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.
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