Transcriptome analysis of five different tissues of bitter gourd (Momordica charantia L.) fruit identifies full-length genes involved in seed oil biosynthesis.

The bitter gourd seed oil, rich in conjugated fatty acids, has therapeutic value to treat cancer, obesity, and aging. It also has an industrial application as a drying agent. Despite its significance, genomics studies are limited, and the genes for seed oil biosynthesis are not fully understood. In this study, we assembled the fruit transcriptome of bitter gourd using 254.5 million reads (Phred score > 30) from the green rind, white rind, pulp, immature seeds, and mature seeds. It consisted of 125,566 transcripts with N50 value 2,751 bp, mean length 960 bp, and 84% completeness. Transcript assembly was validated by RT-PCR and qRT-PCR analysis of a few selected transcripts. The transcripts were annotated against the NCBI non-redundant database using the BLASTX tool (E-value < 1E-05). In gene ontology terms, 99,443, 86,681, and 82,954 transcripts were classified under biological process, molecular function, and cellular component. From the fruit transcriptome, we identified 26, 3, and 10 full-length genes coding for all the enzymes required for synthesizing fatty acids, conjugated fatty acids, and triacylglycerol. The transcriptome, transcripts with tissue-specific expression patterns, and the full-length identified from this study will serve as an important genomics resource for this important medicinal plant.

Selection and validation of appropriate reference genes for real-time quantitative PCR analysis in Momordica charantia.

Real time quantitative reverse transcription PCR (RT-qPCR) has been attracting more attention for its high sensitivity in gene expression analysis. Given the widely use of RT-qPCR in normalization, it is playing a pivotal role for seeking suitable reference genes in different species. In current work, 12 candidate reference genes including Actin 2 (ACT2), Cyclophilin 2 (CYP2), Glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2), Elongation factor 1-α (EF1-α), Nuclear cap binding protein 20 (NCBP20), Serine/threonine-protein phosphatase PP2A (PP2A), Polypyrimidine tract-binding protein 1 (PTBP1), SAND family protein (SNAD), TIP41-like protein (TIP41), Tubulin beta-6 (TUB6), Ubiquitin-conjugating enzyme 9 (UBC9) and Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) were screened from the transcriptome datasets of M. charantia. Afterwards, GeNorm, NormFinder and BestKeeper algorithms were applied to assess the expression stability of these 12 genes under different abiotic stresses including drought, cold, high-salt, hormone, UV, oxidative and metal stress. The results indicated that 12 selected genes exhibited various stability across the samples under different external stress conditions, but TIP41, PTBP1 and PP2A presented high stability among all the reference genes. To validate the suitability of the identified reference genes, the results of hormone subset were compared with RNA sequencing (RNA-seq) data, and the relative abundance of Ascorbate peroxidase 1（APX1）was used to confirm the reliability of the results. This work assesses the stability of reference genes in M. charantia under different abiotic stress conditions, which will be beneficent for accurate normalization of target genes in M. charantia.

Real-Time Quantitative PCR Analysis of the Expression Pattern of the Hypoglycemic Polypeptide-P Gene in Momordica charantia.

This study was designed to establish a real-time quantitative polymerase chain reaction (qPCR) method to rapidly and reliably analyze the hypoglycemic polypeptide-P gene expression pattern in Momordica charantia (MC) and to examine its expression changes in different MC accessions, harvesting seasons, and tissue types. The qPCR results were further verified by using Western blotting (WB). A total of 10 MCs with different accessions were collected and tested in this study. Among the tested accessions, RU5H showed the highest expression level of the polypeptide-P gene. The expression level of the polypeptide-P gene was not only season-related (with the highest in early July) but also tissue-related (with the highest in the seed tissue). In addition, the expression characteristic of the polypeptide-P gene was maturity-related, with the highest expression level in the tender MC. The WB results show that the transcription level of this gene shows an almost similar trend to the corresponding protein expression level. In conclusion, the established qPCR method can rapidly and effectively detect the expression levels of the polypeptide-P gene in MCs with different accessions; furthermore, various factors, including the accessions, harvesting seasons, and tissue types can affect the expression level.

Two Paralogous Genes Encoding Auxin Efflux Carrier Differentially Expressed in Bitter Gourd (Momordica charantia).

