花生栽野种间杂交异源多倍化早期世代基因表达变化分析

为了进一步认识花生种间杂交异源多倍体进化过程中的基因表达变化规律,采用cDNA-HFO-TAG技术,研究花生种间杂交组合(四倍体栽培种‘仲恺花4号’×二倍体野生种Arachis. diogio)杂种F_1和早期多倍体世代S0~S3的基因表达变化情况。14条HFO-TAG引物共扩增出121条cDNA片段,其中差异片段84个,主要包括三种类型:亲本转录物完全沉默(3个),双亲转录物在后代部分材料中沉默(59个)和新转录物激活(22个),上述变化在F1代即开始发生。筛选其中大小为500~2 000 bp的35个TDFs进行克隆测序,有27个和NCBI数据库中已录入的基因具有较高的相似性,包括抗逆相关基因(10条)、未知功能蛋白基因(8条)、能量与代谢相关基因(7条)和转录因子相关的基因(2条)。这些研究结果进一步表明在花生种间杂交异源多倍化早期发生着快速、剧烈的基因表达变化,从中获得的差异基因片段,有助于了解花生属种间杂交异源多倍化早期分子机制变化,这对有效利用野生花生种质优异基因具有重要意义。cDNA-HFO-TAG技术简单、有效且实用,完全适用于花生属基因表达变化研究,可以作为花生属及其它物种基因表达变化分析技术的有效补充。     

Using antisense nucleic acid technology to study the influence of POLκ on genetic stability

AIM:To establish cell line FL-POLκ^- and to study the role ofPOLκ(polymerase kappa) on genetic stability.METHODS:A mammalian expression vector expressing antisense POLκ gene fragment pMAMneo -amp^--POLκ was constructed by cloning the 1 690-1 918 fragment of POLκ gene into the mammalian expression vector pMAMneo-amp^- in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made.RESULTS:The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL- POLκ^- was 11.2×10^-4, while it was 4.9×10^-4 and 3.7×10^-4 in the control cells FL and FL-M, respectively.CONCLUSION: POLκ playes an important role in maintenance of genetic stability.     

Detection,cloning and expression of bone morphogenetic protein-1 from human osteosarcoma cell lines

AIM:To manufacture recombinant protein of the highly conserved domain in human bone morphogenetic protein-1(BMP-1) using gene engineering methods as antigen for making wide spectrum antibody to BMP-1.METHODS:We analyzed the gene sequences and protein structures of BMP-1 and its related proteins, and chose a highly conserved fragment as target gene. Total RNA was prepared from human osteosarcoma cell line Saos-2, then the target gene was amplified with RT-PCR. The PCR product was cloned into prokaryotic expression vector pMAL c2 to get recombinant vector BMP-1(322-588aa)-pMAL c2. After transforming the recombinant plasmid into DH5-alpha and screening, several prositive clones were got for sequencing. Finally the transformed cells was induced with IPTG to get fusion protein.RESULTS:The BMP-1 gene fragment was successfully cloned into vector pMAL c2, and was able to express efficiently with IPTG inducement. The amount of expressed fusion protein is about 66%-72% in total volume of bacterial proteins.CONCLUSIONS:The recombinant protein contains several key domains(2 CUB domains and 1 EGF domain), which are shared by BMP-1 and its related proteins. Specific wide spectrum antibody to human BMP-1 and its related proteins may be generated with this recombinant protein antigen.     

Cloning of p15INK4b related gene CCRG and expression analysis in the renal carcinoma

AIM and METHODS:To analysis the factor that involved in renal carcinogenesis, we used the bait gene AK001518 to screen GenBank. To understand the relationship between cell cycle related gene(CCRG) and p15, we did RT-PCR and Northern Blot experiments. Then we examined CCRG expression level in renal carcinogenesis. RESULTS:Gained a function unknown gene CCRG that was 67% a mino acid identical with the gene AK001518 that was regulated by p15. It was shown that the CCRG mRNA was dramatically decreased when p15 gene was over-expressed. CCRG expression level was much higher in tumor tissues and cells than normal tissues and cells. CONCLUSION:The novel gene CCRG expressed highly in the renal carcinoma, which might play a significant role in the renal carcinogenesis.     

