Construction And Canker Resistance Evaluation Of Three Polyprotein Genes Containing Two Different Antimicrobial Components In Transgenic Citrus (Citrus Sinensis Csbeck)

Citrus canker disease is a devastating bacterial diseases because of its great economic threatens to citrus industry. Genetic breeding has lots of advantages, such as short cycle, strong controllability, high efficiency and so on. So it has become an important method to cultivate disease-resistant varieties of citrus. Citrus heterologous genes such as animal or insect antimicrobial peptides have been successfully used in citrus disease-resistant genetic breeding. However, this heterologous peptides always show low efficacy because of its low express abundance, unsteadiness and easy to be degradated in plant cells. In this study, in order to improve the resistance of antibacterial peptide gene to citrus canker, three cleavable chimeric polyprotein genes containing two different antimicrobial peptides and lysozyme which are linked by an intervening sequence (linker peptide) was constructed. Theoretically, under the direction of the linker peptide, the chimeric polyproteins can be cleaved into different functional antibacterial peptides and human lysozyme in plant. The cleaving efficacy of linker peptide and canker-resistance of cleavable chimeric polyproteins were investigated in detail in transgenic citrus (Citrus Sinensis Csbeck). The main research results are as follows:1. Analysis of cleaveing efficiency of linker peptides 2 A and LP4 in citrus.In order to evaluate effect of linker peptides 2A and LP4 on cleaving chimeric polyprotein, two hybrids GFP-2A-GUS and GFP-LP4-GUS was constructed using GFP and GUS as reporter genes. The two fusion genes was placed under the control of CaMV 35S promoter, and was delivered into citrus genome by Agrobacterium-mediated mothod. GFP fluorescence and GUS histochemical analysis showed that GFP-2A-GUS and GFP-LP4-GUS constructs have GFP and GUS bifounctional activity in transgenic citrus. Western blot analysis showed that both 2A and LP4 linker peptide can effectively cleave chimeric polyprotein, but the cleaving efficacy of the 2A linker is bettter than that of the LP4 in transgenic citrus.2. Construction of chimeric resistant genes of CB-2A-HL, CB-2A-AP6 and CB-2A-AP5.Based on the above results, using 2A as a linker peptide and using the peptide genes CecropinB (CB), AP00355 (AP5), AP01065 (AP6) and human lysozyme (HL) as targeted protein, we constructed three fusion genes CB-2A-HL, CB-2A-AP6 and CB-2A-AP5. An extracelluar signal peptide sequence of AAT was fused into the 5’end of CB sequence, and the other signal peptide PR1a was fused into the 5’end of AP6、 AP5 and HL sequence.3. Construction of plant expression vector p35S:CB-2A-HL, p35S:CB-2A-AP6 and p35S:CB-2A-AP5 and citrus genetic transformantion.the CB-2A-HL、CB-2A-AP6、CB-2A-AP5 genes was placed separately at the downstream of the CaMV35S promoter in the expression vector pLGNL, and named these resulted vectors were named as p35S:CB-2A-HL, p35S:CB-2A-AP6 and p35S:CB-2A-AP5, respectively. These vectors was delivered into into citrus genome (Citrus sinensis osbeck) by agrobacterium tumefaciens-mediated transformation. 36 p35S:CB-2A-HL、25 p35S:CB-2A-AP6, and 16 p35S:CB-2A-AP5 transgenic plants were obtained an last. qRT-PCR analysis showed that the target genes had been expressed successfuly in transgenic plants.4. Evaluation of canker resistance of transgenic plants.In vitro citrus canker disease resistance evaluations showed that 6 out of 77 transgenic plants exhibited enhanced resitance to citrus canker.