Xiling Wang

Isolation and expression analysis of anthocyanin biosynthetic genes in Morus alba L.

Anthocyanins from mulberry fruits are used in medicine. However, little anthocyanin can be detected in other tissues and sometimes also mulberry fruits are colorless. The aim of this study was to investigate which gene or genes have the strongest correlation with the anthocyanin biosynthesis. The expression of several anthocyanin synthesis genes were determined in different tissues of two white and two purple fruit cultivars. Genes encoding dihydroflavonol reductase (MaDFR) and anthocyanidin synthase (MaANS) showed a high expression only in fruit tissue of purple-fruit cultivars. During the development of mulberry fruits, the anthocyanin content was well correlated with the transcripts abundance of MaDFR, MaANS, and MaCHS (encoding chalcone synthase). The skin of female mulberry flowers turns red under irradiance because of up-regulated expressions of MaCHS, MaDFR, and MaANS. These three genes may control the anthocyanin biosynthesis in mulberry and up-regulation of them may greatly increase the anthocyanin content.

Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1–methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

Characterization of Stilbene Synthase Genes in Mulberry (Morus atropurpurea) and Metabolic Engineering for the Production of Resveratrol in Escherichia coli

Stilbenes have been recognized for their beneficial physiological effects on human health. Stilbene synthase (STS) is the key enzyme of resveratrol biosynthesis and has been studied in numerous plants. Here, four MaSTS genes were isolated and identified in mulberry (Morus atropurpurea Roxb.). The expression levels of MaSTS genes and the accumulation of trans-resveratrol, trans-oxyresveratrol, and trans-mulberroside A were investigated in different plant organs. A novel coexpression system that harbored 4-coumarate:CoA ligase gene (Ma4CL) and MaSTS was established. Stress tests suggested that MaSTS genes participate in responses to salicylic acid, abscisic acid, wounding, and NaCl stresses. Additionally, overexpressed MaSTS in transgenic tobacco elevated the trans-resveratrol level and increased tolerance to drought and salinity stresses. These results revealed the major MaSTS gene, and we evaluated its function in mulberry, laying the foundation for future research on stilbene metabolic pathways in mulberry.

Cloning, Overexpression, and Functional Characterization of a Phytase from the Genus Bacillus

Bacillus strain WYCQ02 was obtained from soil samples by high-temperature screening. A 1,327-bp DNA fragment containing a 1,152-bp long open reading frame named phyC-WYCQ02 was amplified from the genomic DNA of strain WYCQ02 by PCR. The ORF encoded a polypeptide of 383 amino acid residues with a putative signal peptide of 26 amino acids. The 1,089-bp fragment encoding the mature peptide of neutral phytase and a 6 × histidine tag was cloned into the plasmid pPIC9K. The expression vector, pPIC9K-phyC, was linearized and transformed in Pichia pastoris. The molecular weight of phytase was estimated to be approximately 53 kDa by electrophoresis. The optimal temperature of the purified phytase was 55°C and the optimal pH value was between 7.0 and 8.0. After incubation at 70°C for 10 and 30 min, the relative activity was still over 80 and 62%, and over 70% of enzyme activity remained in the pH range of 5.0-10.0. There was no significant difference in enzymatic activity after incubation for 30 or 60 min in buffer with different pH values, therefore the purified phytase had some acid and alkali resistance. The phytase gene and the engineered yeast strain may have value in industrial applications.

Isolation and characterization of a novel chalcone synthase gene family from mulberry

Chalcone synthase (CHS) is the pivotal enzyme that catalyzes the first committed step of the phenylpropanoid pathway leading to flavonoids. Here, five CHS genes were determined in mulberry (Morus atropurpurea Roxb.). Interestingly, phylogenetic analysis tended to group three MaCHSs in the stilbene synthase (STS) family and initially annotated these as MaSTSs. A co-expression system that harbored a 4-coumarate:CoA ligase gene and one of the candidate genes was established to determine the functions of this novel gene family. The fermentation result demonstrated that MaSTS in fact encoded a CHS enzyme, and was consequently retermed MaCHS. Tissue-specific expression analysis indicated that MaCHS1/MaCHS2 was highly abundant in fruit, and MaCHS4 had significant expression in root bark, stem bark and old leaves, while MaCHS3 and MaCHS5 were more expressed in old leaves. Subcellular localization experiments showed that MaCHS was localized to the cytoplasm. Transcription levels suggested MaCHS genes were involved in a series of defense responses. Over-expression of MaCHS in transgenic tobacco modified the metabolite profile, and resulted in elevated tolerance to a series of environmental stresses. This study comprehensively evaluated the function of MaCHS genes and laid the foundation for future research on MaCHS in mulberry.

Screening, cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis, Ciboria shiraiana.

A cellulase gene (KJ700939, CsCelA) from Ciboria shiraiana that is highly expressed during the infection of mulberry fruit was screened by quantitative real-time PCR (qRT-PCR). Using cDNA isolated from infected mulberry fruits as template, the full-length 1170-bp sequence of CsCelA was obtained, which encodes a 390-amino acid protein with a putative signal peptide of 24 amino acids. The 998-bp fragment encoding the mature peptide of CsCelA was cloned into the multiple cloning site of the pPIC9K vector and overexpressed as an active protein of 55.3kDa in the methylotrophic yeast Pichia pastoris. The specific activity of induced supernatants of the recombinant cellulase (CsCelA) was 17.44U/ml and 135U/g for freeze-dried powder. The Kmax and Vmax of CsCelA for sodium carboxymethylcellulose (CMC) were 4.6mg/ml and 107.2U/mg, respectively. The supernatant and freeze-dried powder of the recombinant cellulase exhibited stable activity from pH4.0 to 9.0, and at temperatures ranging from 30°C to 55°C. Finally, the activity of the recombinant cellulase was assessed by enzymatic hydrolysis of the cell walls of mulberry leaves. CsCelA showed an endo-cellulase mode of cleavage, as assessed by thin layer chromatography (TLC).

Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.)

Abstract1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACSor ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and bothof them were strongly up-regulated by abscisic acid (ABA) and ethephon.