Activity Analysis of Tomato Fruit-specific Promoter 2A11 in Citrus and Isolation of Different Expression Gene in Citrus Fruit
|Keywords||Fruit-specific promoter 2A11 Orange (Citrus. sinensis) Kumquat (Fortunella crassifolia Swingle) Genetic transformation Real-time PCR cDNA-AFLP Gene expression Fruit maturation|
The availability of fruit specific promoters makes great benefits to modify the fruit quality by genetic transformation. To make it clear that if the other original fruit-specific promoters can be used to regulate the genes specifically in citrus fruit, a tomato fruit-specific promoter 2A11 was cloned successfully and its regulation function in kumquat (Fortunella crassifolia Swingle) fruit was identified by transient expression and genetic transformation system.Analysis of differential gene expression during fruit ripening/maturation is not only an efficient way of revealing the molecular mechanisms for fruit development and ripening/maturation, but an important prerequisite for isolation of some specific promoters as well. cDNA-AFLP was adopted to identify transcripts differentially expressed in peel and juice vesicle of immature and ripe orange (Citrus sinensis) fruits. The study also included analysis of selected TDFs with real-time PCR to present quantitative information of both specificity and magnitude of expression as well as to confirm cDNA-AFLP results. The main results were summarized as followings:(1) Construction of an expression vector suitable for transient gene expression and apply to identify the function of 2A11 promoter in kumquat fruitApply the strategy that using two adaptors to ligate striction fragments, an intron-GUS gene from vector pCambia1301 was inserted to the pBI121 vector to replace its GUS gene in order to prevent GUS expression in Agrobacterium cells, which results in interference in plant transient expression study. By transient expression study carried out with kumquat tissues as samples, it was found that the vector without interfere by the adaptors added. Moreover, 35S promoter was changed instead by 2A11 promoter. The transient assay had been used to study the activity of 2A11 promoter in different tissues of kumquat. The results showed there is very low or no activity in nutritious tissues, but higher activity in fruit and ovary, indicating 2A11 promoter probably specific for its function in kumquat fruit.(2) Agrobaterium-mediated transformation of tomato fruit-specific gene promoter 2A11A modified GUS gene fused a CaMV 35S promoter and a normal GUS gene driven by the fruit-specific promoter were constructed to the same one plant expression vector pLZ16. An efficient genetic transformation system of kumquat which had been already established was applied to identify the function of promoter 2A11 in kumquat. The results of the normal PCR, MPCR and Real-time PCR analysis showed that the 2A11 promoter fused GUS gene was integrated into kumquat genomes. Three resistant plants incorporating GUS gene under the fruit-specific promoter of tomato were obtained to evaluate the expression pattern of the promoter.(3) Identification and characterization of transcripts differentially expressed in peel and juice vesicle of immature and ripe orange (Citrus sinensis) fruitsFruit tissue preferential genes, especially the fruit ripening/maturation specific ones, are not only necessities for isolating specific promoters but for understanding the molecular mechanisms of fruit ripening/maturation as well. By applying cDNA-amplified fragment length polymorphism (cDNA-AFLP), the current study was aimed to identify the transcripts differentially expressed in peel and juice vesicle of immature and ripe orange (Citrus sinensis) fruits. A total number of 66 primer combinations were applied in cDNA-AFLP screening, which generated products ranging in size from approximately 100 bp to 800 bp. Products were analysed on 3.5% Amresco 3:1 HRB agarose gel, and from which 40 differentially expressed transcript-derived fragments (TDFs) were cloned, sequenced, and homologous information obtained by BLASTing GenBank. The putative functions of the TDFs were related to photosynthesis, respiration, phospholipid biosynthesis, gene transcription as well as stress responses and plant defenses. The expression patterns of five selected TDFs among various tissues and fruits from different developmental stages were analysed by real-time PCR. One of these TDFs, with specific expression in ripe juice vesicle, was found to putatively encode a novel type of invertase/pectin methylesterase inhibitor. These TDFs are ideal candidate genes for further ripening specific promoter isolation as well as fruit maturation investigation for citrus fruits.