Integration and expression of Xa21 in transgenic rice CX8621 / 生物工程学报

Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.

Application of competitive PCR for screening selectable marker-free Xa21 transgenic rice / 生物工程学报

Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa2l transgenic rice plants. The competitive template of Xa21 was the endogenous Xa2l homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.

Study on ConA promoting phagocytosis, cytotoxicity and producing effectors of macrophages / 免疫学杂志

Objective　To study　how　ConA actives macrophages in vivo to produce cytotoxic effectors and its phagocytic functions,and　 cytotoxicity. Methods　 ConA was intraperitoneally injected(ip). Cock red blood cells(cRBC) were used to evaluate MΦ phagocytic activity,and S180 cells as target cells to analyze MΦ dependent cytotoxicity(MTC).Nitric oxide(NO),TNF-α　and IL-1 levels of MΦ cultural supernatant were measured using griess reagent,L929 cells MTT method and thymocytes proliferation test respectively. Results　 ConA could promote MΦ　to phagocytize cRBC and kill S180 cells,enhance the production of such factors as NO,TNF-α　and IL-1 by MΦ. There was significant difference compared with PBS　control group(P＜0.01). Conclusions　 ConA could stimulate MΦ to produce effectors, which mediate immune regulation of ConA to MΦ.

Effects on macrophages activity transfected transiently with pcDNA3-HBV / 中华传染病杂志

Objective To explore the mechanism of immune tolerance in chronic B hepatitis, the effects of HBV infection on the functions of macrophages and related mechanism of signal transduction by transfection in vitro were studied. Methods Peritoneal macrophages of mice were isolated regularly and cultured, transfected transiently with pcDNA3-HBV or pcDNA3 plasmid DNA and cultured under the stimulation of LPS. After 72 h, RT-PCR was performed to detect the expression of PreS1, TNF-? or IL-1? mRNA. To detect the expression of NF-?B RelA protein by FCM, and the level of nitric oxide in cultural supernatant was measured with Griess reaction. Results After being transfected with pcDNA3-HBV,peritoneal macrophages had the expression of PreS1 mRNA, but have lower level of TNF-?、IL-1? mRNA and transcriptional factor NF-?B, compared with pcDNA3-transfected control group; the level of nitric oxide in pcDNA3-HBV group was also decreased. Conclusions Transient transfection of pcDNA3-HBV could decrease the function of macrophages directly by inhibiting NF-?B activity and effector molecules production, which may be one of the mechanisms of immune tolerance in chronic B hepatitis.

Effect of TNF-related apoptosis inducing ligand on the biological activity of hepatocarcinoma cell line / 中国病理生理杂志

AIM: To explore the effect of TNF-related apoptosis inducing ligand (TRAIL), a new apoptotic inducing molecule on the biological activity of hepatocarcinoma cell line. METHODS: The expression of membrane binding TRAIL on HepG2 cells was detected by immuno-cytochemistry. Quantity of secretory TRAIL was assayed by ELISA method. The cytotoxicity and apoptosis induced by TRAIL was detected by MTT and TUNEL method, respectively. The telomerase activity of HepG2 cells was detected by TRAP-PCR assay kit. The expression of hTERT, the catalytic subunit of telomerase, was detected by FCM. RESULTS: TRAIL was constitutively expressed on the membrane of HepG2 cell line. Soluble TRAIL was also expressed to a certain degree. Cytotoxicity assay showed that TRAIL significantly inhibited the growth of hepatocarcinoma cells. TUNEL assay indicated that TRAIL induced apoptosis in hepatocarcinoma cells. Detection of telomerase activity showed that TRAIL inhibited telomerase activity and the expression of telomerase catalytic subunit. CONCLUSION: TRAIL is an effective molecule to inhibit the growth of hepatocarcinoma through multiple pathways, such as inducing apoptosis and inhibiting the activity of telomerase.

Molecular mechanisms involved in the evasion of Legionella from the killing effect of macrophages / 中国病理生理杂志

AIM: To explore the relationship between caspase activation and the evasion of Legionella from macrophage elimination through a Legionella-infected macrophage model. METHODS: After infected by Legionella, the activity of caspase 3 in macrophages was analyzed by confocal microscopy as well as fluorescence reader. Growth and replication of Legionella in macrophage was assayed. Replication of Legionella was analyzed again to see the effect of caspase 3 inhibition on the growth of Legionella after use of caspase 3 inhibitor. RESULTS: Both confocal microscopy and caspase 3 fluorescent substrate analysis showed that Legionella virulent strain had powerful capability of activating caspase 3 while the mutant non-virulent strain did not have this capability. The virulent strain highly replicated in macrophages and the replication was significantly inhibited by caspase 3 inhibitor. CONCLUSION: Our results indicate that the intracellular caspase 3 is activated shortly after infection by Legionella virulent strain. The evasion of Legionella from the elimination of macrophages may be mediated by caspase 3 activation to a great degree.