| Tropane alkaloids(TAs),including hyoscyamine,anisodamine,and scopolamine,are traditional medicines with a history of thousands of years,which are widely used as anticholinergic drugs in treatment of anesthesia,analgesia,pesticide poisoning,and relief of Parkinson’s disease in modern medicine.TAs have extremely high medicinal value and huge market demand,but currently rely entirely on extracting from TAs-producing plants with extremely low content of TAs,which is expensive due to scarce resources.Atropa belladonna is one of the most important commercial medicinal plants of TAs.Therefore,cultivating A.belladonna plants with high-yield of TAs can alleviate the increasingly prominent situation of TAs supplied and demand.The relevant research mainly focuses on the cultivation of high-yielding TAs by genetically modifying biosynthesis of metabolic engineering techniques,but there are limits of the accumulation of TAs as secondary metabolites in plants,so in order to cultivate more high-yielding TAs,there is a need to find another way.Studies have shown that increasing the biomass of resource plants is also one of the effective means of increasing the yield of its active ingredients,and can also be used to cultivate high-yielding TAs of the plant,but research on increasing the yield of TAs by increasing the biomass of A.belladonna plants have been largely unreported.As plant hormone,auxin regulates numerous aspects of plant growth and secondary metabolism,which can also to mediate by the early auxin response genes.The Small Auxin Up-regulated RNA(SAUR)gene family is the largest family of early auxin response genes.Recently,molecular,genetic,biochemical,and genomic studies have implicated SAURs in the regulation of a wide range of cellular,physiological,and developmental processes.However,the effects of SAUR on TAs biosynthesis and plant growth and development in A.belladonna had not been reported.Based on the transcriptome database,this study selected an A.belladonna SAUR gene(named AbSAUR1)as a candidate gene through bioinformatics analysis.The expression pattern analysis of AbSAUR1 was performed,furthermore,AbSAUR1-overexpressing transgenic plants of A.belladonna were established by stable genetic transformation for field experiments,and molecular detection were performed to investigate its effect on TAs biosynthesis.The horticultural and TAs content were further statistically analyzed,and function of AbSAUR1 in A.belladonna was studied.The relevant research results are as follows:1.Acquisition and expression pattern analysis of candidate gene AbSAUR1By analyzing the transcriptome database of A.belladonna,a total of 40 AbSAURs genes was obtained in this study.A Pfam search analysis showed that each of the 40SAUR unigenes had an auxin-inducible signature domain(PF02519)that was conserved in the SAUR family.The phylogenetic tree analysis was performed with the amino acid sequence of AtSAURs as control,which is the most complete study,together with the amino acid sequence of AbSAURs.It was found that an AbSAUR protein,annotated as aba＿locus＿23134＿iso＿1＿len＿1028＿ver＿2,was in the same branch as AtSAUR41.Related studies have shown that AtSAUR41 has a unique expression pattern in meristems,and that overexpression of it led to pleiotropic auxin-related phenotypes,including long hypocotyls,lateral root development and increased biomass,which can speculate that AbSAUR1 may have similar functions of AtSAUR41.Further analysis of gene expression correlations between Ab SAURs and TAs biosynthetic genes was performed,and it was found that aba＿locus＿23134＿iso＿1＿len＿1028＿ver＿2 was specifically expressed in roots,which was similar to the correlation of TAs biosynthetic gene expression.Based on the analysis of phylogenetic and gene expression correlation,aba＿locus＿23134＿iso＿1＿len＿1028＿ver＿2 was selected as the candidate gene,and it was named AbSAUR1 for further research.AbSAUR1 has a full-length cDNA of 1028 bp,its coding region is 510 bp,which can encode a polypeptide of 170 amino acid residues,the relative molecular weight of the protein is 19.1 kDa,and the isoelectric point pI is 9.65.The tissue expression pattern of AbSAUR1 in primary roots,secondary roots,stems,leaves,flowers and fruits of A.belladonna plants were analyzed by qPCR.AbSAUR1 was specifically expressed in roots and not expressed in stems,leaves,flowers and fruits.AbSAUR1 expression in secondary roots was higher than that of primary roots,and its tissue expression pattern was highly similar to the specific/high expression pattern of known TAs biosynthetic genes in secondary roots.The time gradient treatment of wild-type A.belladonna plants with auxin were used,and the transcript accumulation of AbSAUR1 was significantly increased 2 h after auxin treatment,which confirmed that AbSAUR1 was induced by