Selection Of Reference Genes In Morinda Officinalis How.and Excavation Of Related Genes In Anthraquinones Biosynthesis

Morinda officinalis How.is a dicotyledonous herbaceous plant with dry roots as the main medicinal site.Morinda officinalis How.and Areca catechu L.,Alpinia oxyphylla Miq.and Amo mum villosum Lour.are also known as the four major southern medicines in China.It was included in the "Shen Nong Ben Cao Jing" and was listed as a top grade.It tastes sweet,pungent,lukewarm.Function for kidney-replenishing,strong bones and muscles,antimutagenesis,anticancer,strengthen intelligence,anti-aging,diuresis pl diureses and diarrhea.Morinda officinalis How.has been used for impotence,uterus cold and infertility,rheumatic arthralgia,irregular menstruation,the lower abdomen pain and chills and tendon weakness in clinical therapy.The main medicinal components of Morinda officinalis How.are anthraquinones,iridoids,and polysaccharides.In this study,the key genes of anthraquinone biosynthesis were explored by comparing the differences in the roots,stems and leaves of Morinda officinalis.High-throughput sequencing technology was used to sequence transcriptomes of roots,stems and leaves of Morinda officinalis,and the differentially expression genes were compared.The key enzyme genes related to anthraquinones biosynthesis were screened to study the medicinal value of Morinda officinalis’ roots as its main medicinal site.It lays the foundation for the research on the molecular aspects of the Morinda officinalis,and has corresponding theoretical significance and application value for the deeper development and research of the Morinda officinalis.The main results of this study are:1)High-throughput transcriptome sequencing was performed on RNA samples from the roots,stems and leaves of Morinda citrifolia through Illumina HiSeqTM 2000.Obtained 6.36G,7.99G and 7.18G data respectively.After rigorous screening,the root 42401952 reads,stem 53270758reads,and leaf 47777718 reads were obtained.A total of 249,557 transcripts were obtained by using Trinity(Grabherr et al.,2011)for the mixed splicing of root,stem,and leaf clean reads,and transcripts were hierarchically clustered using Corset(Nadia M Davidson,Alicia Oshlack,2014),yielding a total of 163,168 unigenes.These Unigenes were compared in seven databases:NR,NT,KO,SWISSPORT,PFAM,GO,and KOG.There were 122,349 Unigenes can be compared in at least one of the seven databases,accounting for 74.98%.By KEGG metabolic pathway analysis,there are 237 genes involved in the biosynthesis of anthraquinones.2)A total of 11051 differential genes were obtained by comparing the transcriptome data of the roots,stems and leaves of Morinda officinalis.Stem and root contrast,there were 3,256 differential genes,of which 1,204 were up-regulated and 2052 genes were down-regulated.Leaf and root contrasts,there are 7,049 differential genes,of which 3,165 are up-regulated and 3,884 are down-regulated.Leaf and stem contrasts,there were 746 differential genes,of which 352 were up-regulated and 394 genes were down-regulated.And then,they were proformed to GO functional analysis and KEGG enrichment analysis.3)Screening reference genes of Morinda officinalis How.,selecting 9 commonly used reference genes ACTIN,18S,24S,EF2,UBC,UBQ4-1,APTI,GAPDH,and EF1-a from the transcriptome of Morinda officinalis and analyzing their relatively expression level.Then analysis was performed by four methods:Delta Ct,GeNorm,NormFinder,and BestKeeper.Genes with relatively stable expression levels were obtained.Finally,the results of the four analysis methods were comprehensively analyzed using RefFinder.At last,the most stable genes GAPDH and UBC were selected as the reference genes.4)The FPKM value of the related enzyme Unigenes in the biosynthesis pathway of Morinda officinalis was analyzed by multiple test method.It was found that the expression level of MenB-1 in the root was 4 times higher than that in the leaf.The expression levels of APG1 and MenB-2 in their leaves were 2.9 times and 6.9 times higher than that in their roots.Using the reference genes to verify the expression level that in roots,stems and leaves of target genes MenB-1,APG1 and MenB-2 by qPCR.The results are consistent with the transcriptome data.The expression of MenB-1 in roots far exceeds the expression in leaves.MenB-1 is heavily active in the roots and is involved in the metabolic processes of anthraquinone biosynthesis.Therefore,MenB-1 is likely to be a key enzyme gene for anthraquinone biosynthesis.