Cloning And Analysis Of Cmeif4Ei CmeIF(Iso)4E Gene From Chrysanthemum Morifolium And Study On The Interaction With CVB Coat Protein

Chrysanthemum morifolium is perennial root herb of the genus Chrysanthemum in the composite family, which has a huge ornamental value and economic value. In producing aspect, mainly Adopts Cuttings so that lead to the accumulation and dissemination easily of the virus, and greatly reduces the quality and economic value of the chrysanthemum. About more than20varieties of the virus has been reported at home and abroad which infected chrysanthemum. Eight varieties have been reported in China, Chrysanthemum B virus is most damaging viruses. The study found that eukaryotic translation initiation factor4E is a subunit of translation initiation factor eIF4F. It is the most effective speed limit adjustment factor in the course of mRNA translation. eIF4E plays an important role in the regulation of plant gene expression, in addition to eIF4E is the sensitivity factor of the RNA virus proliferation, plays a key role in the infection process of plant viruses. Therefore, mutant eIF4E protein can affect the interactions of virus and plant, thus effectively blocking viral infection, and the resistance has a broad spectrum for use of plant gene. The chrysanthemum translation initiation factor4E gene has not been reported. Therefore, the CP of CVB and CmeIF4E and CmeIF(iso)4E is for the object of this study. Study on the the CVB pathogenic mechanisms and the resistance of Chrysanthemum was carried out. We cloned CmeIF4E and CmeIF(iso)4E gene from chrysanthemum, analyzed the expression characteristics and to study the interactions between the CVB CP with CmeIF4E or CmeIF(iso)4E, which layed the foundation for the study of translation initiation factor4E on expression regulation and virus infection. The main findings are as follows:1. We cloned eIF4E and eIF(iso)4E gene from Chrysanthemum, named CmeIF4E (Genbank:JQ904591) and CmeIF(iso)4E (Genbank:JQ904592). Sequence analysis showed that these two genes are typical translation initiation factor4E gene which have IF4E family domain. CmeIF4E has total length of914bp,654bp ORF encoding218amino acids, which showing79%sequence identity with that from lettuce. The sites which combined with eIF4G and the cap structure of mRNA are high conservation in the process of protein translation initiation. CmeIF(iso)4E has length of818bp,573bp ORF encoding191amino acids, the deduced amino acid sequence with90%sequence identity with that from lettuce. The amino acid sequence similarity of CmeIF4E and CmeIF(iso)4E is45.66%.2. The Real-time PCR results show that the CmeIF4E and CmeIF(iso)4E genes are expressed at root, stem, leaf, tubular flowers and ligulate flower’Jinba’. Expression level of CmeIF4E from the highest to the lowest is the order: roots, leaves, tubular flowers, stems and ligulate flower. Expression level of CmeIF(iso)4E from the highest to the lowest is the order:roots, tubular flowers, ligulate flower leaves and stems.3. Green fluorescent protein (GFP) and CmeIF4E or CmeIF(iso)4E fusion expression vector was constructed and transformed into onion epidermal cells for transient expression by particle gun. The results showed that CmeIF4E and CmeIF(iso)4E protein are distributed in the nucleus, cytoplasm and cytomembrane.4. Yeast expression vector pGBKT7-CVBCP and pGADT7-CmeIF4E and pGADT7-CmeIF(iso)4E was constructed to prove the interaction between the CVB CP and CmeIF(iso)4E by yeast two-hybrid system. The interaction between CmeIF(iso)4E and CVB CP was confirmed by BiFC, the results showed that they are interacted in the whole cell..5. We have screened the proteins which interact with CVB CP, CmeIF4E or CmeIF(iso)4E in the chrysanthemum’Jinba’ by the yeast two-hybrid system, respectively and found the sequence of E3ubiquitin ligase ARIADNE-like protein and ATP-binding protein initially interact with the coat protein of CVB. Some proteins were screened which interacted with CmeIF4E or CmeIF(iso)4E, which involve in protein synthesis, photosynthesis, resistance stress and defense-related protein, such as translation initiation factor4A, ribosome inactivating protein and ribulose-1,5-phosphatase and so on.