Effects of hypoxia and hypercapnia on expression of soluble guanylate cyclase in rat pulmonary artery

AIM:To investigate the expression of soluble guanylate cyclase protein and its mRNA in rat pulmonary artery after exposure to hypoxia and hypercapnia.METHODS:Male Sprague-Dawley rats were randomly split into 4 group, which were hypoxic hypercapnic (HH 1 week, HH 2 weeks, HH 4 weeks) group and control group, to copy pulmonary hypertensive animal model. The expression of sGCα₁ and β₁ subunits protein of medial and small pulmonary artery was performed by immunohistochemistry with a polycolonal antibody. In situ hybridization was performed on the rat lung tissue using sGC oligonuclear probe to assay the expression of sGCα₁subunit mRNA.RESULTS:The sGCα₁ and β₁ subunits protein and sGCα₁ subunit mRNA were faint staining in the pulmonary small and medium artery in HH1 week, HH 2 weeks and HH 4 weeks groups compared with control group (all P＜0.01).CONCLUSION:sGC subunit mRNA and protein expression in pulmonary small and medium artery were decreased after exposure to hypoxia and hypercapnia, which took part in the development of the pulmonary hypertension.     

Analysis of the distribution and clonality of TCR Vβ T cells in cord blood

AIM:To investigate the distribution and clonality of TCR Vβ subfamily T cells in cord blood. METHODS:The CDR3 of TCR Vβ 24 subfamily genes were amplified in mononuclear cells from 13 cases of cord blood. To observe the usage of TCR Vβ repertoire, the PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Vβ T cells. Peripheral bloods from 10 cases of normal individuals and T cell line Molt-4 and Jurkat served as controls. RESULTS:Only 38.78%±16.26% of 24 Vβ subfamily T cell were selectively expressed in cord blood, predominantly in Vβ 3, 5, 8, 9 and 13, whereas all 24 Vβ subfamilies could be detected in T cells from peripheral blood of normal individuals. Genescan analysis showed that all PCR products of TCR Vβ subfamilies from cord blood or normal individual peripheral blood displayed multi-peaks. CONCLUSION:Some TCR Vβ subfamily T cells were absent in cord blood. All TCR Vβ subfamily T cells in cord blood displayed polyclonality.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Ginkgo biloba extract enhances c-jun expression and attenuates motoneuron death induced by root avulsion

AIM: To investigate the influence of Ginkgo biloba extract (EGb761) on c-jun expressions and motoneurons survival following root avulsion. METHODS: One hundred and eighty adult Sprague-Dawley female rats were randomly divided into control and EGb761 groups. Immediately after avulsion of C5-T1 nerve roots, the rats were injected ip with either 1 mL of EGb761 25 mg·kg^-1·d^-1 or the same volume of normal saline, and the treatment repeated everyday. At 4 h to 6 weeks following avulsion, the C7 spinal segments of all rats were collected and prepared for c-jun immunocytochemistry and neutral red stain. The numbers of c-jun positive and survival motoneurons were counted and compared between two groups at each time point. RESULTS: In control rats following avulsion, c-jun positive motoneurons appeared at 4 h, reached its maximum at 1 d and declined to 2 weeks. Avulsion-induced motoneurons death started at 2 weeks, climbed to its maximum at 4 weeks-6 weeks. In EGb761 treated rats, both numbers of c-jun positive and survival motoneurons were more than that in control group at each time point. CONCLUSION: EGb761 attenuates avulsion-induced motoneurons death, and this effect may be related to up-regulation of c-jun gene in avulsed motoneurons.     

Antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes

AIM: To observe the antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes. METHODS: Rat thymus lymphocytes were separated by Ficoll-Urografin density gradient centrifugation. Lipofectin was used to introduce antisense, sense and mismatched oligonucleotides for c-myc to rat thymus lymphocytes. The antiproliferative effect was assayed by incorporation of [³H]-TdR and MTS cell proliferation assay. TR-PCR was used to detect the expression of c-myc mRNA. RESULTS: c-myc antisense oligonucleotide inhibited ratthymus lymphocytes proliferation[(0.14±0.03)A vs(0.32±0.16)A,P<0.05],but this ef ect had no relationship with the concentration of c-myc antisense oligonucleot ide.c-myc antisense oligonucleotide decreased the expression of c-myc mRNA in rat thymus lymphocytes. CONCLUSION: c-myc antisense oligonucleotide inhibited rat thymus lymphocyte proliferation.