| Real-time quantitative PCR (qPCR) is a new type of nucleic acid quantitative technique which developed on the basis of traditional PCR technique, it is widely used in gene expression analysis with advantages of high sensitivity, specificity, rapidity, quantification and accurately. In order to remove some of the differences between the types of tissue or cell sample may exist on yield, quality and efficiency of the reverse transcription of RNA, and then obtain the target gene-specific expression of the true differences. It is usually need to use the express relatively stable housekeeping gene as a reference gene, correction and standardization of the target gene. An ideal reference gene is expressed stable or relatively stable whether the role of the various test environment, and factors influencing or constant or in different types of tissue or cell. However, a lot of research results show that no one reference gene expression is very stable or rather stable. It should be verified reference genes when doing target gene expression analysis before, so it need to select one or two more stablereference genes for correcting purpose gene expression to gain more believable results.Atropa belladonna is the only commercial plant species authorized by pharmacopoeia of the the people’s republic of China. So far, there was no report about selection of the reference genes in A. belladonna. In this study, A. belladonna as the research object, it was subjected to various environmental stresses, including4℃,42℃, methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), NaCl and UV-B treatments, respectively. The expression of eight potential reference genes are selected, including18S, GAPDH, ACT, TUB, PGK, EF-1α, CYC and FRU, which come from different organs (root, stem and leaf) of A. belladonna. In order to screened more stable reference genes and make sure the number of reference genes, it analyzed by geNorm, NormFinder and BestKeeper software. Then, it can give more accurate target genes for the expression analysis of A. belladonna, and also provided guidelines for reference gene selection in other solanaceae plants.This study suggested that:PGK was more stable in A. belladonna with different organs (untreated) or variety of treating by geNorm, NormFinder and BestKeeper analysis, so PGK will be a reliable reference gene when qPCR analysis on target gene’s expression in future. And then, besides the heat-stressed analysis by geNorm in root, the V2/3is higher than0.15, all other pairwise value V2/3is lower than0.15, which it suggested that two reference genes is need when qPCR analysis target gene’s expression of A. belladonna. However, the sond reference gene selecting with specific conditions.Hyoscyamine and scopolamine belong to the tropane alkaloids, are the anticholine drugs with commonly and widely use in clinical, the need demand of market is large. Scopolamine is much more useful and valuable because of its higher physiological activities and fewer side-effects, so the need demand of market is larger. However, scopolamine contents in the wild type A. belladonna was much lower than hyoscyamine, only0.01%～0.08%of dry weight, so it makes the price of scopolamine is expansive. In order to improve the TAs content in A. belladonna, metabolic engineering is a significant method, which can cultivate high-yield scopolamine source plant. Now the analysis of biosynthetic pathways of TAs is clear, but there were no relative reports about the tropinone reductase Ⅱ (TR Ⅱ) gene cloning and metabolic engineering research in A. belladonna. Tropinone reductase Ⅱ is the control enzyme in TAs biosynthetic pathway on the branch, which can impact the TAs metabolism shunt. This study utilized RACE method cloning the TRⅡ gene cDNA full-length in A. belladonna, and conducted a detailed analysis of bioinformatics. Then it is useing the sutable reference genes to analyze the AbTRⅡ expression in different plant tissuees and different treatmed hairy root.The cDNA of A. belladonna TRⅡ gene (accessing number:KC012910) is1124bp and contains a length of783bp of CDS,5’UTR of64bp and64bp in3’UTR,12bp’s polyA, and the TAA as termination codon. AbTRⅡ encoded a protein containing260amino acid sequence (AbTRⅡ), the similarity were over93%compared with other reported TRⅡs amino acid sequence in solanaceae plant. Evolutionary tree analysis showed that AbTRⅡwith TRⅡ in differnt genus Hyoscyamus niger had the nearest genetic relationship. AbTRⅡ proteins and reported from the D. Stramonium and H. niger TRⅡ both belong to short-chain dehydrogenase/reductase (SDR) family, its C side includes SDR needed NADP+binding sites (Ile192, Thr194and Ser195), and substrate tropinone binding sites are mainly in the C terminal (Glu156Val191and Leu210).A. belladonna of wild type genes expression analysis showed TRⅡ in each parts have expressed, but expression abundance of each are not identical, it is mainly expressed abundant in branch root, followed by old leaves. A. belladonna in blank hairy root induction expression analysis showed that the expression of AbTRⅡ up regulation by UV-B induced, compared with control has a significant difference (p<0.05). But the expression of AbTR Ⅱ down regulation by ABA and SA induced, compairing with EtOH control has a significant difference (p<0.05).The contents of scopolamine and hyoscyamine in belladonna hair roots suggested that the content of scopolamine up to0.88mg.g-1DW when treated by ABA, which relative to blank control (0.12mg·g-1DW) was the most obvious, it enhanced6.3times compared with blank control. And it also enhanced2.1times compared with EtOH treatments.Gene expression analysis of AbTT Ⅱ and the scopolamine contents of hair root analysis after elicitor treatment will help us select suitable inducer for treating A. belladonna plant to improve its content of scopolamine. |
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