High-level expression of modified gene encoding human adiponectin in transgenic rice

Adiponectin is a polypeptide specifically secreted from human adipocytes, and its deficiency is closely linked to increased obesity and type II diabetes. There is an urgent demand for large-scale production of human adiponectin for pharmaceutical applications. Here, we report that we have successfully obtained a high-level of expression of modified genes encoding human adiponectin in transgenic rice. The 735 bp cDNA of the native human sequence was adopted to rice codon usage, fused to the translation initiation sequence in the N terminus and to the KDEL signal sequence in the C terminus. An amplification promoting sequence acting as an enhancer of transcription was also introduced to enhance gene expression. The presence of the transgene and mRNA transcripts was confirmed by PCR, Southern blot and RT-PCR. Western blot analysis revealed that a protein of approximately 30 kDa was produced in rice leaves. ELISA analysis was used to determine the amount of recombinant adiponectin in transformants with the modified gene in up to 0.32% of total soluble leaf protein. Our results establish the feasibility of high-level expression of recombinant human adiponectin in transgenic rice.

Single nucleotide polymorphism of human APM1 detected by using allelic 3′-terminal specific primers real-time PCR / 基础医学与临床

Objective To develop an analyzing technique of single nucleotide polymorphism(SNP) based on non-probe real-time quantitative PCR, and to explore the relationship between SNP in human adiponectin (APM1) gene and type 2 diabetes (T2DM).Methods Design primers with specific 3′-terminal nucleotides and perform Real-time PCR using SYBR Green dye. Genotypes were determined by comparing the amplification efficiency of each pair of primer and verified by sequencing. SNPs 45 and 276 in APM1 of 20 T2DM, 24 obesity and 28 healthy persons were analyzed. Results This technique could discriminate genotype as efficient as sequencing. The genotype of APM1 at +45 site was significantly correlated with T2DM (P