- Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. - rebaudiana and play important roles in the synthesis of steviol glycosides.. - With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCN A) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results.. - rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - 1 Agronomy College, Sichuan Agricultural University, Chengdu 611130, China Full list of author information is available at the end of the article Zhang et al. - Stevia rebaudiana (Bertoni) belongs to the Asteraceae family and is also one of the only two members (S.. - Consequently, the value of the SG mar- ket is expected to exceed $1 billion USD by 2021 [3]. - Owing to the interest in the properties of SGs, there has been extensive transcriptome research in- vestigating stevia. - An early report used expressed se- quence tags (ESTs) to identify candidate UGTs involved in the glucosylation of SGs and successfully collected more than 5500 fully annotated ESTs from S. - Subse- quent RNA-Seq analysis (Illumina platform) of the stevia leaves in two different growing stages yielded twenty-three upregulated SrUGTs, but none of them had desired activity and the name &. - Because of technical limitations, the reported average lengths of the isotigs from the Illumina platform are <. - 500 bp and generally need assembly to obtain full-length transcripts, resulting in redundancy and distortion of the data [15]. - In the current study, we combined NGS and SMRT sequencing approaches to sequence six stevia varieties with different accumulation levels of SGs and then generated a full-length transcriptome of S. - Moreover, a composite phylogenetic tree containing all SrUGTs was also constructed and the can- didate SrUGTs involved in the glycosylation of steviol glucosides were analysized.. - The contents of steviol glucosides in the leaves of all detected samples are shown in Table 2, and the HPLC chromatograms are also shown in Fig. - The results demonstrated that all of the analyt- ical glucosides in the experimental genotypes were clearly varied and provided a potential basis for WGCNA co-expression network analysis to uncover the glucosyltransferases involved in the biosynthesis pathway of the corresponding glycosides. - Furthermore, data ob- tained separately from the leaves of the seedling, adult, and budding stages of the ‘023’ genotype also revealed that the accumulation of steviol glycosides in S. - rebaudi- ana peaked in the budding period.. - rebaudiana and identify the potential genes involved in the biosynthesis pathway of steviol glycosides, both NGS (ILLUMINA) and SMRT (PACBIO) sequencing plat- forms were combined to sequence six different stevia ge- notypes. - Then, high-quality full-length cDNAs from the pooled RNA sample of the ‘023’ genotype were se- quenced on the PacBio Sequel platform, and a total of subreads (approximately 29.1 billion bases) were obtained. - Secondly, mapping of the clean reads to the corrected contigs and resulting a high alignment rate of 85.42%.. - further- more, the repeat numbers of the mononucleotides, dinu- cleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides were and 130, respectively.. - In addition, approximately 69.4% of the assembled contigs from NGS reads were <. - 1000 bp, whereas only 13.4% of the contigs from PAC- BIO reads were <. - Phylogenetic analysis of the UDP-glycosyltransferase multigene family. - In Arabidopsis thaliana, a molecular phylogenetic tree was constructed consisting of ninety-nine UGT sequences and a compos- ite phylogenetic tree that also includes all of the add- itional plant UGTs with known catalytic activities [22].. - This work has significantly promoted the prediction of the evolutionary history, substrate specificities and structure-function relationships of UGTs in Arabidopsis.. - This ana- lysis provided stronger statistical confidence (bootstrap from 64 to 88%) to two of the ancestral genes, corre- sponding to groups M and N, which are likely to share a more recent common origin. - Interestingly, a similar AtUGT78D1 gene has been found in Arabidopsis [22], indicating that the UGT78D genes in the glycosyltrans- ferase family may have evolved more rapidly.. - In an attempt to predict the structure-function re- latedness of the SrUGT family, numerous UGTs identi- fied from a wide range of plant species and having different biochemical functions were aligned with the ninety-eight SrUGTs and constructed a composite phylogenetic tree. - Among these additional plant UGTs, seven of the corresponding UGTs were successfully clus- tered within the fourteen groups identified by this study (Fig. - Interestingly, PdUGT94AF1 and PdUGT94AF2 derived from Prunus dulcis involved in the formation 1, 6-β-D-glucosidic linkage of Prunasin [24] were clustered in group A but had a long genetic distance between them and SrUGTs of this group, implying that the SrUGTs in this group may have no related specificity.. - 1 HPLC-UV profiles of the analytical samples and standards (steviolbioside, Reb B, ST, Reb F, Reb A, Reb D and Reb M). - a HPLC-UV profiles of the seven standards. - b HPLC-UV profiles of the analytical samples. - forming 1,6-glucosidic bonds, this tree cannot predict the