Cloning And Activity Analysis Of The F3′5′H Gene Promoter From Lycium Ruthenicum Murr. In Qaidam Basin

Lycium ruthenicum Murr.mainly distributs in Qinghai,Ningxia,Xinjiang and other western remote places in China,it is an ideal plant to alleviate soil salinization and water conservation in northwest China.The large amount of anthocyanins contained in its black fruit has an important health care effect.Flavonoid-3’,5’-hydroxylase(F3’5’H)is a key enzyme for the synthesis of petunia pigment which make the fruits of Lycium ruthenicum Murr.black.To clarify the F3’5’H gene expression mechanism and the function of F3’5’H gene in the corlor formation of Lycium ruthenicum Murr.Fruit,determine F3’5’H gene promoter activity in Lycium ruthenicum Murr.and its albino fruit,reveal the color difference between Lycium ruthenicum Murr.and its albino fruit.In this study,fruits of Lycium ruthenicum Murr.was taken as the object,Lycium ruthenicum Murr.albino fruit was taken as a control,first the expression pattern of F3’5’H gene in both was determined,then the full length of the Lycium ruthenicum Murr.F3’5’H gene was cloned,the promoter of the gene in the Lycium ruthenicum Murr.and its albino fruit were cloned,then combined the promoters with the GUS reporter gene to construct the plant transient expression vector,and then transient transformed tobacco by Agrobacterium-mediated Method,histochemical staining was performed after tobacco incubation to determine the promoter activity.The results are as follows:1.The expression pattern of F3’5’H gene was determined,it showed that its expression in ripe Lycium ruthenicum Murr.fruit was significantly higher than that in albino fruits.2.The full length of the F3’5’H gene of the Lycium ruthenicum Murr.was cloned by RACE.The results showed that the total length of the F3’5’H gene was 1689 bp.The open reading frame is 1527 bp in length and can encode 508 amino acids.The characteristic of the amino acids and proteins it encodes was determined by bioinformatics analysis.3.The promoter fragment of the F3’5’H gene of Lycium ruthenicum Murr.and its albino fruit was cloned by 3 times of genome walking by Genome Walking Kit(TaKaRa)and sequenced.The bioinformatics analysis showed that the sequence homology of two promoters was 90.3%,the sequences were submitted to PlantCare to predict the promoter elements.The results showed that both promoters have a number of typical eukaryotic promoter elements TATA-Box and CAAT-Box.In addition,there were cis-acting element involved in defense and stress responsiveness TC-rich repeats,wound responsive element WUN-motif and a number of elements related to light response,Sp1,Box I,G-box and so on.Also there were cis-acting regulatory element required for endosperm expression skin-1 motif and anaerobic induction element ARE in both promoters.But in Lycium ruthenicum Murr.promoter,element involved in the MeJA-responsiveness TGACG-motif was predicted,which was not predicted in the albino fruit promoter.4.Combined 2 promoters with the GUS reporter gene to construct the plant transient expression vector,and then transient transformed tobacco by Agrobacterium-mediated Method,histochemical staining was performed to determine the promoter activity.The results showed that both promoters had activities.Then the GUS gene expression level was analyzed by Real-time PCR,the results showed that the GUS gene expression level driven by Lycium ruthenicum Murr.promoter was 3.09 times of its albino fruit.