Molecular Mechanism Of Anthocyanin Biosynthesis In Tuber Epidermis Of Jerusalem Artichoke

Jerusalem artichoke(Helianthus Tuberosus L.)is also known as ginger and guizijiang,and its tubers are rich in nutrients such as s fructan.The tuber epidermis of Jerusalem artichoke is usually white,and the anthocyanin content of some varieties aggregates to produce a purple phenotype.The high anthocyanin content is not only conducive to improving stress resistance of Jerusalem artichoke,but also a special molecular genetic marker.At present,the key genes regulating anthocyanin biosynthesis of Jerusalem artichoke tuber epidermis have not been reported,and the relevant molecular mechanism is still unclear.In this study,the Jerusalem artichoke resources of QY1(purple epidermis)and QY3(white epidermis)were used as materials,using RNA-seq technology comparative analysis of violet skins Jerusalem artichoke and white skin all the transcript expression differences in Jerusalem artichoke,select candidate genes control the purple skin traits,and separation of cloning and functional verification,for resolving Jerusalem artichoke purple leather traits reliable molecular genetic basis.The main research results are as follows:(1)RNA-seq technology was used to sequence the epidermis of purple Jerusalem artichoke(QY1)and white Jerusalem artichoke(QY3).The sequenced Clean data and Raw data were assembled and spliced.After three repetitions,a total of 50.04 GB of raw data and 333.72 Mb reads were obtained.The splicing yielded 197769 Unigenes with an average length of 1140 bp and a total of 164780 single genes were annotated.55,354 Unigenes were differentially expressed in white and purple Jerusalem artichoke,of which 28,113 Unigenes were up-regulated in purple epidermis and27,241 Unigenes were down-regulated.The expression of all structural genes involved in anthocyanin biosynthesis was higher in purple epidermis than in white epidermis.Unigene44371?All(Ht MYB2),a transcription factor that regulates anthocyanin synthesis,is expressed in purple epidermis with a log2 Fold Change value of 8.07,and is hardly expressed in white epidermis.Therefore,Ht MYB2 is predicted to be a candidate gene that mainly regulates anthocyanin synthesis in purpleJerusalem artichoke epidermis.RT-PCR showed that most of the structural genes involved in the regulation of anthocyanin biosynthesis were up-regulated.Among them,the CHS,F3 H,and DFR genes had the highest up-regulation differences in the purple epidermis,and the results were consistent with transcriptome data.(2)Ht MYB2 gene is 1066 bp in total length and 744 bp in CDS length,encoding246 amino acids.The gene sequence contains 2 introns and 3 exons.The genome sequence of Ht MYB2 in QY1 and QY3 is identical to that of CDS.Sequence analysis revealed that Ht MYB2 was a hydrophilic protein.Ht MYB2 gene in phylogenetic tree is closely related to Ha MYB90 gene which regulates anthocyanin biosynthesis in sunflower.Ht MYB2 has a complete SANT / MYB Domain and MYB-like DNA-binding domain.(3)Construct a plant overexpression vector PJAM1502-Ht MYB2,and obtain an overexpressing Ht MYB2 tobacco transgenic line by means of Agrobacterium tumefaciens to obtain purple phenotype transgenic tobacco.Anthocyanin determination showed that the anthocyanin content of transgenic lines was significantly higher than that of wild-type tobacco.The expression levels of all the structural genes involved in the anthocyanin metabolism pathway in transgenic purple tobacco were significantly up-regulated,DFR was most significantly up-regulated,and Ht MYB2 expression was significantly different.Anthocyanin accumulation was found in root,stem,leaf,flower and tuber epidermis to varying degrees,and anthocyanin content in root and tuber epidermis of QY1 was significantly higher than that of QY3.The expression of Ht MYB2 gene in the root and tuber epidermis of QY1 was significantly higher than that of QY3,and its expression abundance was consistent with the concentration of anthocyanins in different tissues.(4)Ht MYB2 upstream promoter fragments isolated from QY1 and QY3 by tail-pcr were 1487bp(Ht MYB2p)and 1508bp(Ht MYB2w),respectively.Among them,QY3 has 21 nucleotide sequences more than QY1 promoter at-1360 bp ?-1342 bp.GUS transient expression verification of the promoter showed that Ht MYB2 p activity was stronger than Ht MYB2 w.Allelic mutation primers were designed with 21 bp difference,and allelic mutation scans were carried out in naturalpopulations with 90 copies of purple and 90 copies of white respectively.It was found that all purple epidermis had Ht MYB2 p characteristics.