Explore The Anthocyanin Synthesis Mechanism Of Colored Potatoes Based On Full-length Transcriptome Sequencing And Extensive Targeted Metabolome

Potato is an important agricultural crop in the world.The reason why plants show different colors has always been the focus of research in the field of plant science.The synthesis and accumulation of secondary metabolites such as anthocyanins is an important reason for plants to exhibit different colors.The anthocyanin synthesis regulatory pathway and the mechanism of anthocyanin synthesis in potato tubers have been studied in depth.However,in the case of potato,a crop that harvests underground tuber parts,there is very little research on the anthocyanin synthesis and regulation mechanism in potato leaves,which is the main organ for anthocyanin synthesis,which senses light signals.In addition,whether there is a molecular mechanism in potato such as transcript fusion and other transcriptome-level regulation of anthocyanin accumulation is also worthy of further study.In this study,the bioinformatics method was used to identify R2R3-MYB and WD40 family genes related to potato anthocyanin synthesis,and evolutionary analysis was performed.Then the potato varieties Shepody,Red Rose 3 and Purple Rose 2 with green,red and purple leaves as testmaterials were used as testmaterials.The potato leaves of plant tissues,which are both anthocyanin-synthesizing organs and anthocyanin-accumulating organs with extremely active biological metabolism,were selected for nanopore full-length transcriptome sequencing and extensive targeted metabolome analysis.The nanopore full-length transcriptome sequencing was used to supplement and improve the existing annotation information of the potato genome.Combined with the results of transcriptomics and metabolomics experiments,the mechanism of anthocyanin synthesis and accumulation in potato leaves of different colors was studied in depth.This study lays a solid theoretical foundation for the study of potato anthocyanin synthesis and regulation related functional genes,the discovery of potato specific germplasm resources,and potato molecular design breeding.Through the above research,the following results are obtained:(1)A total of 101 potato R2R3-MYB family genes and 33 WD40 family genes were identified by regrouping and reanalyzing the original data of the potato transcriptome in the NCBI-SRA database.Semi-quantitative RT-PCR and q RT-PCR experiments showed that the R2R3-MYB family gene stAN1,WD40 family gene stTTG1-3 and CCS52 B all showed a significant upward trend with the deepening of potato leaves and tubers.The key regulatory gene for anthocyanin synthesis,PAL,also showed that the gene expression increased as the color of potato leaves and tubers deepened,but the increase in expression was not significant.Among them,stAN1 n is mainly expressed in potato roots,stems and tubers.The evolutionary analysis of the WD40 gene family shows that the WD40 family genes related to color change were mainly selected by environmental factors in ancient times.With the passage of time,purification selection also plays an increasingly important role.(2)By applying nanopore,a third-generation nucleic acid sequencing technology,transcriptome sequencing of potato leaves of different colors was conducted to study the physiological mechanism of potato anthocyanin synthesis and color changes.The nanopore full-length transcriptome sequencing data was analyzed,and the annotation information of the potato genome was supplemented and improved.The annotation information of the potato genome was optimized to 2360 loci,and 1833 new gene loci were detected.114 genes with significant differential expression were detected in the three color potato leaves.Lnc RNA1 a,Lnc RNA2,Lnc RNA3,Lnc RNA1 b,Lnc RNA4 target gene is PAL gene,Lnc RNA6 target gene was F3H;PONTK.938 gene and F3’5’H gene transcript fusion;DFR gene existed in color potato two kinds of m RNA splicing forms DFRa and DFRb.Combining with the results of Nanopore full-length transcriptome sequencing,a new transcript stAN1 n of R2R3-MYB family gene stAN1 was cloned.Overexpression of stAN1 n in tobacco makes the transgenic tobacco purple.Subcellular localization experiments show that stAN1 n functions in the nucleus,and the PAL gene mainly exerts its biological function in the cell membrane.(3)Extensive targeted metabolome analysis was performed on potato leaves of different colors,and the results of extensive targeted metabolome analysis were used to verify the results of nanopore full-length transcriptome analysis,and to understand the synthesis mechanism of potato anthocyanins.Through extensive targeted generation test,a total of 80 metabolites with significantly different contents in three different color potato leaves were found.In the comparison of the differential metabolites of Red Rose 3 and Purple Rose 2potato leaves,it was found in cyanidin 3-O-galactoside,pelargonin,hydroxymethoxycinnamate,pelargonidin 3-O-glucoside accumulated in large amounts in Red Rose 3potato leaves;but in Purple Rose 2 potato leaves,metabolites such as malvidin 3-Ogalactoside,malvidin 3-O-glucoside,petunidin 3-O-glucoside,cyanidin 3-O-glucoside,delphinidin 3-O-glucoside and tricin 5-O-hexosyl-O-hexoside were mainly accumulated.The synthesis and accumulation of anthocyanins in colored potato leaves resulted in a significant reduction in the content of caffeic aldehyde and coniferyl alcohol,which inhibited the accumulation of lignin in colored potatoes.At the same time,the changes of the metabolite content determined by the extensive targeted metabolome were consistent with the regulation of gene expression measured by the full-length transcriptome.(4)The nanopore full-length transcriptome sequencing results and the results of the extensive targeted metabolome testwere used for joint analysis to explain the colorful molecular regulation mechanism of potato leaves.Lnc RNA1a(PONTK.13936.1)and Lnc RNA1b(PONTK.13936.3)are anti-sense lnc RNA,which interact with PAL gene in cis.In the Red Rose 3 potato leaves,the F3’5’H and CYP75B1 genes were significantly up-regulated;at the same time,the expression of DFRa type transcripts of the DFR gene was more than that of the DFRb type transcripts retained by alternative splicing introns.This caused the Red Rose3 potato leaves to accumulate a large amount of pelargonidin 3-O-glucoside,cyanidin 3-Ogalactoside and pelargonin.In Purple Rose 2 potato leaves,F3’5’H gene was significantly upregulated,but the expression of CYP75B1 did not change significantly;only in the Purple Rose2 leaves were F3’5’H and PONTK.938 transcript fusion,this phenomenon was not found in the other two potato varieties;the expression of DFRb in the leaves of Purple Rose 2 was higher than that of DFRa,which was the opposite of Red Rose 3.The above-mentioned regulatory mechanisms were only detected in the leaves of Purple Rose 2 potato,so that Purple Rose 2 contained pelargonidin,cyanidin and delphinidin.Among them,metabolites in the form of delphinidin glycosides such as petunidin 3-O-glucoside,malvidin 3-O-glucoside,delphinidin 3-O-glucoside were mainly abundant in Purple Rose 2 potato leaves,resulting in purple leaves of purple rose 2 potatoes.In Red Rose 3 leaves,cyanidin mainly accumulates in the form of cyanidin 3-O-galactoside,in Purple Rose 2 leaves,cyanidin mainly accumulates in the form of cyanidin 3-O-glucoside.