| Santolina chamaecyparissus L.is a perennial herbaceous aromatic plant that grows on the Mediterranean coast and south of the Yangtze River.It contains a variety of volatile active ingredients and has significant pharmacological effects such as antibacterial,antitumor and antiviral effects.In China,S.chamaecypatissus is used to make insect repellent or as a garden landscape decoration.Although S.chamaecypatissus have pharmacologically active ingredient,it is mostly wild and lacks professional cultivation and germplasm resource conservation,herb morphology is susceptible to many unfavorable factors such as climate change,pests and diseases,so that the in-depth study of the plant resources and active constituents of S.chamaecypatissus has been neglected,resulting in slow progress in the industrialization development and utilization of S.chamaecypatissus plants.In order to solve these problems,the tissue culture and regeneration technology system of S.chamaecypatissus was established,and the increase of volatile constituents was promoted by hormone induction.The transcriptome sequencing technology was used to functionally annotate the differentially expressed genes in the geranium-producing plants after hormone induction.The volatile component biosynthetic pathway and the expression of its candidate enzyme gene were verified in this paper.The main research contents and results of this thesis are as follows:1.Established a GC-MS Detection and Analysis Method for the Volatile Components of S.chamaecypatissusChromatographic conditions:The capillary column used was DB-5MS 30 m x 0.25 mm x 0.25 ?m;the carrier gas was high purity helium at a flow rate of 1 mL/min.In splitless mode,the inlet temperature is 250 0C.The temperature rise procedure for the column oven is:40 0C for 2 min,then ramp up to 180? at 5?/min for 2 minutes,then ramp up to 240? at 5?/min for 5 min.Mass spectrometry conditions:ionization mode EI,ion energy 70 eV;mass spectrometry interface temperature 250?,ion source temperature 230?,mass scan range 35-550 U.The results showed that 48 volatile substances were identified in S.chamaecypatissus,which was rich in sesquiterpenoids.The method has good repeatability and high precision,and was suitable for subsequent analysis and detection of volatile components in S.chamaecypatissus.2.Established the Tissue Culture Regeneration Protocol of S.chamaecypatissus and Detected and Analyzed the Volatile Components in the Leaves of S.chamaecypatissus.(1)The optimal disinfection method was sterilized the stems of S.chamaecypatissus by using 75%ethanol and 1%sodium hypochlorite solution in sequence.After 10 days of inoculation in MS medium,the survival rate of S.chamaecypatissus explants was 95%.(2)The optimal induction medium for callus of S.chamaecypatissus was MS+0.2 mg L"1 NAA+1.0 mg L-1 BA+30 g L-1 sucrose+8 g L-1 agar,and the pH of the medium was adjusted to 5.8.The callus induction rate was 82%.(3)The optimal shoot induction medium for S.chamaecypatissus was MS+0.04 mg L-1 BA+0.1 mg L-1 NAA,+30 g L-1 sucrose+8 g L-1 agar,and the pH of the medium is adjusted to 5.8.A cool white fluorescent lamp at 25±1?,with an illumination intensity of 1800 Lx,cultured for 6 weeks under 16 hours/day of light to obtain the best and most abundant S.chamaecypatissus shoots.(4)The effects of different concentrations of NAA on the rooting of S.chamaecypatissus shoot were studied by using the length,number,rooting induction rate and biomass of the shoots.The experimental results showed that the optimal rooting induction medium of S.chamaecypatissus was MS+0.1 mg L-1 NAA+30 g L-1 sucrose+8 g L-1 agar,and the pH of the medium was adjusted to 5.8.A cool white fluorescent lamp at 25±1?,with an illumination intensity of 1800 Lx under 16 hours/day of light.Under the above optimal conditions,the rooting rate of S.chamaecypatissus shoot has reached the maximum.(5)Transfer the S.chamaecypatissus regenerative plants to a gravel and sandstone to perlite volume ratio of 1:1:1(v/v)for refining.After 30 days of cultivating the plants,the regenerated plants of the S.chamaecypatissus which grew well in the greenhouse were transferred to the natural environment of the field for growth.After 2 months,the survival rate of the S.chamaecypatissus-regenerated plants grown in the field was 100%.(6)The GC-MS analysis method was used to compare and evaluate the volatile components in the tissue cultured plants of the S.chamaecypatissus and the growth of the plants.The results showed that the accumulation of monoterpenes and sesquiterpenoids in the regenerated plants was higher than that in the field plant.3.Effects of Hormone Induction on Volatile Components in Regenerated Plants of S.chamaecypatissus(1)The effects of IAA on the volatile components in the regeneration of S.chamaecypatissus tissue culture plants were investigated by using indole-3-acetic acid(IAA)to induce the regeneration of S.chamaecypatissus..The results showed that the accumulation of volatile components in the regeneration of S.chamaecypatissus was increased by 1 mmol L-1 IAA induction treatment,and the biosynthesis of sesquiterpenoids was significantly affected.The Tricyclo[5.4.0.0(2,8)]undec-9-ene,2,6,6,9-tetramethyl-,(1R,2S,7R,8R)was rapidly accumulation by IAA treatment.