De novo sequencing, assembly and characterisation of Aloe vera transcriptome and analysis of expression profiles of genes related to saponin and anthraquinone metabolism

- characterisation of Aloe vera transcriptome and analysis of expression profiles of genes related to saponin and anthraquinone.
- Background: Aloe vera is a perennial, succulent, drought-resistant plant that exhibits many pharmacological characteristics such as wound healing ability against skin burns, anti-ulcer, anti-inflammatory, anti-tumor, anti-viral, anti- hypercholesterolemic, anti-hyperglycemic, anti-asthmatic and much more.
- Despite great medicinal worth, little genomic information is available on Aloe vera .
- This study is an initiative to explore the full-scale functional genomics of Aloe vera by generating whole transcriptome sequence database, using Illumina HiSeq technology and its progressive annotation specifically with respect to the metabolic specificity of the plant..
- Results: Transcriptome sequencing of root and leaf tissue of Aloe vera was performed using Illumina paired-end sequencing technology.
- Conclusions: This is the first transcriptome database of Aloe vera and can be potentially utilized to characterize the genes involved in the biosynthesis of important secondary metabolites, metabolic regulation, signal transduction mechanism, understanding function of a particular gene in the biology and physiology of plant of this species as well as other species of Aloe genus..
- Keywords: Aloe vera , Next generation sequencing, Transcriptome, De novo assembly, Secondary metabolism, Differential gene expression.
- Among all the species, Aloe vera Linne Synonym Aloe barbadensis Miller is considered to be medicinally most potent, therefore, it is most popular [2, 3].
- Aloe vera shows innumerable medicinal attributes such as skin burn healing [4], anti-inflammatory [5], hepatoprotective [6], anti-tumor [7], anti-ulcer [8], antihypercholesterolemic [9], anti-hyperglycemic [10], anti-asthmatic[11] and antioxidant activities [12].
- Aloe vera extracts have been demonstrated to be advanta- geous in the treatment of even AIDS [13].
- Aloe vera contains numerous active ingredients including anthraquinones, polysaccharides, alkylben- zenes, dehydrabietic acid derivatives, salicylic acid, lectin, carotenoids, lignin, saponins etc.
- Many of the medicinal prop- erties of Aloe vera are ascribed to secondary metabolites though they are relatively minor in their concentration in the plant (<.
- Aloe vera exhibits antibacterial property because of anthraquinones that behave like tetracycline by blocking the ribosomal A site, thus, interrupting bacterial protein synthesis [17]..
- Saponins present in Aloe vera also act as an effective anti-microbial agent against various bacteria, viruses, fungi, and yeasts [21].
- Aloe vera extract act as an anti- oxidant due to carotenoids present in it [22].
- Lignins present in Aloe vera penetrate deep into the skin and help in introducing other medicinal ingredients to pene- trate into the skin.
- Therapeutic effects of Aloe vera have not been correlated well with individual metabolite and it remains unknown whether the biological activities of the plant are due to a single component or the collab- orative efforts of many components [23]..
- An increasing trend where consumers tend towards a healthy lifestyle, coupled with the increased use of Aloe vera extracts as an ingredient in food, pharmaceutical and cosmetics products, increases its market growth across the globe.
- International Aloe Science Council (IASC) estimated that the global consumption of Aloe vera will surpass 60,720.4 tonnes in 2016, accounting for revenues worth US$ 1.6 billion [24].
- Presently, a large industrial sector is attempting to exploit the great bio- logical potential of Aloe vera.
- The present study used NGS technology for whole transcriptome sequencing of Aloe vera root and leaf tissues, performed using Illumina Hi-Seq technology..
- It provides valuable sequence information from Aloe vera root and leaf tissues with special emphasis on genes re- lated to secondary metabolic pathways.
- The transcriptome sequencing and analysis provides a strong platform of genomic sequences, to serve biosynthetic pathway and metabolic engineering programs of basic as well as applied research on Aloe vera and catalyze path of its change of use from extracts to molecules..
- The figure summarizes the steps undertaken and tools used during Aloe vera transcriptome sequencing.
- In Aloe vera transcriptome KEGG analysis, 6114 CDS for root and 6527 CDS for leaf were found enriched in 24 different KEGG pathway categories.
- biosynthesis have been identified from Aloe vera root and leaf tissue by KEGG Pathway functional annotation.
- Sequencing of Aloe vera transcriptome reveals many putative genes (number of different transcripts obtained is given in bracket with each gene) related to saponin biosynthesis pathway including acetyl-CoA acetyltrans- ferase (ACT: root 3.
- Anthraquinones are another important secondary metabolites present in Aloe vera.
- De novo sequencing of Aloe vera in this study has provided many unigenes encoding octaketide/polyke- tide synthase (8 from root and 12 from leaf.
