Cloning And Expression Of Calmodulin And Aux/IAA In Developing Ovaries/fruitlets Of Pear And Citrus, And In Citrus Somatic Embryogenesis

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.