Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression of chsA gene in transgenic Petunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal of chsA gene, and transferred the fusion gene into Petunia hybrida via Agrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNA in situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.

The Influence of Co-Suppressing Tomato 1-Aminocyclopropane-1-Carboxylic Acid OxidaseⅠon the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase Ⅰ on the expression of fruit ripening-related and pathogenesis-related protein genes, and on the biosynthesis of endogenous ethylene and storage ability of fruits. Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l-aminocyclopropane-1-carboxylic acid oxidase 1 gene （LeAC01）, 1- aminocyclopropane-l-carboxylic acid oxidase 3 gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5）, and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato Ailsa Craig （AC＋＋） and the LeAC01 co-suppression tomatoes （V1187 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carded out. The hybridization results indicated that the expression of fruit ripening-related genes such as LeACO3 and LeEBF1, and pathogenesis-related protein genes such as LePR1, LePR5, and LeNP24, were reduced sharply, and the ethylene production in the fruits, wounded leaves decreased and the storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.

Identification of microRNAs and messenger RNAs involved in human umbilical cord mesenchymal stem cell treatment of ischemic cerebral infarction using integrated bioinformatics analysis

In recent years,a large number of differentially expressed genes have been identified in human umbilical cord mesenchymal stem cell(hUMSC)transplants for the treatment of ischemic cerebral infarction.These genes are involved in various biochemical processes,but the role of microRNAs(miRNAs)in this process is still unclear.From the Gene Expression Omnibus(GEO)database,we downloaded two microarray datasets for GSE78731(messenger RNA(mRNA)profile)and GSE97532(miRNA profile).The differentially expressed genes screened were compared between the hUMSC group and the middle cerebral artery occlusion group.Gene ontology enrichment and pathway enrichment analyses were subsequently conducted using the online Database for Annotation,Visualization,and Integrated Discovery.Identified genes were applied to perform weighted gene co-suppression analyses,to establish a weighted co-expression network model.Furthermore,the protein-protein interaction network for differentially expressed genes from turquoise modules was built using Cytoscape(version 3.40)and the most highly correlated subnetwork was extracted from the protein-protein interaction network using the MCODE plugin.The predicted target genes for differentially expressed miRNAs were also identified using the online database starBase v3.0.A total of 3698 differentially expressed genes were identified.Gene ontology analysis demonstrated that differentially expressed genes that are related to hUMSC treatment of ischemic cerebral infarction are involved in endocytosis and inflammatory responses.We identified 12 differentially expressed miRNAs in middle cerebral artery occlusion rats after hUMSC treatment,and these differentially expressed miRNAs were mainly involved in signaling in inflammatory pathways,such as in the regulation of neutrophil migration.In conclusion,we have identified a number of differentially expressed genes and differentially expressed mRNAs,miRNA-mRNAs,and signaling pathways involved in the hUMSC treatment of ischemic cerebral infarction.Bioinformatics and interaction analyses can provide novel clues for further research into hUMSC treatment of ischemic cerebral infarction.

Co-suppressed glutamine synthetase2 gene modifies nitrogen metabolism and plant growth in rice

A full-length cDNA that encodes the rice chloroplastic glutamine synthetase 2 gene was isolated from a Minghui 63-normalized cDNA library; and GS2 rice transformants were obtained by an Agrobacterium tumefaciens-mediated transformation method. Transcripts of the GS2 gene were shown to accumulate at higher levels in the primary transgenic plants in the T0 generation; whereas plants in the T1 generation exhibited a co-suppressed chlorosis phenotype (yellow leaves) accompanied by decreased plant height, few tillers and decreased dry weight. The plants with yellow leaves also displayed a significant decline in GS2 messenger RNA (mRNA) transcriptional level and chlorophyll content; a decrease in total GS activities of ~50% was also found. Although there was no decrease in the concentration of total free amino acids, a change in the concentration of individual amino acids was observed. Our result also indicates a decreased metabolic level (soluble protein content and ammonium concentration) in GS2 co-suppressed plants. A correlation between chlorophyll content and GS2 mRNA expression level was also observed. The GS2 co-suppressed plants showed better performance when complemented with exogenous glutamine, indicating that the lack of an organic nitrogen pool inside the cell is the possible reason for the chlorosis phenotype of the transformants.

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.)

Rice (Oryza sativa) glutamate synthase (GOGAT,EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency,but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored.In this research,we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database,and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants.Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs,which suggested that they have different roles in vivo.Compared with the wild-type,tiller number,total shoot dry weight,and yield of GOGAT co-suppressed plants were significantly decreased.Physiological and biochemical studies showed that the contents of nitrate,several kinds of free amino acids,chlorophyll,sugars,sugar phosphates,and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants,but the contents of free ammonium,2-oxoglutarate,and isocitrate in leaves were increased.We conclude that GOGATs play essential roles in carbon and nitrogen metabolism,and that they are indispensable for efficient nitrogen assimilation in rice.