Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (5): 112-119.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1223
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ZHANG Na(), LIU Meng-nan, QU Zhan-fan, CUI Yi-ping, NI Jia-yao, WANG Hua-zhong()
Received:
2023-12-29
Online:
2024-05-26
Published:
2024-03-21
Contact:
WANG Hua-zhong
E-mail:zn1999227@126.com;skywhz@tjnu.edu.cn
ZHANG Na, LIU Meng-nan, QU Zhan-fan, CUI Yi-ping, NI Jia-yao, WANG Hua-zhong. Alternative Translation of Wheat Enolase-encoding Gene ENO2 and Its Prokaryotic Expression[J]. Biotechnology Bulletin, 2024, 40(5): 112-119.
引物名称 Prime name | 引物序列 Primer sequence(5'-3') |
---|---|
ENO-m-F | AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC |
ENO-m-R | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
Δ93-m-F | AAGTCCGGAGCTAGCTATGGTTCAGCAGCTTGATGGA |
Δ93-m-R | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
M94A-F1 | AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC |
M94A-R1 | AACAGCAAAGTTGTCGAGCTCAGTTTGAG |
M94A-F2 | GCTCGACAACTTTGCTGTTCAGCAGCTTGATGGAACCAAG |
M94A-R2 | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
ENO-GST-F | GATCTGGTTCCGCGTGGATCCATGGCGGCGACGATCCAGTC |
ENO-GST-R | GTCACGATGCGGCCGCTCGAGGTATGGCTCCACCGGTGCAC |
Table 1 Primers used in this study
引物名称 Prime name | 引物序列 Primer sequence(5'-3') |
---|---|
ENO-m-F | AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC |
ENO-m-R | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
Δ93-m-F | AAGTCCGGAGCTAGCTATGGTTCAGCAGCTTGATGGA |
Δ93-m-R | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
M94A-F1 | AAGTCCGGAGCTAGCTATGGCGGCGACGATCCAGTC |
M94A-R1 | AACAGCAAAGTTGTCGAGCTCAGTTTGAG |
M94A-F2 | GCTCGACAACTTTGCTGTTCAGCAGCTTGATGGAACCAAG |
M94A-R2 | GCCCTTGCTCACCATGGCGTATGGCTCCACCGGTGCAC |
ENO-GST-F | GATCTGGTTCCGCGTGGATCCATGGCGGCGACGATCCAGTC |
ENO-GST-R | GTCACGATGCGGCCGCTCGAGGTATGGCTCCACCGGTGCAC |
Fig. 1 Protein sequence alignment of wheat and Arabidopsis enolases TaENO2-5A, TaENO2-5B, and TaENO2-5D are wheat enolases of homeologous ENO2 proteins, and AtENO1, AtENO2, and AtENO3 are Arabidopsis enolase proteins. Solid red box indicates the serine residue(S)responsible for binding of Mg2+ in the catalytic center. Dashed red box indicates the second methionine residue(M)in the ENO2 sequences. The ATG codon encoding this residue is an internal start codon of alternative translation
Fig. 2 Alternative translation products of TaENO2 A: Schematic representation of the three forms(I, II, and III)of mCherry-fused TaENO2 coding sequences. B: Subcellular localization of the alternative translation products of TaENO2. Different mCherry fusion genes shown in(A)were respectively transformed into protoplasts for expression. The subcellular distribution of red fluorescence and the Hoechst 33342-stained nuclei in protoplasts were observed at 24 h after protoplast transformation. Scale bar indicates 10 μm. C: Western blot analysis on the alternative translation products of TaENO2 expressed in protoplasts
Fig. 3 Prokaryotic expression and purification of recombinant GST-TaENO2 protein A: Prokaryotic expression of recombinant GST-TaENO2 protein. The E. coli strain BL21(DE3)carrying the fusion gene GST-TaENO2 was grown to the log growth phase. The heterologous protein expression was then induced with IPTG for the indicated time periods. Cells were lysed by boiling and an equal aliquot of protein was resolved by SDS-PAGE. Arrows indicate the bands of recombinant protein. B: Solubility analysis on the prokaryotically expressed recombinant GST-TaENO2 protein. E. coli culture induced by IPTG was lysed by sonication. The soluble and insoluble protein fractions were separated by centrifugation and resolved by SDS-PAGE. Arrows indicate the bands of recombinant protein GST-TaENO2. C: Purification of recombinant GST-TaENO2 protein. M: Protein marker; 1: Total extracted protein from the E. coli culture which was not subjected to IPTG induction of gene expression; 2: Total extracted protein from the E.coli culture which had been subjected to IPTG induction of gene expression for 4 h; 3-6: Collected flow-through(3)and successive elution fractions(4-6)in the process of protein purification by affinity chromatography. D: Western blot analysis of the purified recombinant GST-TaENO2 protein. Arrows indicate the bands of recombinant protein
Fig. 4 In vitro assays on the enolase activity of the recombinant protein GST-TaENO2 The catalytic product of enolase, PEP, has maximum optical absorption at a wavelength of 230 nm. The figure shows the time-dependent changes in the optical density of the in vitro enolase assay solutions at 230 nm(OD230). Red dash box indicates the near linear increase stage of OD230. The data of this stage were used for calculation of enolase activity
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