Analysis of AN2 Upstream Promoter Activity Based on Tail-PCR
Lycium ruthenicum is rich in anthocyanin,and AN2 is the main gene regulating anthocyanin anmeta-bolism.In order to analyze the activity difference of AN2 gene promoter,the upstream sequence of AN2 gene start codon about 1686 bp(LrAN2p)and 1495 bp(LbAN2p)of L.barbarum were cloned by Tail-PCR method,respectively.Plant CARE predicted that there were 133 and 137 cis-acting elements in sequences of LbAN2p and LrAN2p,respectively,among which 11 and 15 cis-acting elements were involved in photoregulation,and 13 and 16 cis-elements involved in hormone response,respectively.The plant expression vectors pKGWFS7:LbAN2p and pKGWFS7:LrAN2p were constructed,and the transgenic tobacco was obtained by using Agrobacterium-mediated tobacco genetic transformation system.The results of GUS staining showed that LrAN2p could drive the expression of GUS in tobacco,and the leaves were blue,with stronger starting activity than LbAN2p.The results of qRT-PCR showed that GUS gene in LrAN2p transgenic tobacco had high transcription level,which might lead to high expression of AN2 gene in L.ruthenicum,and activating the anthocyanin anabolic pathway.Thus,these would provide a theoretical basis for understanding the mechanism of fruit color formation and AN2 gene expression.
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