scholarly journals Multi-tissue transcriptome analysis using hybrid-seq revealed potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachtin indica)

2020 ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Analysis Data ◽  
Sequencing Data ◽  
Neem Tree ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The manufacture of azadirachtin A depends on extraction from neem tissues, which is not ecofriendly and sustainable. The low yield and discontinuous supply impeded the further application. The biosynthetic pathway of azadirachtin A is still well-known.Results: We attempted to explore azadirachtin A biosynthetic pathway and identified key involved genes by analyzing transcriptome data of five neem tissues through hybrid-seq (Illumina HiSeq and Pacific Biosciences Single Molecule Real Time (PacBio SMRT)) technology. A total 219 and 397 up-regulated differentially expressed genes (DEGs) in leaf and fruit tissues than other tissues (root, stem and flower) were isolated. After phylogenetic analysis and domain prediction, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase (ADH), cytochrome P450 (CYP450), acyltransferase (ACT) and esterase (EST) proposed to be involved in azadirachtin A biosynthesis were finally selected. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis.Conclusions: By integrating and analysis data from Illumina HiSeq and PacBio SMRT platform, 22 DEGs were finally selected as candidates involved in azadirachtin A biosynthesis. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on the understanding of other triterpenoids biosynthesis in neem trees and provide reference for exploring other valuable natural product biosynthesis in plants.

scholarly journals Multi-tissue transcriptome analysis using hybrid-seq revealed potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachtin indica)

2020 ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
Transcriptome Data ◽  
Quantitative Real Time Pcr ◽  
Sequencing Data ◽  
Neem Tree ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The manufacture of azadirachtin A depends on extraction from neem tissues, which is not ecofriendly and sustainable. The low yield and discontinuous supply impeded the further application. The biosynthetic pathway of azadirachtin A is still unknown. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified key involved genes by analyzing transcriptome data of five neem tissues through hybrid-seq (Illumina HiSeq and Pacific Biosciences Single Molecule Real Time (PacBio SMRT)) approach. Candidates were firstly screened by comparing expression level within five tissues. After phylogenetic analysis, domain prediction and molecular docking, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase (ADH), cytochrome P450 (CYP450), acyltransferase (ACT) and esterase (EST) were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homolog of MaOCS1 and MaCYP71CD2 were identified. An unigene encoding complete homolog of MaCYP71BQ5 was first reported. Accuracy of the assemblies were verified by Quantitative Real-Time PCR (qRT-PCR) and full-length cloning PCR. Conclusions: By integrating and analysis transcriptome data from hybrid-seq technology, 22 DEGs were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on the understanding of other triterpenoids biosynthesis in neem trees and provide reference for exploring other valuable natural product biosynthesis in plants.

scholarly journals Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (azadirachta indica)

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
Docking Studies ◽  
Transcriptome Data ◽  
Sequencing Data ◽  
Pcr Cloning ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

scholarly journals Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica)

2020 ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
Docking Studies ◽  
Transcriptome Data ◽  
Sequencing Data ◽  
Pcr Cloning ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

scholarly journals Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica)

2020 ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
Docking Studies ◽  
Transcriptome Data ◽  
Sequencing Data ◽  
Pcr Cloning ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

scholarly journals Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (Azadirachta indica) 

2020 ◽  
Author(s):  
Huiyan Wang ◽  
Ning Wang ◽  
Yixin Huo
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Biosynthetic Pathway ◽  
Docking Studies ◽  
Transcriptome Data ◽  
Sequencing Data ◽  
Pcr Cloning ◽  
Illumina Hiseq ◽  
Oxidosqualene Cyclase ◽  
Azadirachtin A

Abstract Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.

