| The early bolting of Angelica sinensis(Olive.)Diels refers to a phenomenon that the cultivated A.sinensis plant enters the reproductive growth stage early in the second year,its effective compounds cannot accumulate in the vegetative growth stage,and the fleshy roots of the medicinal parts atrophy and fibrosis,completely losing the medicinal value.Early bolting seriously affected the medicinal properties and output of A.sinensis,and has become a key factor restricting the sustainable of A.sinensis production in recent years.Since traditional agronomic measures cannot solve the problem of early bolting of A.sinensis fundamentally,people try to explore the regulation mechanism of early bolting at the molecular level.However,the genetic background of A.sinensis is complicated and the genome information is currently not perfect.Research on the molecular mechanism of early bolting is still in the exploratory stage.In order to reveal the changes in gene expression during early bolting in A.sinensis,and to find out the key genes and signal pathways which regulate the early bolting,the study uses high-throughput third-generation full-length transcriptome combined with second-generation sequencing technology to compare and analyze the dynamic characteristics of the transcriptome of early bolting of A.sinensis of Min County,the discovery of major genes involved in the regulation of early bolting of A.sinensis,combined with the physiological and biochemical analysis of the Medicinal Value Formative Period,and preliminarily constructed the molecular regulation network of early bolting of A.sinensis,and perfecting the early bolting theory,effectively protect the sustainable of A.sinensis production in Gansu Province,and provide theoretical support for the subsequent breeding of new bolting resistant varieties.The main findings are as follows:1.Under same natural environmental conditions,the two traditional farming sources of A.sinensis from Min County and Zhang County were in the regreening stage(June 27),bolting stage(July 14)and the late bolting stage(August 3),which follow up the growth and development of seedlings.The results showed that the individual growth and development indicators of the two sources of A.sinensis were basically the same with phenological changes in the Medicinal Value Formative Period;Plants from Min County had lower death rate,but the bolting rate was higher;plants from Zhang County had lower bolting rate but the death rate was higher;the growth and development indexes of the early bolting plants are higher than that of the normal plants during whole Medicinal Value Formative Period;the early bolting plants show a phenotype of larger plant size and darker color of the whole plant(leaf and stem)in the regreening stage.2.The anthocyanin content and four antioxidant enzyme activities of A.sinensis from Min County and Zhang County were tracked and detected in whole Medicinal Value Formative Period.The anthocyanin content was pre-enhanced by 21.91%(Min County)and 22.24%(Zhang County)in bolting plant,and the Catalase(CAT)activity was also pre-enhanced by 48.33%(Min County)and 60.50%(Zhang County)in bolting plant,respectively.It drops to a certain level during the bolting stage,then rises again in the late bolting stage;while the anthocyanin content and CAT activity of the normal plants showed a completely opposite trend,reaching a peak during bolting stage;The activities of Superoxide Dismutase(SOD),Peroxidase(POD)and Ascorbate Peroxidase(APX)have the same variation trends,however,the activities of these three enzymes in early bolting plants were higher than those in normal plants,indicating that the stronger the plant’s antioxidant enzyme activity,the higher the survival rate,but the early bolting rate is also higher;the correlation analysis results showed that the correlation between anthocyanin and CAT gradually increases with individual phenological changes,indicating that the content of anthocyanin and catalase in the young leaves of A.sinensis at regreen stage Pre-enhancement of activity can induce early bolting,and this process is simultaneously regulated by SOD,POD and APX.3.Ultra-high performance liquid chromatography determined the content of 24 endogenous plant hormones in A.sinensis at the initial stage of bolting.The results showed that the content of abscisic acid and trans-Zeatin in early bolting plants is significantly higher than normal plants,while the content of dihydro-Jasmonic acid and Kinetin is significantly lower than that of normal plants.4.By using the method of combining the third-generation high-throughput full-length transcriptome and the second-generation general transcriptome sequencing,transcriptome analysis is performed on the early bolting stage of A.sinensis and the normal medicine A.sinensis,and finally the third-generation full-length transcriptome sequencing Generated43,438 full-length transcripts of A.sinensis,with an average length of 1996 bp;a total of2.61 billion clean reads were obtained by second-generation sequencing.Through gene function annotation and differential main expression analysis,a total of 475 differentially expressed genes(DEGs)were identified;metabolic pathway enrichment analysis showed that DEGs are mainly involved in semi terpenoid and triterpenoid biosynthesis and secondary metabolites biosynthesis,cutin/corking and wax biosynthesis,unsaturated fatty acid biosynthesis and other processes.Co-expression network analysis classified the18,685 transcripts obtained by second-generation sequencing into 24 modules,selected 8modules that were significantly differentially expressed in early bolting plants,and intersected with 475 DEGs to obtain 124 co-expressed DEGs.Finally,51 DEGs related to the early bolting were screened,which were mainly enriched in plant hormone signal transduction pathway,age-regulated flowering pathway regulation,early floral organ development pathway and very-long-chain fatty acid biosynthesis pathway.5.By using bioinformatics methods,25 As SPL genes were identified from the full-length transcriptome of A.sinensis.Phylogenetic analysis showed that As SPLs of A.sinensis can be divided into 4 subfamilies.The gene structure and conserved motifs of As SPL genes under the same phylogenetic tree branch are very similar.The As SPL promoter sequence contains multiple cis-acting element related to light signal response or hormone response.The expression pattern and q PCR analysis showed that some As SPLs genes may play an important role in the early bolting process of A.sinensis.The 12 As SPL genes are all highly expressed in the early bolting plants,and their abundances are relatively consistent.It also proves that Functional redundancy of As SPLs.The construction of Arabidopsis overexpressing As SPL transgenic plants showed that35S::As SPL5 plants bolted in advance,which proved that As SPL5 may play a leading role in the early bolting process of A.sinensis. |
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