- L-phenylalanine ammonia-lyase (PAL), caffeoyl CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), 4-coumarate:coenzyme A(CoA) ligase (4CL), cinnamoyl-CoA reductase (CCR), hydroxycinnamoyl transferase (HCT), cinnamate-4-hydroxylase (C4H), 4-coumarate 3-hydroxylase (C3H), ferulate 5-hydroxylase (F5H), cinnamyl alcohol de- hydrogenase (CAD) were identified from the Aloe vera data- base by KEGG pathway mapping and the results were then further validated by differential gene expression and real-time expression analysis..
- root involved in lignin, saponin and anthraquinone biosynthesis pathway in Aloe vera.
- Genes encoding lignin biosynthesis pathway were also expressed at higher level in Aloe vera root as compared to leaf tissue as presented in DGE data (Fig.
- Aloe vera is one of the most popular medicinal plant used worldwide nevertheless, hardly any work has been conducted on its functional genomics.
- There- fore, results of this investigation on whole transcriptome sequencing of Aloe vera root as well as leaf tissues are important in the perspective of functional genomics of the plant..
- A number of high quality reads, fine transcripts and CDS length obtained from Aloe vera next generation transcriptome sequencing indicates the functionality of Aloe vera sequencing at first sight.
- shoot for the selected 16 unigenes involved in lignin, saponin and anthraquinone biosynthesis pathway in Aloe vera which represents the higher expression of these unigenes in root as compared to shoot.
- into the significant blast hits for root and leaf samples respectively, which represented a big coverage of Aloe vera genome.
- The majority of hits were found to be against Elaeis guineensis followed by Phoenix dactylifera for both the samples, showing closer relationship with Aloe vera genotypically..
- Transcription factors identified in Aloe vera database may play a crucial role in modifying.
- the levels of secondary metabolites, to stimulate the pro- duction of valuable secondary metabolites and to reduce the level of an undesirable metabolite that have an ad- verse effect on the quality of Aloe vera..
- It was found that 194 genes in root and 192 genes in leaf were related to environmental adaptation which proves well adapted feature of Aloe vera in diverse conditions..
- The major secondary metabolite related constituents of Aloe vera include anthraquinones, saponin, lignin, ste- rols, polysaccharides, alkylbenzenes, dehydrabietic acid derivatives, lactin and salicylic acid [52, 53] which attri- bute for its pharmacological activity.
- Biosynthetic path- way of secondary metabolites in Aloe vera remains undiscovered till date.
- Several putative genes related to saponin, lignin, anthraquinone, carotenoid and phenyl- propanoid biosynthesis have been identified from Aloe vera root and leaf tissue by KEGG Pathway functional annotation.
- steroid are preferentially formed via MVA pathway, whereas, monoterpenes, diterpenes, and carotenoid are formed predominantly via the MEP pathway [55] There is no information as of today that which of the pathways (MVA or MEP) or both are involved in the biosynthesis of saponins precursors and subsequently the number of genes that are involved in the final biosynthesis of sapo- nins in planta / Aloe vera.
- Ferulate 5-hydroxylase EC: 1.14.
- HMG-CoA synthase EC:2.3.3.10 CDS_12664_Unigene .
- Phosphomevalonate kinase EC:2.7.4.2 CDS_24841_Unigene .
- Isopentenyl-PP isomerase EC:5.3.3.2 CDS_24864_Unigene .
- Squalene synthase EC:2.5.1.21 CDS_3000_Unigene .
- Cycloartenol synthase EC:5.4.99.8 CDS_16290_Unigene .
- DOX Phosphate synthase EC:2.2.1.7 CDS_29944_Unigene .
- Octaketide synthase EC:2.3.1.- CDS_8049_Unigene E-76.
- UDP glycosyltransferase EC:2.4.1.- CDS_5315_Unigene E-62.
- HMG-CoA synthase EC:2.3.3.10 CDS_14177_Unigene .
- Phosphomevalonate kinase EC:2.7.4.2 CDS_25395_Unigene .
- Squalene synthase EC:2.5.1.21 CDS_463_Unigene .
- Cycloartenol synthase EC:5.4.99.8 CDS_11153_Unigene .
- DOXP(1-deoxy-D-xylulose-5-phosphate)synthase EC:2.2.1.7 CDS_22770_Unigene DOXP(1-deoxy-D-xylulose-5-phosphate).
- EC:1.1.1.267 CDS_20571_Unigene .
- From Aloe vera transcriptome sequencing data no unigene encoding β-amyrin synthase was found, it denotes the lack of triterpenoid saponin in our research plant.
- However downward steps towards sap- onin biosynthesis are still unknown for Aloe vera..
- Anthraquinones, another important group of second- ary metabolites in Aloe vera are tricyclic aromatic qui- nines having strong antibacterial, antiviral, antifungal activity.
- Aloin, an anthraquinone glycoside is one of the most active metabolite of this group present in Aloe vera [67].
- To date, very little is known about the biosynthetic steps leading to the formation of aloin/anthraquinones in Aloe vera.