auxin.2.Establishment and molecular detection of AbSAUR1-overexpressing transgenic A.belladonna plantsIn this study,a p1305~+-AbSAUR1 overexpression vector was constructed and the explants were infected with agrobacterium to finally obtain a stable genetically transformed transgenic A.belladonna plant.PCR detection of fusion markers NPTⅡand35S::AbSAUR1 fusion fragments confirmed that the overexpression vectors p1305~+-AbSAUR1 and p1305~+empty plasmids were successfully integrated into the genome of A.belladonna,respectively,and successfully obtained transgenic plants.qPCR analysis was used to detect the expression of AbSAUR1 and TAs biosynthesis genes.The expression of AbSAUR1 in the AbSAUR1-overexpressing line was increased to 23.75 times compared to the control.The TAs biosynthesis genes,including AbODC,AbPMT,AbTRI,AbCYP80F1 and AbH6H,have no significant changes.It showed that overexpression of AbSAUR1,the auxin downstream response gene,would not inhibit the expression of TAs biosynthetic pathway genes.3.Analysis of horticultural of AbSAUR1-overexpressing A.belladonnaIn field experiments,AbSAUR1-overexpressing plants showed a series of phenotypic changes,including increased plant height,thickened stems,increased number of leaves and branches,and increased roots.Analysis of horticultural showed that the plant height of the AbSAUR1-overexpressing line was 1.23 times that of the control line,and the stem thickness was 2.14 times that of the control line.AbSAUR1-overexpressing lines showed mature branches at the lower end of the stem,with an average of five branches,while the control lines had no mature branches.The biomass of AbSAUR1-overexpressing lines has also been significantly improved.The fresh and dry weights of the entire control line were125.46±22.43 g and 35.59±6.75 g,respectively.The average fresh and dry weights of AbSAUR1-overexpressing lines were significantly higher.The weights were495.05±36.88 g and 113.84±7.36 g,which reached 3.79 times and 3.20 times of the control lines.The fresh and dry leaves of the control lines were 34.42±5.39 g and7.90±2.51 g,and the average fresh and dry weights of the AbSAUR1-overexpressing lines were 133.90±13.36 g and 24.66±2.92 g.The fresh and dry weights of the control lines were 39.36±8.38 g and 11.22±2.84 g,and the average fresh and dry weights of the AbSAUR1 overexpressing lines were 90.21±12.58 g and 22.80±1.41 g.The analysis of horticulture analysis showed that overexpression of AbSAUR1 can positively regulate the growth and development of A.belladonna,moreover,it can significantly promote biomass accumulation.4.Analysis of TAs in AbSAUR1-overexpressing A.belladonnaThere were no significant differences in the content of hyoscyamine,anisodamine,and scopolamine in the AbSAUR1-overexpressing and control lines,neither in the roots or leaves.It was confirmed that the biosynthetic ability of TAs was not inhibited due to the overexpression of AbSAUR1,and with the significant increase in the biomass of AbSAUR1-overexpressing lines,the yield of TAs was significantly improved.In the roots of AbSAUR1-overexpressing plants,the production of hyoscyamine,anisodamine and scopolamine were 37.37±11.16 mg,40.69±12.62 mg,and 5.42±2.21 mg,respectively.In the control line it was 10.55±3.06 mg,16.24±3.07 mg and 1.56±0.43 mg.The average production of hyoscyamine,anisodamine and scopolamine in the leaves of p1305~+-AbSAUR1-overexpressing lines were 38.48±16.02 mg,8.69±3.54 mg,and 14.37±2.19 mg,which were 6.25 times,6.30 times and 2.92 times of control,respectively.In terms of the whole plant,the yields of hyoscyamine,anisodamine and scopolamine in the p1305~+-AbSAUR1 overexpression lines were 4.54 times,2.80 times,and 3.06 times that of the control lines,respectively.The results of TAs analysis showed that overexpression of AbSAUR1 had basically no effect on TAs content,however,which could significantly increase TAs production by increasing biomass.In this study,AbSAUR1 was cloned from A.belladonna,and then establishment of AbSAUR1-overexpressing transgenic plants for field experiments,in order to evaluate the effects of AbSAUR1 on plant growth and development.The results showed that overexpression of AbSAUR1 could promote plant growth and development,resulting in a significant increase in biomass,without inhibiting TAs biosynthesis,and ultimately significantly increasing TAs yield.In this study,the SAUR gene was reported and identified in A.belladonna for the first time,moreover,the effects of AbSAUR1 on the growth and development and TAs biosynthesis were systematically studied of A.belladonna.It provided new ideas and methods for cultivating high-yield TAs plants,which can fill in the blank of biomass research in A.belladonna and has certain theoretical and application value. |
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