The phytohormone auxin regulates various developmental programs in plants, including cell growth, cell division and cell differentiation. The auxin efflux carriers are essential for the auxin transport. To show an involvement of auxin transporters in the coordination of fruit development in bitter gourd, a juicy fruit, we isolated novel cDNAs (referred as McPIN) encoding putative auxin efflux carriers, including McPIN1, McPIN2 (allele of McPIN1) and McPIN3, from developing fruits of bitter gourd. Both McPIN1 and McPIN3 genes possess six exons and five introns. Hydropathy analysis revealed that both polypeptides have two hydrophobic regions with five transmembrane segments and a predominantly hydrophilic core. Phylogenetic analyses revealed that McPIN1 shared the highest homology to the group of Arabidopsis, cucumber and tomato PIN1, while McPIN3 belonged to another group, including Arabidopsis and tomato PIN3 as well as PIN4. This suggests different roles for McPIN1 and McPIN3 in auxin transport involved in the fruit development of bitter gourd. Maximum mRNA levels for both genes were detected in staminate and pistillate flowers. McPIN1 is expressed in a particular period of early fruit development but McPIN3 continues to be expressed until the last stage of fruit ripening. Moreover, these two genes are auxin-inducible and qualified as early auxin-response genes. Their expression patterns suggest that these two auxin transporter genes play a pivotal role in fruit setting and development.

Accumulation of Charantin and Expression of Triterpenoid Biosynthesis Genes in Bitter Melon (Momordica charantia).

Charantin, a natural cucurbitane type triterpenoid, has been reported to have beneficial pharmacological functions such as anticancer, antidiabetic, and antibacterial activities. However, accumulation of charantin in bitter melon has been little studied. Here, we performed a transcriptome analysis to identify genes involved in the triterpenoid biosynthesis pathway in bitter melon seedlings. A total of 88,703 transcripts with an average length of 898 bp were identified in bitter melon seedlings. On the basis of a functional annotation, we identified 15 candidate genes encoding enzymes related to triterpenoid biosynthesis and analyzed their expression in different organs of mature plants. Most genes were highly expressed in flowers and/or fruit from the ripening stages. An HPLC analysis confirmed that the accumulation of charantin was highest in fruits from the ripening stage, followed by male flowers. The accumulation patterns of charantin coincide with the expression pattern of McSE and McCAS1, indicating that these genes play important roles in charantin biosynthesis in bitter melon. We also investigated optimum light conditions for enhancing charantin biosynthesis in bitter melon and found that red light was the most effective wavelength.

Draft genome sequence of bitter gourd (Momordica charantia), a vegetable and medicinal plant in tropical and subtropical regions.

Bitter gourd (Momordica charantia) is an important vegetable and medicinal plant in tropical and subtropical regions globally. In this study, the draft genome sequence of a monoecious bitter gourd inbred line, OHB3-1, was analyzed. Through Illumina sequencing and de novo assembly, scaffolds of 285.5 Mb in length were generated, corresponding to ∼84% of the estimated genome size of bitter gourd (339 Mb). In this draft genome sequence, 45,859 protein-coding gene loci were identified, and transposable elements accounted for 15.3% of the whole genome. According to synteny mapping and phylogenetic analysis of conserved genes, bitter gourd was more related to watermelon (Citrullus lanatus) than to cucumber (Cucumis sativus) or melon (C. melo). Using RAD-seq analysis, 1507 marker loci were genotyped in an F2 progeny of two bitter gourd lines, resulting in an improved linkage map, comprising 11 linkage groups. By anchoring RAD tag markers, 255 scaffolds were assigned to the linkage map. Comparative analysis of genome sequences and predicted genes determined that putative trypsin-inhibitor and ribosome-inactivating genes were distinctive in the bitter gourd genome. These genes could characterize the bitter gourd as a medicinal plant.

Constitutive expression of McCHIT1-PAT enhances resistance to rice blast and herbicide, but does not affect grain yield in transgenic glutinous rice.

To produce new rice blast- and herbicide-resistant transgenic rice lines, the McCHIT1 gene encoding the class I chitinase from Momordica charantia and the herbicide resistance gene PAT were introduced into Lailong (Oryza sativa L. ssp. Japonica), a glutinous local rice variety from Guizhou Province, People's Republic of China. Transgenic lines were identified by ß-glucuronidase (GUS) histochemical staining, PCR, and Southern blot analyses. Agronomic traits, resistance to rice blast and herbicide, chitinase activities, and transcript levels of McCHIT1 were assessed in the T2 progeny of three transgenic lines (L1, L8, and L10). The results showed that the introduction of McCHIT1-PAT into Lailong significantly enhanced herbicide and blast resistance. After infection with the blast fungus Magnaporthe oryzae, all of the T2 progeny exhibited less severe lesion symptoms than those of wild type. The disease indices were 100% for wild type, 65.66% for T2 transgenic line L1, 59.69% for T2 transgenic line L8, and 79.80% for T2 transgenic line L10. Transgenic lines expressing McCHIT1-PAT did not show a significant difference from wild type in terms of malondialdehyde (MDA) content, polyphenol oxidase (PPO) activity, and superoxide dismutase (SOD) activity in the leaves. However, after inoculation with M. oryzae, transgenic plants showed significantly higher SOD and PPO activities and lower MDA contents in leaves, compared with those in wild-type leaves. The transgenic and the wild-type plants did not show significant differences in grain yield parameters including plant height, panicles per plant, seeds per panicle, and 1000-grain weight. Therefore, the transgenic plants showed increased herbicide and blast resistance, with no yield penalty.

