Isolation and Expression of Glucosinolate Synthesis Genes CYP83A1 and CYP83B1 in Pak Choi (Brassica rapa L. ssp. chinensis var. communis (N. Tsen & S.H. Lee) Hanelt)

Abstract

CYP83A1 and CYP83B1 are two key synthesis genes in the glucosinolate biosynthesis pathway. CYP83A1 mainly metabolizes the aliphatic oximes to form aliphatic glucosinolate and CYP83B1 mostly catalyzes aromatic oximes to synthesis corresponding substrates for aromatic and indolic glucosinolates. In this study, two CYP83A1 genes named BcCYP83A1-1 (JQ289997), BcCYP83A1-2 (JQ289996) respectively and one CYP83B1 (BcCYP83B1, HM347235) gene were cloned from the leaves of pak choi (Brassica rapa L. ssp. chinensis var. communis (N. Tsen & S.H. Lee) Hanelt) “Hangzhou You Dong Er” cultivar. Their ORFs were 1506, 1509 and 1500 bp in length, encoding 501, 502 and 499 amino acids, respectively. The predicted amino acid sequences of CYP83A1-1, CYP83A1-2 and CYP83B1 shared high sequence identity of 87.65, 86.48 and 95.59% to the corresponding ones in Arabidopsis, and 98.80, 98.61 and 98.80% to the corresponding ones in Brassica pekinensis (Chinese cabbage), respectively. Quantitative real-time PCR analysis indicated that both CYP83A1 and CYP83B1 expressed in roots, leaves and petioles of pak choi, while the transcript abundances of CYP83A1 were higher in leaves than in petioles and roots, whereas CYP83B1 showed higher abundances in roots. The expression levels of glucosinolate biosynthetic genes were consistent with the glucosinolate profile accumulation in shoots of seven cultivars and three organs. The isolation and characterization of the glucosinolate synthesis genes in pak choi would promote the way for further development of agronomic traits via genetic engineering.

Comparative Transcriptome Analysis between Broccoli (Brassica oleracea var. italica) and Wild Cabbage (Brassica macrocarpa Guss.) in Response to Plasmodiophora brassicae during Different Infection Stages

“…In the glucosinolate biosynthesis pathway, four cytochrome P450s were found to be down-regulated at T14 compared with T7, among which CYP79B2 and the homolog CYP79B3 catalyze the conversion of tryptophan to indole-3-acetaldoxime (IAOx) (Mikkelsen et al, 2000), and CYP83A1 and CYP83B1 are oxime-metabolizing enzymes (Zhu et al, 2012). Previous studies have found correlations between resistance and a low indole GSL content (Butcher et al, 1974; Ockendon and Buczacki, 1979; Chong et al, 1985), whereas Mullin et al (1980) were not able to correlate indole GSL and resistance to clubroot.…”

Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

“…In B . rapa , two CYP83A1 genes named BrCYP83A1-1 and BrCYP83A1-2 were respectively cloned from pak choi, but no functional analysis was conducted with the two genes [ 35 ]. Thiohydroximates are in turn S-glucosylated by glucosyltransferases of the UGT74 family, UGT74B1 and UGT74C1, forming desulfoglucosinolates in Arabidopsis [ 36 – 38 ].…”

Glucosinolate Accumulation and Related Gene Expression in Pak Choi (Brassica rapa L. ssp. chinensis var. communis [N. Tsen & S.H. Lee] Hanelt) in Response to Insecticide Application

“…The RNA extraction and RT-PCR procedure were performed according to our previously described protocols. 23 Briefly, total RNA was extracted from samples using TRIzol reagent RNAisoTM Plus (Takara, Japan) following the manufacturer's protocols. Transcription levels were analyzed by quantitative realtime PCR (qRT-PCR), and the gene-specific primer sets were designed as described previously.…”

“…Transcription levels were analyzed by quantitative realtime PCR (qRT-PCR), and the gene-specific primer sets were designed as described previously. 23 Gene expression was normalized using Brassica actin (EX087730) as the housekeeping gene. Real-time PCR reactions were performed in triplicate on an ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR Green qPCR kit (Takara, Japan).…”