| Zhe-Maidong?Ophiopogon japonicus?Linn.f.?Ker-Gawl.?,a famous traditional Chinese medicine,the planting period is usually 3 years,“Zhejiang Bawei”one.The tubers of Zhe-Maidong contain rich flavonoids,polysaccharides and saponins.It is found that Zhe-Maidong showed promising on the treatment for cardiovascular diseases,respiratory diseases,and regulation of immunomodulatory activity.The medicinal value of Zhe-Maidong has been realized since ancient times.Zhe-Maidong was used in the traditional decoction Maimendong Decoction and now developed modern Chinese medicine injection“Shenmai Injection”.The main results are as follows:?1?Different years of tubers of Zhe-Maidong were collected from the same field in Cixi,Zhejiang.After RNA sequencing,we obtain 16.4G datas,and ambigious sequence were filtered,and fowllowed by assembling steps,we got 96,738 Unigenes eventually,with the average of length of which is 605.7 bp and N50 length is 859 bp.The assembled sequences were compared with the NR,Swiss-Prot,NT,GO and KEGG database,and get 65,536,57,705,22,870,63,324,57,157 and 29,010 hits respectively in the mentioned database.DEGseq software package was applied to differential expression analysis,and found 9810 unigenes significantly differentially expressed between different years of Zhe-Maidong samples.SSR and SNP analysis showed a total of 25,140SSR markers from 20,981 transcripts.The three types of SSR markers:di-riucleotide repeats,mono-nucleotide repeats and tri-nucleotide repeats account for 97.56%of the total markers.252,831 SNP polymorphic site and 8238 In-Del loci were detected from 50566 transcripts by SNP and In-del analysis.Furthur analysis of 252,831 SNP polymorphic sites implicated that the transiton type accounted for 62%of the total variation,while the transversion types accounted for 38%.?2?The experiment determined the content of total flavonoids,methylophiopogonanone A,total saponins and total polysaccharides in different years of Zhe-Maidong.The results showed that the content of methylophiopogonanone A were 3.09×10-3%,3.95×10-3%and 4.37×10-3%for one year old Zhe-Maidong,two years old Zhe-Maidong and three years old Zhe-Maidong,respectively.The content of total flavonoids were 0.0280%,0.0393%and 0.0464%for one year old Zhe-Maidong,two years old Zhe-Maidong and three years old Zhe-Maidong,respectively.The content of total saponins was 0.0485%,0.0780%and 0.0965%for one year old Zhe-Maidong,two years old Zhe-Maidong and three years old Zhe-Maidong,respectively.The total polysaccharide was 5.44%,8.98%and 10.98%for one year old Zhe-Maidong,two years old Zhe-Maidong and three years old Zhe-Maidong,respectively.?3?Pathway analysis base on swiss-prot database annotation indicated 254 unigenes were related to flavonoids biosynthesis,135unigenes were related to saponins biosynthesis,236 unigenes were related to polysaccharides biosynthesis,respectively.The expression levels of above unigenes were normalized to FPKM for each sample.We found that most of unigenes which related to flavonoids and saponins biosynthesis are down-regulated with the years of growth,and most of unigenes related to polysaccharides biosynthesis are up-regulated at first but followed by a down-regulation.At last,17 unigenes were selected to RT-qPCR analysis,and 11 unigenes were found to be consistent with the change of FPKM value. |
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