Purification and characterisation of an antifungal protein, MCha-Pr, from the intercellular fluid of bitter gourd (Momordica charantia) leaves.

An antifungal protein, designated MCha-Pr, was isolated from the intercellular fluid of bitter gourd (Momordica charantia) leaves during a screen for potent antimicrobial proteins from plants. The isolation procedure involved a combination of extraction, ammonium sulphate precipitation, gel filtration on Bio-Gel P-6, ion exchange chromatography on CM-Sephadex, an additional gel filtration on HiLoad 16/60 Superdex 30, and finally, HPLC on a SOURCE 5RPC column. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that the protein had a molecular mass of 25733.46Da. Automated Edman degradation was used to determine the N-terminal sequence of MCha-Pr, and the amino acid sequence was identified as V-E-Y-T-I-T-G-N-A-G-N-T-P-G-G. The MCha-Pr protein has some similarity to the pathogenesis-related proteins from Atropa belladonna (deadly nightshade), Solanum tuberosum (potato), Ricinus communis (castor bean), and Nicotiana tabacum (tobacco). Analysis of the circular dichroism spectra indicated that MCha-Pr predominantly contains α-helix and ß-sheet structures. MCha-Pr had inhibitory effects towards a variety of fungal species and the 50% inhibition of fungal growth (IC50) for Alternaria brassicae, Cercospora personata, Fusarium oxysporum, Mucor sp., and Rhizoctonia solani are 33 µM, 42 µM, 37 µM, 40 µM, and 48 µM, respectively. In addition, this antifungal protein can inhibit the germination of A. brassicae spores at 12.5 µM. These results suggest that MCha-Pr in bitter gourd leaves plays a protective role against phytopathogens and has a wide antimicrobial spectrum.

Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

BACKGROUND: Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. RESULTS: Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. CONCLUSION: Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P.

Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia.

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel-nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo's fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC(50) values of 6.23 and 4.15 µM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC(50) value of 0.59 µM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

RNase MC2: a new Momordica charantia ribonuclease that induces apoptosis in breast cancer cells associated with activation of MAPKs and induction of caspase pathways.

Ribonucleases (RNases) are ubiquitously distributed nucleases that cleave RNA into smaller pieces. They are promising drugs for different cancers based on their concrete antitumor activities in vitro and in vivo. Here we report for the first time purification and characterization of a 14-kDa RNase, designated as RNase MC2, in the seeds of bitter gourd (Momordica charantia). RNase MC2 manifested potent RNA-cleavage activity toward baker's yeast tRNA, tumor cell rRNA, and an absolute specificity for uridine. RNase MC2 demonstrated both cytostatic and cytotoxic activities against MCF-7 breast cancer cells. Treatment of MCF-7 cells with RNase MC2 caused nuclear damage (karyorrhexis, chromatin condensation, and DNA fragmentation), ultimately resulting in early/late apoptosis. Further molecular studies unveiled that RNase MC2 induced differential activation of MAPKs (p38, JNK and ERK) and Akt. On the other hand, RNase MC2 exposure activated caspase-8, caspase-9, caspase-7, increased the production of Bak and cleaved PARP, which in turn contributed to the apoptotic response. In conclusion, RNase MC2 is a potential agent which can be exploited in the worldwide fight against breast cancer.

Gene cloning and expression of a novel hypoglycaemic peptide from Momordica charantia.

