Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

Quantitative Screening of Secretory Protein Genes in <i>Candidatus</i>Liberibacter Asiaticus

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by Candidatus Liberibacter spp., which is vectored by the psyllids Diaphorina citri Kuwayama and Trioza erytreae. Secretory proteins are important in bacterial pathogenesis and structure components. Some of them are expressed at a high level. To obtain the highly-expressed secretory protein genes (SPGs) for antiserum preparation, six candidate SPGs were chosen from Candidatus Liberibacter asiaticus by bioinformatic analysis and were further tested by qPCR and RT-qPCR methods, respectively. The result showed that two SPGs, 408 and pap (both are Flp pilus assembly protein genes), have relative high amounts of DNA and RNA transcripts of early HLB-infected green orange leaves. The 408 and pap genes were further constructed into the plant expression vector pCAMBIA1300 (GV1300: GFP) and expressed in tobacco leaf epidermal cells for subcellular localization analysis. The transient expression results indicated that the 408 protein is located in the nuclei and cytoplasm of tobacco leaf cells. However, the pap protein is located in the cytoplasm of tobacco leaf cells, which may help the pathogen invade into plant cells. This research is an important foundation for the preparation of the antiserum against Candidatus Liberibacter asiaticus and the early detection of HLB disease.

Bioinformatics Analysis of the Interaction between Coat Protein and Nuclear Shuttle Protein in <i>Babuvirus</i>

Protein and protein interactions play important roles in many biological processes and are responsible for carrying out the function of biological regulatory network in living organisms. Previous study indicated that Banana bunchy top virus (BBTV) coat protein (CP) interacted with BBTV nuclear shuttle protein (NSP). However, the protein and protein interaction and the binding affinity of CP and NSP in Babuvirus are remaining unclear. In this study, the CPs and NSPs proteins of BBTV, Abaca bunchy top virus (ABTV) and Cardamom bushy dwarf virus (CBDV) were used for bioinformatic analysis. The binding free energy and the dissociation constant of the possible interaction proteins were tested in PPA-Pred2, and the results confirmed CP interaction with NSP in Babuvirus. The study will help us to understand the interaction between viral protein and viral protein, and the pathogenesis mechanism of Babuvirus in host plants.