glycosyltransferases involved in the synthesis of ste- viol glycosides containing 1,6-glucosidic bonds (such as Reb L). - therefore, it is reasonable to be- lieve that the SrUGT91 subfamily is responsible for the formation of the 1,2-β-D-glucosidic linkage in stevia.. - UBGAT from Scutellaria baicalensis is one of the few glycosyltransferases identified in plants that could use UDP-GlcUA as the sugar donor to catalyse a glucurono- sylation reaction [27]. - In the composite tree, UBGAT was clustered in group C and had a closer relationship with the SrUGT88 subfamily. - moreover, the similarity of the PSPG box between the UBGAT and SrUGT88 sub- family was more than 65%. - SrUGT76G1) involved in the synthesis of steviol glyco- sides: SrUGT85C2 and SrUGT74G1 glucosylate the C13-hydroxyl and C19-carboxylic acid functional groups of the steviol backbone, forming a β-D-glucoside, re- spectively, while SrUGT76G1 is capable of catalysing 1, 3-β-D-glucosylation at both the C13 and C19 positions of steviol. - therefore, we hypothesized that the SrUT85C subfamily may be responsible for glucosylating the C13-hydroxyl position of the steviol backbone. - Similar to the SrUGT85 subfamily, in group J, the SrUGT74G subfamily could theoretically be the glucosylated C19-carboxylic acid functional group of the steviol backbone. - Furthermore, the SrUGT76G and SrUGT76I subfamilies not only have a high degree of support (100% bootstrap) but also have a close genetic distance, indicating that these two sub- families may be responsible for the formation of the 1,3- β-D-glucosidic linkage.. - To date, many steps involved in the biosynthesis path- way of steviol glycosides have been successfully uncov- ered, especially for elucidating four UDP-dependent glucosyltransferases (UGTs) [6, 29], but several glucosy- lation steps of some glycosides that have not been re- solved to date. - In addition, for the large family of glucosyltransferases, we speculate that multiple enzymes with similar functions may participate in the same cata- lytic step in the glucosylation of steviol glycosides. - Therefore, in this study, one co-expression network approach named WGCNA, which was proved to be a powerful tool in systematically describing the correlation relationship between clusters of highly correlated genes or modules and external conditions or sample traits [31, 32], was used to analyse the potential UGTs involved in the glucosylation of steviol glycoside. - First, we per- formed qRT-PCR analysis of the expression levels of. - nine UGTs (SrUGT71H1, SrUGT85B1–2, SrUGT91D2, SrUGT76G1–1, SrUGT91D3, SrUGT85C3–1, SrUGT79A2, SrUGT73G2 and SrUGT71I1) in the leaves of six genotypes to confirm the reliability of the tran- scriptome data, and the primers used for qRT-PCR are shown in Table S2. - 4 Phylogenetic analysis of the S. - The tree shown was derived by neighbour-joining distance analysis of the full-length amino acid sequence described in Additional file 6. - Bootstrap values are listed as percentages of the replications, where values over 50% are indicated above the nodes. - Based on the selected power value, a weighted co-expression network model was established, and 14,995 genes were eventually di- vided into fifteen modules, of which the grey module had no reference significance because of the failure to assign to any module. - The results showed that the genes in the module are both highly correlated with the traits and the eigengenes. - Among these genes in the fourteen modules, two genes belong to the acetylglucosaminyltransferase, fifty-five genes were annotated to be members of the plant UGT superfamily, including SrUGT85C2, SrUGT74G1, SrUGT76G1, SrUGT85A8 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides except for SrUGT85A8 [6, 7], illus- trating the reliability of our results. - expression levels of these genes in the leaves of the six genotypes are shown in Fig. - 8 Heat map of the fifty-seven potential glucosyltransferase genes (in red and black) obtained from the weighted correlation network analysis and the genes (in green) known to be involved in steviol glycoside biosynthesis. - involved in steviol biosynthesis might be expected to ex- hibit a similar co-expression pattern with the SrUGTs involved in the synthesis of steviol glycosides. - Notably, forty-four SrUGTs, includ- ing SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, were then identified as being co-expressed with at least one of the upstream genes (Fig. - 8), and it is reasonable to believe that these SrUGTs may be directly involved in the synthesis of the corresponding steviol glycosides, which warrants further research.. - To confirm the sequence accuracy of the candidate SrUGTs, twenty of these forty-four candidate SrUGTs (randomly selection) were cloned by reverse transcript- ase polymerase chain reaction (RT-PCR) using the de- signed primers (Table S3), and then sequenced with the pClone007 Blunt vector (TsingKe, China). - Because of the self-incompatible in stevia, the sequence difference should be due to genetic heterozygosity [9, 10]. - rebaudiana genome (GenBank: WOUH and found that these genes were located in different posi- tions in the S. - The taste of steviol glycosides relies on glycosylation at the