Abstract
Background
Ophiorrhiza pumila (Rubiaceae) is capable of producing camptothecin (CPT), one monoterpene indole alkaloid extensively employed in the treatment of multiple cancers. Transcription factors (TFs) GATA are a group of transcription regulators involved in plant development and metabolism, and show the feature of binding to the GATA motif within the promoters of target genes. However, GATA TFs have not been characterized in O. pumila.
Result
In this study, a total of 18 GATA genes classified into four subfamilies were identified, which randomly distributed on 11 chromosomes of O. pumila. Synteny analysis of GATA genes between O. pumila and other plant species such as Arabidopsis thaliana, Oryza sativa, Glycine max, Solanum lycopersicum, Vitis vinifera, and Catharanthus roseus genomes were analyzed. Tissue expression pattern revealed that OpGATA1 and OpGATA18 were found to be correlated with ASA, MK, CPR and GPPS, which were highly expressed in leaves. OpGATA7, showed high expression in roots as most of the CPT biosynthetic pathway genes did, suggesting that these OpGATAs may be potential candidates regulating CPT biosynthesis in O. pumila.
Conclusions
In this study, we systematically analyzed the OpGATA TFs, and provided insights into the involvement of OpGATA TFs from O. pumila in CPT biosynthesis.
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Background
Ophiorrhiza pumila is a dicotyledonous plant classified into Rubiaceae family and remains as a sustainable source of camptothecin (CPT). CPT is a type of monoterpene indole alkaloids (MIAs) commonly used in treatment of cancers and was initially isolated from Camptotheca acuminate [1], and subsequently detected in Nothapodytes nimmoniana and other plants [2,3,4]. CPT inhibits tumor growth by blocking DNA topoisomerase I [5, 6]. Topotecan and irinotecan, two drugs developed by CPT derivatives, have been extensively employed in various cancers including lung, colorectal, cervical, and ovarian cancers [7]. The biosynthesis pathway of CPT is complex and remains not fully resolved [8]. Briefly, the terpene section of CPT is derived from the 2-C-methyl-d-erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways. The produced geraniol is hydroxylated to 10-hydroxygeraniol under the catalysis of geraniol 10-hydroxy (G10H) [9,49]. Gene replication events were investigated using the multiple collinear scanning toolkit (MCScanX) and BLASTP method. TBtools software (https://github.com/CJ-Chen/TBtools) was adopted to build syntenic analysis maps for determining the syntenic relationships between OpGATA proteins and GATA proteins from A. thaliana, O. sativa, G. max, S. lycopersicum, V. vinifera and C. roseus.
Expression analysis by quantitative real-time PCR (QRT-PCR)
Total RNA was extracted using a Plant RNAprep Pure Kit (TIANGEN, China). Corresponding sequences of OpGATA genes and key enzyme genes were acquired from the O. pumila genome sequence database (http://pumila.kazusa.or.jp/). Primers used for QRT-PCR analysis were designed using Primer 5 software (Table S6). Relative expression levels were calculated using the 2−ΔΔCt method, with housekee** gene OpActin from O. pumila as the internal control [10]. All QRT-PCR analyses were performed with three biological replicates. The heatmap was constructed by TBtools software base on QRT-PCR analysis. Pearson’s correlation coefficient was analyzed using the OmicStudio tools at https://www.omicstudio.cn, significant difference was tested at significance levels of 0.05, 0.01, and 0.001.
Availability of data and materials
The genomic information of Ophiorrhiza pumila was downloaded from Ophiorrhiza pumila Genome DateBase (http://pumila.kazusa.or.jp/). All data generated or analyzed during this study are included in this published article and its supplementary information files.