(2)The effect of melatonin on the volatile constituents in the regeneration of S.chamaecypatissus tissue culture plants was investigated by using melatonin to induce the regeneration of S.chamaecypatissus.The results showed that 0.5 mmol L-1 melatonin treatment had a more significant effect on the accumulation and composition of volatile constituents in the plants of S.chamaecypatissus,and the delta-elemene sesquiterpene compound was synthesized and accumulated in a large amount after melatonin induction.4.Transcriptome Sequencing and Data Analysis of Hormone-induced Treatment of S.chamaecypatissus Tissue Culture Plants(1)In this study,Illumina sequencing technology was used to obtain the transcriptome data of the plant regeneration from the plant hormone IAA and melatonin.After the assembly,a total of 756826 transcripts were obtained,and the minimum transcript length was 200 bp.(2)By comparing the transcriptome data of the S.chamaecypatissus regenerated shoots before and after IAA hormone treatment,a total of 22326 differentially expressed genes were obtained,of which 10373 were up-regulated and 11953 were down-regulated Through the analysis of GO classification function,it was found that all differentially expressed genes were mainly distributed to Cellular component,Molecular function and Biological process;3,747 differentially expressed genes were co-annotated in 20 significantly enriched KEGG Pathways.In addition,the key enzyme genes in the synthetic pathway of glucosinolates were significantly induced and regulated by exogenous hormones,and the expression levels of 20 key enzyme genes were changed after IAA hormone induction.(3)By comparing the transcriptome data of S.chamaecypatissus regenerated plants before and after treatment with melatonin,a total of 24387 differentially expressed genes were obtained,of which 11705 were up-regulated and 12682 were down-regulated.GO classification analysis of differentially expressed genes obtained from melatonin-treated S.chamaecypatissus regeneration plants showed that differentially expressed genes are mainly enriched in binding,catalytic activity,cellular process,and metabolic processes.(metabolic process)and single-organism process.A total of 3495 differentially expressed genes were annotated by KEGG metabolic pathway enrichment analysis.The expression levels of 19 key enzyme genes in the geranium skeleton synthesis pathway were changed after melatonin induction.5.Expression Verification of Key Regulatory Genes in Volatile Components Biosynthesis Pathway in Regenerated Plant of S.chamaecypatissus by Hormone-induced Treatment(1)The relative expression levels of all genes except HMGS gene were significantly increased by using IAA hormone to induce the regeneration of S.chamaecypatissus plant for 12 h,among which,acetoacetyl-coenzyme A(CoA)thiolase(AACT),mevalonate kinase(MK)and geranylgeranyl diphosphate synthase(FDPS)gene has a significant response effect which was 50.7 times,41.6 times,60.8 times of the blank control treatment group in the biosynthesis of anthraquinones in IAA-induced S.chamaecypatissus regeneration plants.(2)The relative expression levels of HMGS gene,HMGR gene,MK gene,PMVK gene,MVD gene,IDI gene and FDPS gene reached the maximum when induced by melatonin for 6 h,which was 21.5 times,8.2 times,20.2 times,27.1 times,20.6 times,11.5 times and 22.0 times of the blank control treatment group,respectively,indicating that the steroidal biosynthetic enzyme gene in the S.chamaecypatissus regenerated plants can significantly respond to the transcriptional activation of melatonin-induced stimulation.The optimization of the production of volatile components of the S.chamaecypatissus tissue culture regeneration system established by this research and development,for the future use of the isolated organ culture system industrial production of volatile active ingredients laid the theoretical basis.The accumulator technique was used to enhance the accumulation of volatile constituents in the regeneration culture of S.chamaecypatissus.The high-throughput transcriptome sequencing confirmed the key enzyme genes regulating the biosynthesis of volatile constituents in the regeneration of S.chamaecypatissus.The verification of its gene expression provides an important theoretical basis for the use of elicitor technology to regulate the production of secondary metabolically active components in other plant tissue culture systems and to elucidate the key genes of biosynthesis. |
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