Construction And Citrus Transformation Of Novel Cecropin B Gene For Improving Extracellular Secretion Of Antibacterial Peptide

Citrus is one of the most important commodity fruit in the world, and its plantation area and output rank first in all the kinds of fruit, and citrus fruit has become the fifth largest agricultural products in international trade. However, one of the most challenges in citrus industry is the threat of various diseases and insect pests, and Citrus canker disease is devastating disaster for citrus industry. Therefore, it becomes important to research strategies controlling the disaster of citrus canker disease.The preliminary research showed that the antibacterial peptides from tussah could significantly improve citrus resistance to canker bacteria. Here, based on the intercellular parasitizing characteristics of citrus canker bacteria, two new editions of cecropin B gene for secreting antibacterial peptide to exacellular space were synthesized by PCR and transformed into citrus by Agrobacterium-mediated genetic transformation. This will provide a novel strategy and materials for further effectively modifying citrus resistance via antimicrobial peptides. The results are as follows:1. The synthetic of secretory cecropin B genesBased on associated literarures,6 selected signal peptides(PRla、aAM、GRP、PG、SPS、AAT) were subjected to bioinformatics analysis, and PRla, aAM, PG and AAT signal peptides showed the potential of improving intercellular excretion of Cecropin B protein and were used to design new cecropin B gene for targeting Cecropin B peptide to intercellular space. And finally PRla:CB and AAT.CB were successfully obtained by PCR method.2. Sub-cellular localization expression analysis of PRla:CB and AAT:CB genes Using the red fluorescent protein (RFP) gene as reporter gene, plant expression vectors for sub-cellular localization gene expression analysis were constructed and were transferred into onion skin via Arobacterium-mediated transformation. The sub-cellular localization expression of antibacterial peptides was observed with fluorescence microscope.The results showed that either of PR1a or AAT signal peptide could efficiently promote antimicrobial peptide molecule secreting to extracellular space of onion skin.3. Construction of plant expression vectors with overexpressing antibacterial peptides gene and genetic transformation of citrusCaMV 35S promoter was used to drive the expression of antibacterial peptides gene, and p35S-PRla:CB、p35S-AAT:CB and p35S-CB plant expression vectors were constructed and transferred to Citrus sinensis (Linn.) Osbeck cv. Jin Cheng by 4grobacterium-mediated method.72 transgenic plants containing antibacterial peptides gene were obtained by GUS histochemical straining and PCR analysis.4. Real-time PCR analysis of antimicrobial peptide gene expression in transgenic plantsTotal RNA were extracted from different independent transgenic Jincheng plants, and the expression levels of cecropin B in transgene plant were investigated by Real-time PCR using actin gene as reference.The result displayed that the expression of cecropin B was detected in all transgenic plants transformed by PRla:CB, AAT.CB or CB gene, while no cecropin B mRNA was found in pGN transgenic plants without cecropin B.