Cloning and expression analysis of arginine decarboxylase gene (CrADC) from Citrus reticulata‘Shatangju’

【Objective】Shatangju (Citrus reticulata Blanco) is one of the most widely cultivated citrus in southern China and often encounters drought stress during cultivation. Polyamines can reduce drought damage by regulating stomatal closure and promoting root development. The arginine decarboxylase as a rate-limiting enzyme in polyamine synthesis, catalyzes conversion of arginine to putrescines, which is further converted into other polyamines. Therefore, in this study, arginine decarboxylase gene (CrADC) was cloned from Shatangju and its expression pattern was examined under drought stress, in order to provide understanding of the molecular mechanism regulating polyamines synthesis in drought resistance.【Methods】The cDNA sequence of CrADC was obtained by reverse transcription PCR (RTPCR). The coding sequences of CrADC was amplified from cDNA, then cloned into the vector pMD19-T and transformed into DH5α by heat shock. The DH5α was cultured overnight at 37 ℃, then DNA from the plasmid was extracted and sequenced after PCR identification. Bioinformatics tools were used to analyze the characteristics and evolutionary relationship of the CrADC protein. The quantitative real-timePCR (qPCR) was used to detect the expression level of the CrADC gene in different tissues (young leaves, old leaves, flowers, 30d fruit flesh, and 30d fruit peel) and at different times (0, 3, 6, 9, 12, 24, 36 h) after exposure to 10% PEG-6000 solution. Transgenic tobaccos were obtained by leaf disk transformation using Agrobacterium tumefaciens, and the expression level of the CrADC in the transgenic tobacco plants was detected by qPCR. Related physiological parameters, such as water loss (FL), electrolyte leakage (EL), malondialdehyde (MDA), and activities of catalase (CAT) and superoxide dismutase (SOD) were compared between transgenic lines (TL) and non- transgenic lines (CK) after drought stress.【Results】The cDNA sequence of the CrADC had 3076 bp including a 2262 bp open reading frame (ORF) encoding a protein with 753 amino acids. Bioinformatics analysis indicated the relative molecular weight of the CrADC protein was 80.84 ku; the theoretical isoelectric point was 5.13; the instability coefficient was 40.98; and the average hydrophilic coefficient was -0.009. The CrADC protein belongs to an unstable hydrophilic protein. There was no transmembrane domain in CrADC, and there was a pyridoxal binding domain (Orn_Arg_deC_N) between 139th and 414th amino acids. Pairwise sequence alignment of ADC protein sequences from 16 fruit trees species was performed. The results showed that the CrADC protein from Shatangju was highly similar to those of C. sinensis, C. clementina and C. trifoliata, with a sequence identity higher than 96.5%. The sequence identity was the lowest between CrADC and Musa acuminata ADC protein (62%). Phylogenetic analysis showed the amino acid sequences of ADC from the 16 fruit tree species were relatively conservative and could be divided into three clusters. Eight deciduous fruit species, such as M. domestica, Vitis riparia and Ziziphus jujuba, belonged to an evolutionary branch from temperate areas. Six fruit tree species, such as Citrus, Mangifera indica and Carica papaya, belonged to another evolutionary branch from tropical or subtropical areas. The results of qPCR showed the CrADC was expressed in leaves, flowers, fruit flesh and peel. The highest expression level of the CrADC gene was detected in fruit peel at day 30, and the lowest expression was detected in the old leaves. Furthermore, the expression level of CrADC gene in the peel at day 30 was 3.18 folds higher than that in the flesh. The expression level of CrADC in young leaves from spring was 3.41 folds higher than that in old leaves in winter. In total, the CrADC gene has expression specificity at different development stages. The expression level of CrADC gene obviously increased with the extension of drought treatment time, and the highest level was detected at 24 h and 3.82 folds higher than that at 0 h. Transgenic tobacco experiments showed that the CrADC gene was stably expressed in root, stem and leaf of transgenic tobacco. Transgenic physiological experiment showed the EL and MDA in transgenic tobacco were lower than in non-transgenic tobacco (CK), indicating that the cell membrane permeability of transgenic lines was lower than that of CK. The CAT and SOD in transgenic tobacco were higher than in CK, conferring higher ability in scavenging reactive oxidative species (ROS) to the transgenic plants. Therefore, the transgenic tobacco has greater drought resistance than CK.【Conclusion】The amino acid sequence of CrADC is relatively conservative, and the CrADC protein belongs to the evolutionary branch from the tropical or subtropical area. The CrADC gene has expression specificity at different development stages of Shatangju, and the expression level of the CrADC increases with the extension of drought, and the transgenic tobacco has greater drought resistance than non-transgenic tobacco.