Cloning And Specificity Analysis Of The Chinese Cabbage BrCYP79B2/B3 Promoter

Chinese cabbage(Brassica rapa ssp. pekinensis) is one of the leafy cruciferous vegetables. Glucosinolates were important secondary metabolites mainly found in cruciferous plants with multiple functions. In order to analyze the subfunctionalization of BrCYP79B2 (BrCYP79B2-1, BrCYP79B2-2, BrCYP79B2-3) and BrCYP79B3 genes in indolyl glucosinolates pathway, we cloned their promoter from Chinese cabbage ’chiifu’ leaves, constructed GUS expression vector, transformed into Arabidopsis and analyzed their differences in expression levels. The major results were as follows:(1) Defined the length and position of BrCYP72B2 and BrCYP79B3 promoter based on the Brassica Database and the BrGDB Query. Designed the specific primers by Primer Premier5 software.(2) Cloned promoters of BrCYP79B2 (BrCYP79B2-1, BrCYP79B2-2, BrCYP79B2-3) and BrCYP79B3. Analyzed those elements and found that those promoter sequences not only included core elements of TATA-box and GATA box, but also contained a number of specific elements, such as light-inducible element AE-box, hormone-inducible element CGTCA-motif, defense emergency mechanism components MBS and so on.(3) Successfully constructed plant expression vector pBI-B2-1, pBI-B2-3 and pBI-B3 harboring GUS report gene. They were into Arabidopsis thaliana by floring dipping method. GUS histochemical assays were performed for transgenic plants of T2 generation. Expression pattern of different periods and tissues were all analyzed.(4) GUS assay was performed for ProB2-1:GUS and ProB2-3:GUS transgenic plants after 3 h water treatment,2 h dry treatment,200 μM ethylene treatment for 2.5 h, or IAA (25 μM、50μM、100μM) treatment for 3 h. The results showed that ProB2-1:GUS transgenic plants can express in the apical under the water treatment. The expression can be obviously observed in the whole root under the dry treatment. While the gene can not express in the lower end of hypocotyl and cotyledon under the ethylene treatment. Root is most sensitive part under the Auxin treatment, especially in apical part. As the concentration increased, the expression in roots became lower. The maximum expression appear when the concentration is 25 μM. But in apical, the gene always expressed. In constract to other treatments, the expression of ProB2-3:GUS transgenic plants change largest under the ethylene treatment.(5) The effect of 200 μmol/L NaCl,200 μmol/L MeJA,1.5 μmol/L Kyn or 200 μmol/L AOA treatment on expression level of GUS driven by BrCYP79B2-1 and BrCYP79B2-3 promoters in transgenic plants were analyzed using qRT-PCR method. They usually decreased after the first lift, but the time for tip expression varied among treatments.