| Jasmine tea is a kind of Chinese special tea,belonging to reprocessed tea,which is made thro ugh absorbing jasmine(Jasminum sambac(L.)Aiton)flower fragrance into parched green tea leaves.It is favored by consumers,and has broad market prospects.Jasmine(Jasminum Sambac(L).Ait)is a genus of oleaceae and Jasminum and it is a night-flowering plant.Its blossom and aro ma releasing occur together,with unique and rich aroma.It is the main raw material for making sc ented-flower,which has high economic value.Exploring the molecular mechanism of the blossom of jasmine flower provides the theory basis for the clear time of jasmine flower picking;the scient ific and reasonable regulation among the selection of scented tea,fragrance intensity and the time of aroma releasing;and jasmine blossom and interpretation of the genetic improvement of fragran ce.It is of crucial theoretical value and practical significance.This research adopts the GC-MS technique,testing content and its changes of living jasmine flowers and excised flowers’ aroma components;Excavate the best reference genes in real-time fluorescent quantitative PCR from the transcriptome data of double-petal jasmine;Using the petals(Driver)of jasmine that is unopened and not releasing fragrance and opened and fragrance released jasmine petals(Tester)as the materials,to construct DSN mediated suppression subtractive hybridization cDNA library.Using reverse Northern Blotting to sift through the cDNA library,so as to get the positive clones.Using fluorescence quantitative PCR technique to detect dynamic changes of expression quantity in gene involvement of aroma formation in jasmine living flowers and flowers in vitro,the main results are as follows:1.Compare the dynamic changes of fragrance components and contents of living jasmine and excised jasmine:the results of quantitative analysis in volatile aroma components in the flowering process of living jasmine and the 5 periods of excised jasmine by GC-MS technology showed that the aroma components of living jasmine and excised jasmine were basically the same which mainly included benzyl acetate,methyl benzoate,benzoic acid cis-3-hexene ester,α-farnesene and methyl salicylate.The methyl benzoate content in petals of the living jasmine flowers or excised jasmine flower was not high.Germacrene was not detected in living jasmine flowers while detected in flowers in vitro.In the flowering process of flowers in vivo,the volatile aroma components increased with the petals flowering which reached the highest at 00:00 midnight.The total aroma components of excised jasmine flowers were higher than the flowers in vivo,and 65%of the aroma components reached the highest at the 8 hours of in vitro maintenance.2.Determine the best reference gene of the jasmine fluorescence quantitative PCR(Polymerase Chain Reaction)detection:three kinds of software were used which were Genorm,Bestkeeper and NormFinder to fish 13 housekeeping genes from the transcriptome database of jasmine,synthesize the cDNA(complement DNA)first strand using the total RNA extracted from the roots,the stems,the leaves and the petals in different periods of jasmines as template,make the real-time quantitative PCR detection and have the stability evaluation on the detection results.The results show that JsTub2 is the best reference gene of the jasmine in real time quantitative PCR analysis.3.Some differential genes related to fragrance in process of opening and releasing of fragnances from petals of jasmine were obtained by construction and screening of DSN mediated-suppressive subtracted cDNA library:optimum DSN concentration of treatment was 1/4 U,optimum cycling number of amplification of differential cDNAs was 19,the size of amplified products distriduted from 750bp and 2000bp,The dispersion did not appear bright band which means DSN treatment can remove cDNA of high expression and homologous genes effectively to achieve the effect of subtraction and homogenization.The results of electroporation showed that the titer of constructed cDNA library of suppression subtractive hybridization was 1.5×106cfu/mL.The results of bacterial liquid PCR showed that every clone had inserts,and the size of the inserts was at the range of 750bp-2000bp,and the average size of the inserts was 1000bp.The library was well represented which covered the size range of full length cDNA of most plant genes.142 positive clones were screened by reverse Northen-Blotting technology,and the positive rate was 29.89%.The results of the positive clone sequencing showed that 131 of 139 effective nucleotide sequences were not repeated accounted for 94.2%while 8 repeated making the redundancy rate to 5.7%.The cluster analysis of obtained 131 differential genes by BLAST2GO showed that 47.3%of the genes participated in cell composition,26.7%of them participated in molecular function and 26%of them participated in biological processes.Comparing the 131 differential genes with Arabidopsis thaliana database and NR database,4 genes directly related to aroma metabolism which were terpene sythases(TPS),germancrene synthase(GDS),deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)and benzyl alcohol benzene acetyl transferase(BEBT),5 CONSTANS-LIKE(CO)which participated in photoperiod controlling flowering and 7 genes related to hormonal regulation and response in which 5 genes were ethylene-responsive transcription factors were found.4.Bioinformatics analysis and expression of 4 differential genes related to aroma(JsDXR、JsGDS、JsTPS、JsBEBT):The positive clones were sequenced and showed that the full length cDNA of JsDXR(1641 bp)contains a 1436 bp open reading frame(ORF),encoding a protein of 461 amino acid,share 88%homolog with Camellia sinensis,Sesamum indicum,Solanum lycopersicum,Eucommia ulmoides,Actinidia argute,result of real-time fluorescent quantitative PCR showed that JsDXR kept the low expression at 18:00 and then increased at 20:00 to express the highest amount,2 times than 18:00,gradually declined after 20:00.JsDXR relative expression in excised 0 h is low,then continue to rise till reached the highest at 16 h was 10 times higher than 0 h;the full length cDNA of JsGDS(1773 bp)contains a 1658 bp open reading frame(ORF),encoding a protein of 552 amino acid,share 54%homolog with Sesamum indicum,Nicotiana tomentosiformis,Vitis vinifera,result of real-time fluorescent quantitative PCR showed that JsGDS expression reached the highest at 22:00,which was 9 times than 18:00 in the process of jasmine flowers open,then fell slightly but still higher than closed stage;In excised jasmine flowers curing process,the expression of JsGDS amount reached the highest in 4 h is 8 times of closed stage,then decreased;the full length cDNA of JsTPS(1884 bp)contains a 1491 bp open reading frame(ORF),encoding a protein of 552 amino acid,share 78%homolog with Osmanthus fragrans,75%with Olea europaea,51%with Solanum lycopersicum,result of real-time fluorescent quantitative PCR showed that JsTPS remained minimum level when the flower was not opened(18:00),and then came to a high level,reached the highest at 22:00 period about 75 times than initial stage;In excised jasmine flower,the expression of JsTPS showed a trend of rapid increase and reached the highest at 8 h,it was 19 times high than oh,after that fell a little;the full length cDNA of JsBEBT(1924 bp)contains a 1404 bp open reading frame(ORF),encoding a protein of 467 amino acid,share 72%homolog with Nelumbo nucifera,Cucumis melo and Solanum pennellii,result of real-time fluorescent quantitative PCR showed that JsBEBT expression was the minimum in the not open stage,and reached the highest expression at 20:00 of 35 times higher than the closed flower,after that the transcript level went down but still maintain at a high level;In vitro process of excised jasmine flower,the expression of JsBEBT quantity was upregulated obviously at 4 h,reached the highest,2.8 times of 0 h in vitro,then decreased. |
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