Cloning And Expression Analysis Of Vc Biosynthesis-Related Genes And ERF In Citrus

Vitamin C (Vc), as a well-known abundant antioxidant and enzymatic cofactors, plays crucial roles in maintaining human health, plant development and stress tolerance. Ethylene-responsive element binding factors (ERFs), are a member of the AP2/EREBP gene family of plant transcription factors. They have been found to be mainly involved in response to biotic and abiotic stresses and play a important role in plant development progresses.In the present study, five genes involved in vitamin C biosynthesis and ERF genewere isolated from the Citrus unshiu; ascorbate accumulation and expression profiles of these genes were investigated during fruit developing in fruit pulp of'Egan No.2' Satsuma mandarin and'Newhall'orange. In addition, we also investigated the effects of girdling on citrus ascorbate accumulation and genes'expression during fruit ripening in pulp of Satsuma mandarin. The main results were as follows:1. Five genes involved in L-galactose pathway and ERF gene were isolated from the citrus unshiu and the primary information of these genes was listed in the following: GDP-D-mannose pyrophosphorylase (CitGMP) has an open reading frame of 1,086bp, encoding 361amino acids and its GenBank accession number is HQ224946; GDP-Man-3', 5'-epimerase(CitGME), GenBank accession number is HQ224947, has anORF of 1, 134bp, encoding 377 amino acids; GDP-L-galactose phosphorylase (CitGGP), has an ORF of 1,368bp, encoding 455 amino acids and its GenBank accession number is HQ224948; L-galactose-1-P phosphatase(CitGPP) GenBank accession number is HQ224949, has an ORF of 813bp, encoding 270 amino acids and its; L-galactose dehydrogenase(CitGDH), has an ORF of 954bp, encoding 317 amino acids and its GenBank accession number is HQ224950. Citrus Ethylene-responsive element binding factor(CitERF) has an ORF of 874bp, encoding 207 amino acids and its GenBank accession number is HM064500. Further analysis indicated that predicted amino acid sequences of these genes showed high homology with their respective gene in other plant species.2. Ascorbate accumulation in pulp of'Newhall'were higher than those in'Egan No.2' fruits; Gene expression by real-time PCR revealed that CitGME, CitGGP, CitGDH and CitGLDH transcripts in 'Newhall'increased roughly during the whole experimental period which were higher than those in'Egan No.2'.3.'Guoqing No.1'Satsuma mandarins(C.unshiu cv. Guoqing No.1) was chose for girdling treatment at fruit green mature stage. As compared to control, it was found thatVc contents in fruit pulp were increased by BG (branch girdling), but those contents were decreased after FGD (fruit-bear-shoot girdling plus defoliation). Gene expression analysis indicated that transcripts of CitGME, CitGGP, CitGDH and CitGLDH were upregulated by BG treatment,which was positive correlated with Vc accumulation in this treatment;4. CitERF mRNA level in fruit pulp increased obviously by BG, but its expression be reduced significantly by FGD during fruit ripening, which was correlated with total soluble solid(TSS) accumulation in this treatment.