C13-hydroxyl and/or C19- carboxylic acid positions of the diterpenoid steviol backbone. - In this study, we combined short-read NGS and long-read SMRT sequencing of six different ge- notypes of stevia, which are the largest number of materials used for sequencing at all times, and then successfully generated a full-length transcriptome of the stevia leaf (total of contigs = 39,879, average length = 1949 bp). - Similarly, in the transcriptome of S. - These reflect that the contigs in our corrected full-length transcriptome were better than those of the assem- bled transcripts. - rebaudiana gen- ome (GenBank: WOUH was published on January 28, 2020 and available in the public databases, but the genome with no annotation file and the se- quencing work of this study was finished on January 7, 2019. - Among the forty-four candidate SrUGTs, including the four identified SrUGTs (SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2), there- fore, it is reasonable to believe that these forty-four enzymes are mainly involved in the glycosylation of steviol glucosides. - Due to the lack of UDP-Rha and UDP-Xyl, the SrUGTs using UDP-Rha or UDP-Xyl as sugar donors in the stevia were not successfully iden- tified, in this work, we provided a high-quality ana- lysis in the composite phylogenetic tree and obtained potential candidate SrUGTs with full-length se- quences. - rebaudiana and fi- nally forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained using WGCNA, phylogenetic trees, and qRT-PCR methods.. - The full- length transcriptome dataset may also provide useful candidate genes for the elucidation of the mechanism of steviol glucosides biosynthesis.. - The remaining geno- types were obtained by the induction through mutagenic methods as follows: 023 and 110 were obtained from in- duction by 60 Co γ-ray irradiation of the callus of 11–14 leaf tissue. - To obtain sufficient full- length transcriptome sequences, samples of the 3rd leaf tissues of the ‘023’ genotype were separately collected from the seedling and adult stages. - Eighteen samples of the six different genotypes were submitted for second-generation transcriptome sequencing using the Illumina HiSeq X Ten platform (Illumina, USA).. - Therefore, analysis of the eight steviol glycosides in all samples was conducted by reference to the HPLC-UV method in [36]. - The LOD and LOQ were calculated using both the values of the calibration curve and signal-to-noise ratios of 10 and 3, respectively [37].. - Purified cDNA was normalized by end repair, adenylation of the 3 ends and ligation of the adapters. - To validate the quality of the contigs, we used two different approaches. - org), and obtained the annotation information of the contigs with e-values <1e − 5 against the eight databases, including non-redundant protein sequence database (NR. - To verify the reliability of the transcriptional data, qRT- PCR experiments were carried out with ten UGT genes.. - An unrooted phylo- genetic tree was then constructed by the neighbour- joining clustering method with the amino acid sequences of the ORFs using the bootstrap method with 1000 repli- cates. - BUSCO assessment of the 30,859 corrected contigs.. - Primers and annealing temperature of the twenty SrUGTs.. - rebaudiana genome of the forty-four candidate SrUGTs.. - Additional file 7.The nucleotide sequences of the five SrUGTs, which cloned and differed with transcriptome in this study.. - SZ performed most of the experiments and data analysis and wrote the manuscript. - The supporters had no role in study design, data collection, data analysis, the writing of the manuscript or decision to publish.. - The raw data from the Illumina HiSeq X Ten platform have been submitted to the Sequence Read Archive (SRA) of the NCBI under accession numbers SRR10799213, SRR10799212, SRR10799203, SRR10799202, SRR10799201, SRR10799200, SRR10799199, SRR10799198, SRR10799197, SRR10799196, SRR10799211, SRR10799210, SRR10799209, SRR10799208, SRR10799207, SRR10799206, SRR10799205 and SRR10799204.. - The accession number of the raw data from SMRT sequencing was SRR10567116. - The assembled and the corrected transcripts were submitted to the Transcriptome Shotgun Assembly (TSA) of the NCBI under the accession numbers GISI00000000 and GISQ00000000, respectively.. - Insights from the sequencing and annotation of the Stevia rebaudian genome and their application in agronomy and health [C]//international congress of nutrition “ from science to nutrition security. - Functional genomics uncovers three glycosyltransferases involved in the synthesis of the major sweet glycosides of S. - Resolving the complexity of the human genome using single-molecule sequencing [J]. - A single-molecule long-read survey of the human transcriptome [J]. - Phylogenetic analysis of the UDP- glycosyltransferase multigene family of Arabidopsis thaliana [J]. - Elucidation of the amygdalin pathway reveals the metabolic basis of bitter and sweet almonds (Prunus dulcis)[J]. - Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from S.. - Mutations in the uridine diphosphate glucosyltransferase 76G1 gene result in different contents of the major steviol glycosides in S
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