Molecular Mechanism Of Interaction Between SmBICs And SmCRY2 In The Regulation Of Anthocyanin Biosynthesis In Eggplant(Solanum Melongena L.)

Anthocyanins are natural pigments with strong antioxidant activity and highly beneficial for human health.Purple eggplant is one of the few vegetables rich in anthocyanins.Light is the predominant environmental factor affecting eggplant anthocyanin biosynthesis,but weak light conditions such as close planting and greenhouse cultivation often lead to poor coloring of photosensitive eggplants,which seriously affect the quality of eggplant.In the previous study,through a large number of bagging screening,non-photosensitive eggplant variety were obtained,shading almost did not affect its anthocyanin biosynthesis,but its internal regulation mechanism needs to be studied.In order to screen the key regulatory factors involved in the anthocyanin biosynthesis of non-photosensitive eggplant,this study adopted comparative transcriptome and functional analysis methods,and obtained the following research results:1.Through comparative transcriptome analysis,it was found that there were significant differences between photosensitive eggplant ‘Lanshan Hexian’ and nonphotosensitive eggplant ‘145’ in light response and anthocyanin biosynthesis.Some genes that may be involved in non-photosensitive eggplant anthocyanin biosynthesis were screened,and the regulatory roles of SmBIM1,SmAP2,SmHD,SmMYB94,SmMYB19,SmTT8,SmYABBY,SmTTG2 and SmMYC2 in anthocyanin biosynthesis of eggplant were proved.The flowers of two eggplant materials were bagged at full flowering stage.Two weeks later,the transcriptome of the pericarp at 0h,0.5h,4h,and 8h after bag removal were sequenced and analyzed.The transcriptome sequencing analysis showed that most of the anthocyanin biosynthesis structure genes were up-regulated in ‘Lanshan Hexian’,but down-regulated or remained unchanged in ‘145’ after bags removal,and most of the genes were highly expressed in ‘145’ under shading condition.According to the expression pattern of structural genes,regulatory factors that may be involved in anthocyanin biosynthesis in ‘145’ were screened.Furthermore,through transcription factor target gene prediction,yeast one-hybrid and DualLUC experiments,it was found that SmBIM1,SmAP2,SmHD,SmTT8,SmTTG2 and SmMYC2 could bind to the promoter of SmCHS and activate its expression,SmMYB94 and SmMYB19 could bind to the promoter of SmCHS and inhibit its expression,and SmYABBY could bind to the promoter of SmTT8 and activate its expression.At the same time,the interaction networks among these transcription factors were constructed to further clarify their regulatory functions in anthocyanin biosynthesis.2.Genetic transformation and VIGS transient transformation of eggplant showed that SmBIC1 and SmBIC2 negatively regulated the anthocyanin biosynthesis in eggplant.Furthermore,through yeast two-hybrid,bimolecular fluorescence complementary and protein degradation experiments,it was proved that SmBIC1 and SmBIC2 could negatively regulate the anthocyanin biosynthesis of eggplant by inhibiting the activity of SmCRY2.Studies have shown that At BICs can regulate the photosensitivity of Arabidopsis cells through CRY.Transcription analysis showed that SmBIC1 and SmBIC2 were significantly upregulated after the ‘Lanshan Hexian’ was exposed to light,but they were almost not expressed in ‘145’.Therefore,it is speculated that SmBIC1 and SmBIC2 may be related to the photosensitivity of eggplant anthocyanin biosynthesis.In this study,we found that SmBIC1 and SmBIC2 belong to nuclear proteins,which are mainly expressed in the anthocyanin-rich parts of photosensitive eggplant,and different light types affects their expression.Yeast onehybrid analysis showed that SmBIC1 could be bind to the light response elements of SmCHS and SmF3 H promoters.In SmBICs-silenced eggplants,more anthocyanins were accumulated,while SmBIC1-OE and SmBIC2-OE eggplants synthesized less anthocyanin,these plants also had longer hypocotyls.q RT-PCR also revealed that the anthocyanin structural genes,which were down-regulated in SmBIC1-OE and SmBIC2-OE lines,were up-regulated in SmBICssilenced eggplants.In addition,transcriptome sequencing of SmBIC1 and SmBIC2 overexpressed lines also showed that significant differentially expressed genes(DEGs)were mainly concentrated in the pathways relevant to light response and anthocyanin biosynthesis,and the structural genes SmCHS,SmCHI,SmDFR,SmANS,SmF3’5’H,transcription factors SmMYB1,SmTT8 and light signal transduction factors SmCOP1 and SmHY5,which were known to be involved in anthocyanin biosynthesis in eggplant,were down-regulated.Yeast two-hybrid,bimolecular fluorescence complementarity and protein degradation experiments showed that SmBICs could interact with light-activated SmCRY2,inhibit the activity of SmCRY2,down-regulate the expression of genes related to light signal transduction and anthocyanin biosynthesis,and negatively regulate anthocyanin biosynthesis in eggplant.In summary,this study shows that the biosynthesis of large amounts of anthocyanins in non-photosensitive eggplants under shading conditions may be related to the regulation of SmBIC1,SmBIC2,SmBIM1,SmAP2,SmHD,SmMYB94,SmMYB19,SmTT8,SmYABBY,SmTTG2,SmICE1 and SmMYC2.