BACKGROUND: Momordica charantia (MC) is used in many Asian countries as a traditional functional food and medicine. Polypeptide-P, a 166 amino acid (AA) polypeptide isolated from MC seeds, has been reported to show hypoglycaemic effects in patients with type I or type II diabetes. The AA sequence of this peptide has been determined, but its gene sequence has yet to be published. RESULTS: In this study a gene-cloning strategy was employed to obtain the polypeptide-P gene sequence using degenerate reverse transcription polymer chain reaction and genome-walking methods. A complete 498 bp sequence encoding the polypeptide-P protein was cloned from MC seeds. Subsequent assays of the bioactivity of the expressed recombinant protein revealed that it had significant hypoglycaemic activity in alloxan-induced diabetic mice. This result suggests that recombinant polypeptide-P has hypoglycaemic effects. CONCLUSION: This is the first report of cloning and expression of the MC polypeptide-P gene. The cloned gene could be helpful for exploring the mechanisms of polypeptide-P gene expression and regulation in MC. Furthermore, this gene could be used as a potential tool both for screening MC varieties with high hypoglycaemically active substance content and for breeding new varieties of MC with high economic value, which could in turn be beneficial to farmers.

Mining the bitter melon (momordica charantia l.) seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes.

BACKGROUND: Seeds of Momordica charantia (bitter melon) produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. RESULTS: Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. CONCLUSIONS: 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in developing bitter melon seeds. The transcriptomic data presented provide a resource for the study of novel fatty acid metabolism and for the biotechnological production of conjugated fatty acids and possibly other novel fatty acids in established oilseed crops.

The unfolding of alpha-momorcharin proceeds through the compact folded intermediate.

The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2-3 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state.

Expression of a ribosome-inactivating protein gene in bitter melon is induced by Sphaerotheca fuliginea and abiotic stimuli.

The gene encoding a single-chain, ribosome-inactivating protein (SCRIP) was cloned from bitter melon (Momordica charantia L.) leaves infected with the fungus, Sphaerotheca fuliginea, by RT-PCR. The ORF was 861 bp. The ribosome-inactivating protein was expressed in E. coli and, when purified, it inhibited the growth of Fusarium solani [corrected] Northern blot analysis revealed that RIP transcripts rapidly accumulated in leaves 1-day post inoculation with Sphaerotheca fuliginea and reached a peak at 3 d. The expression pattern of RIP induced by methyl jasmonate and salicylic acid were different from that of pathogen-induced expression. Mechanical wounding, silver nitrate and osmotic stress stimulated only a slight accumulation of RIP transcripts. Abscisic acid also induced transcription of RIPs. The signal compounds, ethylene and okadaic acid, induced a moderate accumulation of RIP transcripts.

Antioxidant properties of Momordica Charantia (bitter gourd) seeds on Streptozotocin induced diabetic rats.

The aim of the present study is to investigate the antioxidant activities of the aqueous extract of seeds of two varieties, namely a country and hybrid variety of Momordica charantia (MCSEt1 and MCSEt2) respectively in streptozotocin induced diabetic rats. Oral administration of both the seed extracts at a concentration of 150 mg/kg b.w for 30 days showed a significant decrease in fasting blood glucose, hepatic and renal thiobarbituric acid reactive substances and hydroperoxides. The treatment also resulted in a significant increase in reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase in the liver and kidney of diabetic rats. The results clearly suggest that seeds of Momordica charantia treated group may effectively normalize the impaired antioxidant status in streptozotocin induced-diabetes than the glibenclamide treated groups. The extract exerted rapid protective effects against lipid peroxidation by scavenging of free radicals there by reducing the risk of diabetic complications. The effect was more pronounced in MCSEt1 compared to MCSEt2.

[Cloning and characterization of a homologous gene of plant class V chitinase from balsampear, Momordica charantia Linn].

Balsampear (M. charantia Linn.) is a vegetable crop, highly resistant to pathogens. Chitinases were proposed to play an important role in the defense response of this crop. Based on the N-terminal sequence of a purified balsampear chitinase, a fragment (ChitB), similar to the tobacco class V chitinase gene, was amplified from the leaf RNA using 3'RACE, and the corresponding 5' sequence was further amplified by the Y-RACE method. By joining the two amplified fragments, the full-length cDNA of M. charatica homologous gene of plant class V chitinase (McChi5) was obtained. The 1348 bp cDNA contained an ORF of 1044 bp, which coded for a polypeptide of 347 amino acids. The deduced polypeptide had a predicted molecular weight of 38.3 kD and a pI of 5.77. Homology analysis demonstrated that, McChi5 protein, which contained a conserved domain of family 18 glycosyl hydrolyse, had the sequence similar to tobacco class V chitinases, several putative chitinases and chitinase-like proteins of Arabidopsis thiliania, and some chitinases from mammals, insects and bacteria. Southern blotting suggested that two copies of McChi5 gene and several homologous genes existed in the M. charatica genome. By RNA dot blotting analysis, expression of the McChi5 gene was detected in cotyledons, roots, stems, and leaves, and it was not induced by wounding treatment. The biological functions and the potential applications of Mochi5 gene were discussed.