| Amomum villosum is the main source of Fructus Amomi, which is used as medicine by the dried ripe fruit. Fructus Amomi has the function of appetizer with dampness, warming the spleen and stopping diarrhea, regulating qi and tocolysis. Fructus Amomi is the commonly used traditional Chinese herbs, the market demand is very great. Owing to the narrow cultivated area and low yield, the expansion of drug sources is severely restricted.1ObjectiveUsing the new generation of high-throughput sequencing technology, we conducted a de novo assembly whole-transcriptome of A villosum, which served as a reference for the following. By the sequencing of expression profiles, a larger number of genes at different stages of fruits (30DAF,60DAF and90DAF) were obtained and the genes related to sugar and terpenoid biosynthesis were analyzed in detail.The relationship was established between gene expression and sugar content, enzyme activity of sugar metabolism, so as volatile terpenes content. To provide theoretical basis for improving the fruits yield and active ingredient content, the limiting factors and molecular mechanisms of fruits yield and active ingredient content were discussed.2Methods2.1Transcriptome sequencing of A villosum and data miningTo construct a cDNA library,the total RNA of A villosum whole plant (except the flowers) were extracted respectively and mixed equally. Using high-throughput Illumina HiSeq2000sequencing platform, the transcriptome of A villosum was analyzed.T o obtain the unigene’s sequences, high quality sequences were assembled by SOAPdenovo software. The unigenes were annotated with four public databases (Nr,Swiss-Prot,KEGG and COG) and the sugar and terpenoid metabolic pathways were analyzed in detail. Classify the unigenes of HMGR、DXR and DXS genes in the biosynthesis pathways of terpenoids by the phylogenetic trees. Those unigenes without annotation in the pathways of monoterpenoid and sesquiterpenoid biosynthesis were searched through PSI-BLAST.2.2Differential expression profiles of A villosum fruits and the screening of differentially expressed genes related to sugar and terpenoid metabolismThe expression profiles of three different developmental A villosum fruits stages were examined by RNA-Seq quantification. The expression trends of differentially expressed genes were determined by cluster analysis based on the STEM software. The genes related to sugar metabolism, terpenoid metabolism and transcription factors were screened. Total RNA for qPCR were extracted from A villosum fruits of different developmental stages and different tissue parts (peels and seeds), and reverse transcribed into cDNA. Individual genes of differentially expressed were screened as target genes. The primers of qPCR were designed and synthesised according to the gene sequences. The qPCR was applied to study the change of gene expression in different samples, in order to verify the expression profile data.2.3The research of sugar and terpenoid metabolism of A villosum fruits at different developmental stagesThe fresh fruits at three different development processes were used to investigate the changes of soluble sugar content, the sucrose metabolizing enzyme activities and the volatile terpenoids content.The soluble sugar and crude enzyme were extracted with ethanol and extraction buffer respectively, and determined separately by HPLC and UV spectrophotometry. Microwave extraction method was used to extract volatile terpenoids and determined by GC/MS. By modern analytic determination methods, the content of sugar and terpenoids were analyzed, making the correlation analysis between the gene expression and compounds content, enzyme activity.3Results3.1Obtain the information of A villosum transcriptomeA total of14,4020unigenes were assembled from the whole transcriptome sequencing data of the A villosum whole plant(except the flower).Through blastx and ESTscan software, the protein coding sequences of the annotated unigenes were predicted. Annotating with clusters of orthologous group (COG),26,303unigenes were returned to25functional category. By the GO database annotations,31,978unigenes were classified into44functional category, including the metabolic process of14,456unigenes, which provided sufficient data for the research of the primary and secondary metabolism. By analyzing KEGG metabolic pathways,21,254unigenes were divided into270classes,567unigenes related to starch and sucrose metabolic pathway and154unigenes related to terpenoid backbone biosynthesis pathway. Through the study of expression profiles during fruits development, differential expression genes were screened. The genes related to sugar and terpenoid metabolism were analyzed in detail.The unigenes of HMGR、DXR and DXS genes were classified by phylogenetic analysis and the funtions were predicted. Furthermore,12,715microsatellites(simple sequence repeats) were detected,6,122of the microsatellites were tri-nucleotide motifs. Through the PSI-BLAST protein homology alignment and KEGG database,3genes of monoterpene synthases and2genes of sesquiterpene synthases were aligned.3.2The analysis of gene expression profiles data and the verification of differentially