[Research on construction and expression of recombinant eukaryotic expression plasmid for mouse adiponectin].

OBJECTIVE: To construct the recombinant eukaryotic expression plasmid for mouse adiponectin so as to supply experimental basis for research further on the function of adiponectin. METHODS: Extracting the total RNA from mouse adipose tissue, applying RT-PCR to amplify the complete coding region of mouse adiponectin cDNA and connecting the amplified product with pCDNA3.1(+) vector; then verifying the recombinant plasmid by sequencing and detecting in vitro recombinant expression and cellular proliferation depression to mouse breast cancer cell 4T1. RESULTS: A 744 bp fragment was got after PCR amplification with the recombinant plasmid, which was confirmed by sequence and expression detected by Western-blot. Besides, compared with the control in vitro test, visible morphological changes of 4T1 cell could be observed after recombinant plasmid cell transfection for 48 h, with cells collapsed or shrunk and cellular proliferation inhibited. During the flow cytometry analysis, the transfected 4T1 cells could also be observed obviously to have a greater proportion of cell apoptosis than the control could be detected. CONCLUSION: The recombinant eukaryotic expression plasmid for mouse adiponectin is successfully constructed, which has an obvious inhibition effect on the proliferation of mouse breast cancer cell 4T1.

[Searching for genes interacting with human PCIA1 gene by using the bacterial two-hybrid system].

OBJECTIVE: To search for the genes which could interact with newly found homo sapiens cross-immune reaction antigen (PCIA1) gene and accordingly to provide insights into the study of the gene function. METHODS: The Stratagene's BacterioMatch Two-Hybrid System and BacterioMatch Fetal Kidney Library were adopted and the recombinant bait plasmid pBT-PCIA1 was cotransformated with the target plasmid pTRG-cDNA library DNA into the reporter stain. After screening and isolation of positive pTRG clones, the target genes were identified by DNA sequencing and bioinformation analysis. RESULTS: Among all the seven detected target genes, three genes' function were not known, the other four genes had important functions. Their mutations or abberant expression resulted in severe diseases and overexpression of ACTN4 (actinin, alpha 4), PSAP (prosaposin) or EIF3S10 (eukaryotic translation initiation factor 3, subunit 10 theta) could promote tumor development and progression. CONCLUSION: The bacterial two-hybrid system technique is an efficient method, which can provides insights into the study of novel genes' function by detecting protein-protein interactions. This study indicates that PCIA1 gene expression correlates with tumor formation, invasion and metastasis.

[Thermal dissipation pathway in cucumber seedling leaves under hypoxia stress].

A water culture experiment was conducted to study the relationship between photosynthetic thermal dissipation and xanthophyll cycle in cucumber seedling leaves under hypoxia stress (the dissolved oxygen concentration in nutrient solution was 0.9-1.1 mg x L(-1)). Under the hypoxia stress, there was a significant decrease in the quantum yield of PS II photochemistry rate (phi(PS II)), net photosynthetic rate (Pn) under saturation light intensity, quanta yield (AQY), and maximal photochemical efficiency (Fv/Fm), suggesting that the photoinhibition of the seedling leaves was induced. Meanwhile, the thermal dissipation (NPQ) and the allocation of dissipation energy (D) by antenna increased, but the photochemical quenching apparent (q(p)) decreased, suggesting the enhancement of thermal dissipation in cucumber leaves under hypoxia stress. A positive correlation was observed between NPQ and xanthophyll de-epoxidation state (DEPS), and both of them were promoted by ascorbic acid (AsA) and inhibited by 1,4-dithiothreitol (DTT), suggesting that xanthophyll cycle was the major pathway of photosynthetic thermal dissipation in cucumber seedling leaves under hypoxia stress.