Polygonum Tinctorium Lour Transcriptome Sequencing Used For Expression Analysis Of Genes Related To Indigo And Indirubin Biosynthetic Pathways

Polygonum tinctorium Lour is an important natural plant dyeing material,and it is also known as a precious Chinese herbal medicine.It is widespread in China and has abundant resources.The secondary metabolites,indigo and indirubin,are important materials for ethnic minorities in ethnic handicraft batik and tie-dye processing.In addition,indigo and indirubin have important pharmacological effects.In particular,indirubin-based drugs have significant anti-cancer,anti-viral and anti-tumor effects.These medicinal effects have been confirmed and recognized in traditional Chinese medicine and modern medicine.Experimental studies and practices have demonstrated that the content of indigo and indirubin in Polygonum tinctorium Lour are different at different growth stages.Sorting out the differential genes that regulate the contents of indigo and indirubin in the biosynthetic pathways has a significant effect on the abundance of genetic information of Polygonum tinctorium Lour and is of vital importance to the survival and development of Polygonum tinctorium Lour.This paper has conducted the following research on this issue.The research findings are as follows:1.Qualitative and quantitative indigo and indirubin in Polygonum tinctorium Lour in different growth stages(July,August and October)by high performance liquid chromatography.The chromatographic conditions were determined in the experimental process:the detection wavelength range of indigo and indirubin was 260-290 nm,the elution conditions were confirmed as methanol:water=70:30,the flow rate was 1cm~3/min,and the column temperature was chosen as 30°C.By comparing the spectrum of the sample to be measured with the standard spectrum,the retention times of indigo and indirubin were determined to be 12.5 min and 21.5 min,respectively.By fitting the regression equation and drawing a standard curve,quantitative analysis was performed on indigo and indirubin.It was found that the content of indigo and indirubin had the same changing tendency.Both of the content of indigo and indirubin was the highest in August,followed by October and lowest in July.The content of indigo in August was26.3%higher than that in October,57.2%higher than that in July;the content of indigo in August was 14.6%higher than that in October,24.0%higher than July.At the same time,the accuracy and reliability of the experiment are ensured by the determination of stability and precision experiments.The RSD of the indigo and indirubin standards in the stability experiments were 1.24%and 1.97%,respectively,and the RSD in the precision experiments were 1.18%and 1.24%,respectively.The results demonstrate that the results of high performance liquid chromatography established by this work have high accuracy and good reproducibility,and are suitable for the quantitative analysis of indigo and indirubin in Polygonum tinctorium Lour.2.Based on the above work,transcriptome sequencing of Polygonum tinctorium Lour at three different growth stages was performed.714,264 transcripts and 267,093Unigenes were obtained through transcript spilicing.The length of transcripts and Unigene are concentrated between 200-400 bp.51.6%of Unigenes matched at least to a database by the transcripts annotation,which greatly enriched the Polygonum tinctorium Lour transcriptome information.Through data mining,the correlation coefficient between the samples within the groups and among groups was obtained.It was found that the biological replicates within the specific group were good,and the differences between the samples among the groups were large,which conformed to the basic scientific laws.In the analysis of differential gene expression,indigo in August was selected as the control group.Compared to August samples,there were 3,717up-regulated genes and 3,455 down-regulated genes in the month of July and there were 7,581 up-regulated genes and 5,220 down-regulated in the month of October relative to August.By analyzing the differential expressed genes of indigo at different growth stages and combining with the previously study of indigo and indirubin biosynthetic pathways,it was found that the genes that contribute to the difference between indigo and indirubin may be involved in the synthesis of cytochrome P450monooxygenase.TRINITY_DN59463_c0_g1 and TRINITY_DN59463_c0_g1 were found to be involved in the synthesis of cytochrome P450 monooxygenase,and the change trend of gene expression was consistent with that of indigo and indirubin.Therefore,it can be inferred that these two genes are the key genes that cause differences in indigo and indirubin content.