AStrap: identification of alternative splicing from transcript sequences without a reference genome

2018 ◽  
Vol 35 (15) ◽  
pp. 2654-2656 ◽  
Author(s):  
Guoli Ji ◽  
Wenbin Ye ◽  
Yaru Su ◽  
Moliang Chen ◽  
Guangzao Huang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Alternative Splicing ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Supplementary Information ◽  
Model Organisms ◽  
Sequencing Data ◽  
Extensive Evaluation ◽  
Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Chloroplast Genome Sequence ofArabidopsis thalianaAccession Landsbergerecta, Assembled from Single-Molecule, Real-Time Sequencing Data

2016 ◽  
Vol 4 (5) ◽  
Author(s):  
Kai Bernd Stadermann ◽  
Daniela Holtgräwe ◽  
Bernd Weisshaar
Keyword(s):  
Arabidopsis Thaliana ◽  
High Resolution ◽  
Chloroplast Genome ◽  
Real Time ◽  
Genome Sequence ◽  
Single Molecule ◽  
Sequencing Data ◽  
Data Set ◽  
Chloroplast Genomes ◽  
Chloroplast Genome Sequence

A publicly available data set from Pacific Biosciences was used to create an assembly of the chloroplast genome sequence of theArabidopsis thalianagenotype Landsbergerecta. The assembly is solely based on single-molecule, real-time sequencing data and hence provides high resolution of the two inverted repeat regions typically contained in chloroplast genomes.

scholarly journals Purification and properties of an enzyme duet, phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase, involved in the biosynthesis of purine nucleotides de novo

1973 ◽  
Vol 135 (3) ◽  
pp. 543-545 ◽  
Author(s):  
Carole Anne Hilton Patey ◽  
Gordon Shaw
Keyword(s):  
Single Molecule ◽  
Biosynthetic Pathway ◽  
De Novo ◽  
Dual Function ◽  
Purine Nucleotides ◽  
Adjacent Pair ◽  
Purification And Properties

Purification and some properties of an adjacent pair of enzymes in the biosynthetic pathway to AMP, phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6), are described. The enzymes form a duet, which may be regarded as a single molecule with a dual function, or as two very similar substances.

scholarly journals Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China

2016 ◽  
Author(s):  
Ying Wang ◽  
Kun Liu ◽  
De Bi ◽  
Biao Shou Zhou ◽  
Wen Jian Shao
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
Sequence Similarity ◽  
Molecular Study ◽  
Sequence Information ◽  
Sequencing Data ◽  
Protein Database ◽  
Illumina Hiseq ◽  
Significant Similarity ◽  
Assembly Technology

Background. Resurrection plants constitute a unique cadre within angiosperms. Boea clarkeana Hemsl. (Boea, Gesneriaceae) is a desiccation-tolerant dicotyledonous herb that is endemic to China. Although research on angiosperms with DT could be instructive for crops, genomic resources for B. clarkeana remain scarce. In addition, transcriptome sequencing could be an effective way to study desiccation-tolerant plants. Methods. In the present study, we used the platform Illumina HiSeqTM 2000 and de novo assembly technology to obtain leaf transcriptomes of B. clarkeana and conducted a BLASTX alignment of the sequencing data and protein databases for sequence classification and annotation. Then, based on the sequence information obtained, we developed EST-SSR markers by means of EST-SSR mining, primer design and polymorphism identification. Results. A total of 91,449 unigenes were generated from the leaf cDNA library of B. clarkeana in this study. Based on a sequence similarity search with a known protein database, 72,087 unigenes were annotated. Among the annotated unigenes, a total of 71,170 unigenes showed significant similarity to known proteins of 463 popular model species in the Nr database, and 59,962 unigenes and 32,336 unigenes were assigned to GO classifications and COG, respectively. In addition, 44,924 unigenes were mapped in 128 KEGG pathways. Furthermore, a total of 7,610 unigenes with 8,563 microsatellites were found. Seventy-four primer pairs were selected from 436 primer pairs designed for polymorphism validation. SSRs with higher polymorphism rates were concentrated on dinucleotides, pentanucleotides and hexanucleotides. Finally, 17 pairs with highly polymorphic and stable loci were selected for polymorphism screening. There were a total of 65 alleles, with 2–6 alleles at each locus. Mainly due to the unique biological characteristics of plants, the HE, HO and PIC per locus were very low, ranging from 0 to 0.196, 0.082 to 0.14 and 0 to 0.155, respectively. Discussion. A substantial fraction transcriptome sequences of B. clarkeana were generated in this study, which is the first molecular-level analysis of this plant. These sequences are valuable resources for gene annotation and discovery and molecular marker development. These sequences could also provide a valuable basis for the future molecular study of B. clarkeana.

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