不同灵芝类群药效的特异性研究

通过对10种灵芝的表型性状及亲缘关系进行分析将灵芝聚为3类,对不同类群灵芝菌株的免疫调节、抗疲劳及改善睡眠的药效作用进行了比较研究。结果表明:灵芝的功效具有特异性,不同类群间存在功效差异性。     

砂糖橘上碎叶病毒外壳蛋白基因的克隆及序列分析

利用RT-PCR技术对广东砂糖橘碎叶病毒的外壳蛋白(CTLV-CP)基因进行了克隆和测序分析。DNA序列分析表明,砂糖橘CTLV-CP基因的cDNA序列全长为786bp,编码262个氨基酸。BLAST分析结果显示,所得序列与苹果、梨茎沟病毒(ASGV)外壳蛋白基因及其它柑橘CTLV-CP基因的同源性在87%～91%,证明所克隆的片段为CTLV-CP基因。     

Diploid female gametes induced by colchicine in Oriental lilies

The young flower buds of diploid Oriental cultivars: ‘Con. Amore’ and ‘Acapulco’ (Lilium) were treated with 0.02, 0.05, 0.1, and 0.2% colchicine to induce diploid egg (female gamete). The treated young buds between the two cultivars were crossed reciprocally as female parents with normal pollen respectively. The polyploid plants were identified by measuring stoma size and chromosome number. The results showed: that triploid progenies could be obtained through this way, and the fact that the treated young flower buds were successfully used as female parents indicated the formation of 2n or 2x egg cells. The above results implied that polyploidization by artificially induced diploid female gametes could be a powerful method to create novel variations in the breeding of Oriental lilies.     

Assessment of genetic purity of tomato (Lycopersicon esculentum L.) hybrid using molecular markers

Three DNA molecular marker systems, RAPD, ISSR and SSR, were used to test seed genetic purity of two commercial hybrid tomato (Lycopersicon esculentum L.) cultivars ‘Hezuo 903’ and ‘Sufen No. 8’. Genomic DNA from the two F₁ hybrid cultivars and their corresponding parental lines was screened with 218 RAPD decamer primers, 54 ISSR primers and 49 SSR primers. Among the 321 primers, 4 primers for ‘Hezuo 903’ and 3 for ‘Sufen No. 8’, which could produce both female and male parent-specific markers, were selected for testing the genetic purity. A total of 210 hybrid individuals of each cultivar were analyzed using the identified primers. The combined results of the marker analysis showed that eight of the 210 F₁ plants in ‘Hezuo 903’ and 13 of 210 in ‘Sufen No. 8’ were false hybrids, and the overall genetic purity of the two F₁ hybrid seed lots was 96.2 and 93.8%, respectively. This study showed that RAPD and SSR markers could provide a practical and efficient tool in quality control of the tomato commercial hybrid seeds.     

Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

AIM:To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 (hFGF-10) gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS:HFGF-10 gene was amplified by PCR and ligated with shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hFGF-10, which was linearized with PmeI and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy-hFGF-10. The recombinant adenoviral plasmid was then transfected into HEK-293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF-10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirus on the proliferation of kerotinocytes was checked by MTT. RESULTS:The recombinant adenovirus containing hFGF-10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat cells reacted with hFGF-10 antibody. The recombinant adenovirus stimulated the proliferation of kerotinocytes. CONCLUSION:HaCat cells infected with the recombinant adenovirus expresses and secrets hFGF-10 protein, which promotes the proliferation of HaCat cells.     

VEGF induces HUVECs to produce extracellular H2O2 and its proliferation role

AIM:To study the effect of VEGF on extracellular H₂O₂ production in HUVECs and the role of H₂O₂ in the VEGF-induced proliferation. METHODS:HUVECs was stimulated with 500 μg/L VEGF. Products of extracellular H₂O₂ was detected by H₂DCFDA staining. MTT method was used to value the influences of 3×10⁶ U/L catalase and 5-20 mmol/L H₂O₂ to VEGF function. RESULTS:After treatment for 15 min with VEGF, HUVECs appeared fluorescence, and continued to become stronger, peaked at 45 min then decreased. HUVECs, which was treated simultaneity with VEGF and 3×10⁶ U/L catalase, only appeared very faint fluorescence. The proliferation of HUVECs by VEGF was restrained when treated with 3×10⁶ U/L catalase. The extrinsic H₂O₂ at concentration of 5-10 mmol/L promoted the proliferation of HUVECs but inhibited the proliferation effect of VEGF on HUVECs (P<0.01). CONCLUSION:These findings indicate that VEGF induces HUVECs to produce extracellular H₂O₂ and plays role in proliferation, but extrinsic H₂O₂ restrains VEGF function.     

Lethal effect of VEGFR2shRNA on HL60 cells in vitro using lentiviral vector

AIM: To look for harmfulless anti-leukemia drug with selective high performance, lethal effect of small hairpin RNA (shRNA) on VEGFR₂ gene expression of tumor cell line HL60 in vitro.METHODS: The most effective VEGFR₂ siRNA was designed and screened. The shRNA oligo was designed and pU6/VEGFR₂ entry clone was constructed. HL60 was transfected transiently and vascular endothelial growth factor receptor 2(VEGFR₂) expression was tested with MTT assay, RT-PCR and Western blotting. The expression clone was constructed and cotransfected with ViraPowerTM Packaging Mix into 293FTTM cells to produce Lentiviral vectors harboring Lenti6/shVEGFR₂. The virion supernatant was added into HL60 cells and VEGFR₂ gene inhibitory effect was determined. RESULTS: The inhibitory rates of VEGFR₂ siRNA c were high. VEGFR₂ expression in HL60 was inhibited by using pU6/VEGFR₂ entry clone constructed with shRNA and pENTRTM/U6. For HL60 cells, the inhibitory rate was 84.9%. The expression of VEGFR₂ mRNA and protein decreased significantly. 48 hours after transfection of pU6/shVEGFR₂ entry clone and transduction of Lenti6/shVEGFR₂ expression clone, the cell inhibitory rates were similar. Cell growth inhibitory rate of entry clone descended rapidly after this time point, the expression clone changed slowly, reaching the peak at 96 hours, dropped slightly, having no significance deviation. CONCLUSION: in vitro, VEGFR₂ shRNA using lentiviral vector blocks VEGF/VEGFR₂ self-secretion in HL60 cells, which inhibits leukemia development.     

转基因抗病毒病四倍体西瓜的培育

利用西瓜花叶病毒2号(WMV-2)外壳蛋白(CP)基因、小西葫芦黄花叶病毒(ZYMV)复制酶(NIb)基因和黄瓜花叶病毒(CMV)复制酶基因构建三价基因的植物表达载体,通过农杆菌介导转化四倍体西瓜植株,经分子检测证明目的基因成功地导入西瓜植株,并在后代中稳定遗传。经温室及大田接种鉴定,T3代转基因西瓜抗病毒性达中抗水平,为抗病毒无籽西瓜新品种的培育提供了可能性。