Abbreviations
- O. pumila :
-
Ophiorrhiza pumila
- A. thaliana :
-
Arabidopsis thaliana
- O. sativa :
-
Oryza sativa
- G. max :
-
Glycine max
- S. lycopersicum :
-
Solanum lycopersicum
- V. vinifera :
-
Vitis vinifera
- CPT:
-
Camptothecin
- pI:
-
Point isoelectric
- TFs:
-
Transcription factors
- MIAs:
-
Monoterpene indole alkaloid
- MEP:
-
2-C-methyl-d-erythritol 4-phosphate
- MVA:
-
Mevalonate
- G10H:
-
Geraniol 10-hydroxy
- 10-HGO:
-
10-hydroxy geraniol oxidoreductase
- IO:
-
Iridodial oxidoreductase
- 7-DLGT:
-
7-deoxyloganic acid by glucosyltransferase
- 7-DLH:
-
7-deoxyloganic acid hydroxylase
- SLS:
-
Secologanin synthase
- TDC:
-
Tryptophan decarboxylase
- STR:
-
Strictosidine synthase
- ASA:
-
Anthranilate synthase
- CPR:
-
Cytochrome P450 reductases
- DXR:
-
1-deoxy-D-xylulose-5-phosphate reductoisomerase
- DXS:
-
1-deoxy-D-xylulose-5-phosphate synthase
- HDR:
-
1-hydroxy-2-methyl-2(E)-butenyl-4-diphosphate reductase
- TSB:
-
The beta-subunit of tryptophan synthase
- QRT-PCR:
-
Quantitative real-time PCR
- 8-HGO:
-
8-hydroxy-geraniol oxidoreductase
- AACT:
-
Acetyl-CoA C-acetyltransferase
- CMK:
-
4-(cytidine 5-diphospho)-2-C-methylerythritolkinase
- CMS:
-
4-(cytidine 5-diphospho)-2-C-methylerythritol synthase
- GES:
-
Geraniol synthase
- GPPS:
-
Geranyl diphosphate synthase/geranyl pyrophosphate synthase
- HDS:
-
Hydroxymethylbutenyl 4-diphosphate synthase
- HMGR:
-
3-hydroxy-3-methylglutaryl-CoA reductase
- HMGS:
-
3-hydroxy-3-methylglutaryl-CoA synthase
- IPPI:
-
Isopentenyl diphosphate isomerase
- IS:
-
Iridoid synthase
- LAMT:
-
Loganic acid O-methyltransferase
- MDC:
-
Mevalonate 5-diphosphate decarboxylase/mevalonate (diphospho)-decarboxylase
- MECS:
-
2-C-methylerythritol-2,4-cyclodiphosphate synthase
- MK:
-
Mevalonate kinase
- PMK:
-
Phosphomevalonate kinase
- CSC:
-
Cell suspension cultures
- HR:
-
Hairy roots
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Funding
This work was supported by Zhejiang Provincial Ten Thousand Program for Leading Talents of Science and Technology Innovation [2018R52050]; National Natural Science Fund of China [82073963, 31571735, 81522049]; The Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province [2021C02074]; Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents; Zhejiang Chinese Medical University Research Foundation [2021JKZDZC06]; The Opening Project of Zhejiang Provincial Preponderant and Characteristic Subject of Key University (Traditional Chinese Pharmacology), Zhejiang Chinese Medical University [ZYAOX2018009].
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M.S. and Q.H. performed bioinformatics analysis, sample collection, experiments and wrote the manuscript. Y.W., C.W., R.Z., and S.Z. helped analyze the data. G.K. designed the experiments and conceived the project, provided overall supervision of the study and revised the manuscript. The authors read and approved the final manuscript.
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The Ophiorrhiza pumila materials used in the study were kept as sterile in the greenhouse, which are maintained in accordance with the institutional guidelines of School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, China. This article did not contain any studies with human participants or animals.
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Shi, M., Huang, Q., Wang, Y. et al. Genome-wide survey of the GATA gene family in camptothecin-producing plant Ophiorrhiza pumila. BMC Genomics 23, 256 (2022). https://doi.org/10.1186/s12864-022-08484-x
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DOI: https://doi.org/10.1186/s12864-022-08484-x