expression patterns of calmodulin in Citrus ovaries/fruitletsdevelopment were investigated. Results showed that CuCaM and CsAux expressed highlyin ovaries before and at blossom and decreased after blossom in Guoqing No.4. However,in Bendizao tangerine calmodulin and CsAux expressed at low levels 3 days beforeblossom and increased gradually at blossom and highly expressed after blossom.Expression of CuCaM and CsAux in weakly developed fruitlets were lower than those innormally developed ones. CuCaM and CsAux expression in embryo sac increased 3 dayspre-to post-blossom. CuCaM and CsAux in ovary expressed polarly and higher in chalazathan in micropyle. Distribution of the two genes in the ovaries and fruitlets wereapproximately the same, mainly in the prospertly mitosised organs such as pericarp andovules. At cellular level, CuCaM was located at cell walls, interstitial space andintercellular layer. The results suggested that fertilization could stimulate the expressionof calmodulin and CsAux and probablly these two genes regulated cell mitosis andembryogenesis.3. Calmodulin and CsAux expression in somatic embryogenesis（1） Expression pattern of CuCaM in somatic embryogenesis of Mucortt （C.reticulate×C. sinensis） was the same as that of C. sinensis Osbeck cv. ’Newhall’. CuCaM andCsAux expressed both in embryogenic and non-embryogenic callus but higher in theformer. With the development of somatic embryos, expression of the two genes increased,which was higher in pre-embryo, arche-global embryo, global embryo than inembryogenic callus. They expressed similarly in heart-shape, torpedo and cotyledonembryo, but lower than in global embryo. In the differentiated embryoids, they expressedhighly in prospertly mitosised locus such as in protoderm and protocambium. CuCaM andCsAux in embryoids polarly expressed, trended to accumulate in plumule and radicle. Atcellular level, CuCaM and CsAux expressed mainly in nucleus, cytoplasm, middle lamellaand intercellular space. The results above indicated that calmodulin mRNA and CsAuxregulated the initiation and morphogenesis in somatic embryogenesis.（2） Effects of 2,4-D （2,4-dichlorophenoxy acetic acid） treatment on somaticembryogenesis and CsAux expression of diploid Valencia embryogenic callus.Histological observation indicated that embryogenic capacity was enhanced with 1 week treatment and the non-embryogenic cells decreased. With the increase oftreatment time, the capacity of somatic embryogenesis decreased, embrygenic cellsdegenerated and large numbers of non-embryogenic cells initiated. With the changes ofembryogenesis capacity by 2,4-D treatment, expression of CsAux increased and thendecreased, which was coincident with the embryogenic capacity. The result revealedthat 2,4-D regulate the initiation and morphogenesis in somatic embryogenesis and theexpression of CsAux as well.更多还原