| Citrus is one of the most economically important fruit tree speciese in the world. Its annual global trade value has reached nine billion dolars. Citrus tristeza virus is the causal agent of citrus tristeza, which was considered as the most devastating virus disease in citrus industry. The successful infection of a plant virus depends on the interaction between the virus and its host. The accumulation of virus in the process of infection disturbs the normal metabolism of host and result in the symptom exhibition. The infection of CTV could cause distinct symptoms like stem pitting, seedling yellow and tristeza on its host. However, the mechanism underlying the symptom development induced by CTV is still poorly understood, In this research, the transcriptional changes caused by a CTV severe isolate named N21were evaluated by using SSH, and the expression profiles of some candidate genes associated with defense and energy metabolism were analysed during a time course of CTV infection in leaves at different development stage. The proteins involved in the interactions between CTV and its suscept and resistance hosts were also identified. The main results obtained in this research were list as follows:1. The transcriptional changes of Mexican lime response to CTV-N21infection were identified by SSH and two differentially expressed gene libraries consisted of14000clones were constructed. Screened by reverse southern blot, a total of774clones,501from the forward library and273from the reverse library, showing significant differential hybridization signals were selected and sequenced. A search for homologous genes of the selected EST sequences was performed by using tool of BlastX in NCBI database and the result showed that the657ESTs represent430genes, of which282were from forward library and148were from reverse library. The relative expression levels of25genes,14from forward-and11from reverse-library, in CTV-N21infected lime plants to that in mock inoculated lime were analyzed by qRT-PCR. Results showed that the relative expression levels of14genes from the forward library were up-regulated for1.2-10fold,11genes from the reverse library were down-regulated for1.1-7fold. According to a criterion of fold change≥1.5for positives, the credibility of all differentially expressed ESTs identified by SSH was above80%. According to GO annotation of molecular function, these430genes could be classified into nine main functional categories. The largest gene set involved in primary metabolism (26.0%), and then in protein metabolism (15.1%).351genes of Arabidopsis were found homologous to the screened430genes by BlastX search and their encoding proteins showed a complicated interaction network. The largest set of interactions was involved in energy metabolism.2. Expression profile of six genes in upper and lower leaves were analysed by real-time PCR during a time course of17to177dpi with CTV-N21inoculation. Three of these genes including carbonic anhydrase, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and light-harvesting complex Ⅰ protein Lhca4were functionally involved in energy metabolism and other three genes including acidic class Ⅱ chitinase, miraculin-like protein and rsdvl resistance like protein were involved in defense and response to stress. The expression of these genes fluctuated during the infection of CTV-N21and the expression profiles differed in upper and lower leaves. The two genes encoding miraculin-like protein and acidic class Ⅱ chitinase had a similar induced expression tendency. Both of their expression peaked at97dpi, but showed different induction levels in upper and lower leaves. The expression level of the gene encoding a homolog of rsdvl resistance like protein showed to be persistantly repressed after CTV-N21infection in upper leaves. However, its expression in lower leaves was showed to be induced at137dpi. The induction of light-harvesting complex Ⅰ protein Lhca4gene showed consistently reduced in both upper and lower leaves, and turned to be repressed at137dpi. The expression of genes encoding carbonic anhydrase and RuBisCO was totally reduced during the infection of CTV-N21and showed a different expression profile in upper and lower leaves.3. Moxican lime and C. trifoliata plasmid cDNA libraries were constructed by RT-rPCR and in vitro in fusion technology. The protein-protein interactions between virus and host were identified by cotransformation of the virus bait and library plasmid. The results indicated that CTV viral protein p20and p23may interact with three host proteins, which were homology to carbon-sulfur lyases, protein phosphatase, homeodomain leucine zipper family IV protein (HD-ZIP) in Arabidopsis. p20and HD-ZIP were shown interacted in BiFC analysis in Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaves. The fluorescence signals resulted from their interaction accumulated and showed some bright spots along the cell wall, which implied that they may interact at cell wall and the bright spots may located at plasmodesmata. |
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