| Agarwood is a black-resin wood formed from the stems,branches or roots of trees belonging to the species Aquilaria,Gonystylus and Gyrinops after damage?due to natural causes such as wind,lightning,insect bites or microbial infection?,and it is considered to be a pathological product.As a precious spice,agarwood is not only widely respected by global religions such as Buddhism,Islam and Christianity but also valuable for health care and collection.As a traditional medicine,this resin-containing wood can be used to make antiemetics,sedatives and digestive drugs.However,the current endangered status of wild agarwood resources and the low efficiency of artificial production makes it difficult to meet market demand.For many years,numerous researchers committed themselves to the development of efficient production technology for agarwood;some progress has been made based on the exploration and clarification of the synthetic process of the marker ingredients of agarwood,sesquiterpenes and chromones.In addition,if fresh wood is used as the study material,the experimental cycle is long,and the environmental factors are complex and uncontrollable.The use of suspended cells as the material is not very compatible with simulation of the natural process of agarwood production due to their essential characteristics of heterotrophy and being an in vitro system.The present study attempted to make use of an aeroponic cultivation system with a short cycle and controllable conditions for studying the induction of chromone formation in Aquilaria sinensis.1.ObjectiveThe purpose of this study was to explore molecular basis of MeJA-induced 2-?2-phenylethyl?chromones biosynthesis and speculate the related genes in lateral roots of Aquilaria sinensis by an aeroponic system.2.Methods2.1 The establishment of aeroponic cultivation systemThe starting material of this study was one-year-old Aquilaria sinensis seedlings,The seedlings were removed from the nutrition bag.After cleaning the soil from the roots,all of the lateral roots were cut off,followed by disinfection of the main root with 0.1%KMnO4 for 1 min.The 10-s treatments with different growth regulators?NAA,IBA and ABT1?at different concentrations(0.5,1.0 and 1.5 g · L-1)were designed for the induction of the aerial rooting.After the roots were dipped in the solution,the seedlings were immediately transferred into the aeroponic cultivation system,After rooting had occurred,the water was replaced with MS media of different electrical conductivities(0.6,1.2 and 2.4 dS · m-1)to compare their effects on rooting.Then GC-MS analysis of chromone derivatives in aeroponic roots of A.Sinensis under different concentrations?0?50?100?200?M?MeJA with 7d.2.2 Transcriptome analysis on lateral roots of Aquilaria sinensisIllumina HiSeqTM 2000 sequencing technique was made use to analysis the lateral roots under 50?M MeJA-induced Od and 5d after extraction of total RNA.2.3 A preliminary study on the relationship between PCD and chromones derivatives in lateral roots of Aquilaria sinensisThe microscopic observation,TUNEL&PI testing,DNA degration and GC-MS were used in lateral roots of Aquilaria sinensis under 50?M MeJA-induced 0d?3d?5d?7d.3 Results3.1 The establishment of aeroponic cultivation systemThe experimental results showed that optimal rooting rates?86.67%?and amounts(32.87roots · plant-1)were obtained by cutting off the lateral roots of soil-cultivated Aquilaria sinensis seedlings,treating them with 1.5g · L-1 IBA and placing them in the water aeroponic cultivation system.The lateral roots cultivated in 1.2 dS · m-1 MS medium grew vigorously and accumulated the maximum biomass(1272.6 ± 681.6 mg plant-1).There are 2-?2-phenylethyl?chromone and 6-Methoxy-2-?2-phenylethyl?chromone were produced under 50?M MeJA with 7d and the former' s relative amount was approximately 8-fold than the standard medicine?positive control?,while the latter was 0.45%.However,the relative content of 2-?2-phenylethyl?chromone was only 0.23%?100?M?and 0.65%?200?M?,far less than 50?M.3.2 Transcriptome sequencing of lateral roots of Aquilaria sinensisIllumina HiSeqTM2000 is an advancing next-generation sequencing technique,was carried out to sequence the transcriptome of lateral roots of Aquilaria sinensis.We obtained 128878 unigenes,among which 67604 unigenes were annotated to at least on database,including Nr,Swiss-prot,KEGG,KOG,and accounting to 52.45%of the total unigenes.According to the FDR<0.05 ? |log2FC|>1,we got 48777 differential expressed genes?DEGs?,including 17039 up-regulated unigenes and 31738 down-regulated unigenes.The results of analysis of DEGs,indicated the"Activity of transcription factors" and "binding" function were up regulated,while other GO classification functions were down regulated.In addition,phenylpropane pathway and sesquiterpenes pathway were annotated and significance by KEGG,among which most significant was ribosome pathway.The receptor-like kinase,MAPK cascades and Ca2+ proteins involved in MeJA stress signal transduction,which included that four receptor-like kinase families?LRR-RLK,CRR-RLK,SRK and PERK?annotated as 124 DEGs could control multiple plant growth and development process under MeJA stress;The Ca2+-related genes?CaM,CML,CDPK and Ca2+ ATPase?were the second messenger of signal pathways and able to participate in the plant hormone signaling pathways.Meanwhile,MAPK cascades protein phosphorylation and dephosphorylation in many signal transduction pathways as the important role that helps signal transmission,we found 43 DEGs annotation for the MAPK cascades?43?,including the MAPK?22?,MAPKK?10?and MAPKKK?11?.Defense genes are usually affected by regulation of TFs will therefore TFs in different signaling pathways play a direct or indirect role.MeJA cause the response of the large number of transcription factors,found 313 DEGs annotated as TFs,most significantly down-regulated,including mainly induces the WRKY?23,16 up-regulated and 7 down-regulated?,MYB?31,8 up-regulated and 23 down-regulated?and ERF?38,13 up-regulated and 25 down-regulated?,etc.The result showed they may participate in secondary metabolites biosynthesis regulation of adventitious roots,this information is helpful for our further analysis response genes about MeJA induced chromone.3.3 The relationship between PCD and chromones derivativesThe results indicated that the lysigenous aerenchyma tissue was appeared obviously under MeJA?50?M?-induced 5d and 7d.Besides,TUNEL,PI and DNA degradation showed that cells died gradually during treatment,while 2-?2-phenylethyl?chromone was detected and accumulation in lateral roots of 5th and 7th day.4.ConclusionIn conclusion,we established the aeroponic system of Aquilaria sinensis,preliminarily.The study used the lateral roots induced by MeJA during different times,indicated that MeJA could induce 2-?2-phenylethyl?chromone and the roots could be material used to research chromone biosynthesis by transcriptome sequencing.This will provide theoretical basis for research the formation mechanism of chromone further. |
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