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https://doi.org/10.1105/tpc.110.081695

has been cited by the following article:

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

  AUTHORS: Naitong Yu, Qin Zhou, Hongwei Yuan, Shuli Xian, Jianhua Wang, Yi Yang, Zhixin Liu

  KEYWORDS: Banana, E3 Ubiquitin-Protein Ligase, Antiserum Preparation, Polyclonal Antibody

  JOURNAL NAME: American Journal of Plant Sciences, Vol.13 No.3, March 17, 2022

  ABSTRACT: 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.