- OKS cata- lyzes the iterative condensation of eight molecules of malonyl-CoA, might be involved in the biosynthesis of the octaketide anthrone aloin in Aloe vera.
- Various genes like octaketide/polyke- tide synthase, aldoketoreductase, and UDP-glycosyltransferase have been identified from transcriptome sequencing which can be useful for unravelling the aloin biosynthesis in Aloe vera..
- L-phenylalanine ammonia-lyase (PAL), caffeoyl CoA O-methyltransferase (CCoAOMT), caffeic acid O-methyltransferase (COMT), 4-coumarate:coenzyme A(CoA) ligase (4CL), cinnamoyl-CoA reductase (CCR), hydroxycinnamoyl transferase (HCT), cinnamate-4-hydroxylase (C4H), 4-coumarate 3-hydroxylase (C3H), ferulate 5-hydroxylase (F5H), cinnamyl alcohol de- hydrogenase (CAD), meticulously investigated for their roles in plant development,[49] have been identified from the Aloe vera sequencing database which may be helpful to reveal the steps toward the lignin biosynthesis in Aloe vera..
- Aloe vera has been reported to have anti-aging effect similar to vitamin A derivatives.
- Various genes belonging to carotenoid biosynthesis in- cluding phytoene synthase, 15-cis-phytoene desaturase, zeta-carotene isomerase, prolycopene isomerase, lycopene beta-cyclase, lycopene epsilon-cyclase, zeaxanthin epoxi- dase, violaxanthin de-epoxidaseand abscisic-aldehyde oxi- dase have been isolated from sequencing, may pave the way of carotenoid biosynthesis in Aloe vera.
- In short, the unigenes related to different secondary metabolites bio- synthesis, identified from transcriptome sequencing may unravel the secondary metabolism which is most respon- sible for potent applications of Aloe vera and still undiscovered..
- UDP glycosyl- transferase (UGT) and octaketide synthase genes (OKS) were highly expressed in both root and leaf tissue stipu- late that aloin is synthesized in both root and leaf tissue in Aloe vera..
- Putative genes encoding lignin biosynthesis pathway were also substantially better expressed in Aloe vera root as compared to leaf tissue as presented in DGE data indicating its reliability..
- Aloin biosynthesis pathway related genes were also upreg- ulated and showed maximum expression after 24 h of me- thyl jasmonate induction, indicating the correlation of both aloin and saponin with defense mechanism of Aloe vera.
- De novo assembly of transcriptome data in conjugation with DGE analysis served as a powerful approach for the identification of genes involved in the biosynthesis of important secondary metabolites pertaining to different chemical classes in Aloe vera - a non-model plant.
- Aloe vera is well known plant used worldwide for its medicinal and cosmetic properties due to its specialized metabolic competence.
- Therefore, in the present study, tran- scriptome sequence data for Aloe vera root and shoot was generated using NGS technology.
- The potential genes isolated can be exploited for characterization of metabolic understand- ing and modulation of saponin, aloin, carotenoid and lignin biosynthesis in Aloe vera.
- To our knowledge, this would be the first tran- scriptome sequencing study of Aloe vera until now..
- Aloe vera was grown in the herbal nursery at Guru Jambheshwar University of Science and Technology, Hisar, India.
- Aloe vera was grown in the herbal nursery at Guru Jambheshwar University of Science and Technology, Hisar, India under natural conditions according to institutional and national guidelines..
- Characterization of Aloeride, a new high-molecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity.
- Aloe vera gel and thyroid hormone cream may improve wound healing in Wistar rats.
- Anti-inflammatory effects of aloe vera gel in human colorectal mucosa in vitro.
- Probiotic lactobacillus rhamnosus GG and Aloe vera gel improve lipid profiles in hypercholesterolemic rats.
- Anti-hyperglycemic and anti-hypercholesterolemic effects of Aloe vera leaf gel in hyperlipidemic type 2 diabetic patients: a randomized double-blind placebo-controlled clinical trial.
- Medicinal and cosmetological importance of Aloe vera.
- In vitro antioxidant activities of Aloe vera leaf skin extracts.
- Components of the gel of Aloe vera (L.) Bunn.
- Aloe vera gel: Update for dentistry.
- Evaluation of biological properties and clinical effectiveness of Aloe vera: a systematic review.
- Aloe Vera myth or medicine.
- Composition and applications of Aloe vera leaf gel.
- Screening methods used to determine the anti-microbial properties of Aloe vera inner gel.
- Global demand of Aloe vera extracts to reach 60,720 tonnes in 2016;.
- http://www.futuremarketinsights.com/press-release/aloe-vera- extracts-market.
- https://www.ncbi.nlm.nih.gov/gquery/?term=Aloe +vera.
- Aloe vera: a valuable ingredient for the food, pharmaceutical and cosmetic industries — a review.
- Aloe vera: chemical composition and methods used to determine its presence in commercial products.
- Combined approach using RAPD, ISSR and bioactive compound for the assessment of genetic diversity in Aloe vera (L.) Burm

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