expressed genes related to sugar and terpenoid metabolism using qPCRBy analyzing of gene expression profiles data,22,483significantly differentially expressed genes and3differentially expressed trends were selected. The genes related to sugar and terpenoid metabolism were screened, including genes of SPS,SS,INV,HMGR,AACT,FPSS,DXS,DXR and so on.Otherwise,170transcription factors with significant changes were found, and the expression levels were the highest in the middle and later periods of the fruits development. Among these transcription factors, WRKY were the most types, followed by MYB、bHLH (basic Helix-Loop-Helix).12AP2(APETALA2) type of transcription factors were obtained, and the expression levels of7AP2transcription factors showed gradual increase.Using PCR,the gene expression trends of HMGR、DXR、SS、PK、INV were verified, which were not completely consistent with the result of expression profile. The gene expression trends of SS and INV in sugar metabolism were rising gradually in peels and seeds, which were consistent with the results of expression profile data. The gene expression trend of PK in glycolysis was the highest in young fruit period in seeds, only in peels the genes expression trends (rising gradually) were consistent with the expression profile. In the terpenoid metabolism, the gene expression trend of DXR in peels was not agreed with the result of expression profile, but in seeds the genes expression declined gradually, fitting with the result of expression profile. The genes expression trend of HMGR in peels increased gradually, agreing with the result of expression profile, but in seeds the result was not agreed with expression profile.3.3The changes of soluble sugar content,sucrose metabolizing enzyme activities and volatile terpenoids content were determinedThe content of hexose is higher than the content of sucrose in young fruit. In the middle of the fruit development, hexose was the main sugar in peels but sucrose was the main sugar in seeds. In the later of the fruit development, sucrose content was much higher than hexose both in peels and seeds, and this point was sucrose accumulation phase. Net activity of SS catalyticed the sucrose synthesis, and the activity increased gradually both in peels and seeds. The content of monoterpenes (myrcene, D-limonene) and monoterpenes oxo derivatives (camphor, borneol, bornyl acetate) were highest in the middle and later of the fruits development, but the content of sesquiterpenes (a-copaene, caryophyllene, bicyclogermacrene) increased at first but later decreased.3.4Correlation analysis between the gene expression analyzed by qPCR and sucrose metabolizing enzyme activities, the volatile terpenoids content of terpenoid metabolismThe increase of sucrose concentration and the decrease of hexose concentration were mainly caused by the enzyme activity of SS (synthasizing direction). Net activity of SS increased gradually both in peels and seeds, which fitted with the result of qPCR and expression profile. The gene expression levels of SS and INV and hexose content was negatively correlated, but the gene expression and sucrose content was positively correlated. The correlation analysis between gene expression and volatile terpenoids content showed that most of the terpenoids content in peels had a negative correlation with HMGR, but had a positive correlation with DXR. However, the content of bornyl acetate and most sesquiterpenes had positive correlations with HMGR and DXR, but most monoterpenes content had negative correlations with them.4ConclusionsThe A villosum whole transcriptome and fruits expression profiles of different developmental stages were obtained for the first time by high-throughput RNA-Seq, which provided important reference information for the research of sugar and terpenoid metabolism. Besides, the high throughput sequencing data laid an important foundation for further research the molecular mechanism of fruits development and secondary metabolism. The SSR identification of A villosum transcriptome provided a theoretical basis for studying the genetic diversity and marker assisted breeding of A villosum. Futhermore, the analysis of AP2type of transcription factors laid a solid basis for research the regulatory mechanism of fruits development and secondary metabolism.The differential expression genes of sugar and terpenoid metabolism were screened by analyzing the expression profiles. Through modern analytic determination methods, the changes of sugar and terpenoid metabolism were detected, combining with the gene expression, establishing the relationships between them.The fruits development of A villosum were regulated by the hexose and sucrose concentration, while the activity of sucrose synthase coordinated the source-sink relationship, also increased the fruit production of A villosum. Terpenoids synthesis process was affected by multiple factors, the expression of genes and content of compounds did not align perfectly. However, the peak of genes expression and compounds content of terpenoids biosynthesis pathway were in the middle and later fruit development periods. |
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