甘蓝SRK的S域及SCR酵母表达载体的构建及其相互作用区段的研究

 为了深入研究S位点受体激酶（SRK）和S位点富含半胱氨酸蛋白（SCR）甘蓝自交不亲和（self-incompatibility,SI）信号传导的雌雄决定因子的相互作用机制,以自交不亲和性高的结球甘蓝为材料,采用酵母双杂交体系,构建pGADT7-eSRKs和pGBKT7-SCRs重组载体,并分别转化到Y187和Y2HGold酵母中,通过SD/-Ade-His-Trp-Leu平板上菌落的形成,PCR以及x-α-gal显色反应鉴定转化到酵母中的两个重组质粒相互作用。结果表明,SRK S域的SRK1及SRK4分别与SCR2相互作用,且初步显示其相互作用区域为SRK第1个外显子的第16 ~ 421 bp的片段和SCR第1 876 ~ 2 068 bp的片段。这既为SRK-SCR相互作用提供了具体证据,也为甘蓝自交不亲和性分子机理的深入研究提供了新内容。     

Experimental study of a new hypertension-related gene Slc7a8

AIM: To investigate the disease related genes in SHR.METHODS: The total RNA samples were obtained from second-order mesenteric arteries and kidney of SHR and WKY.Microarray containing over 10 000 genes was used to determine the level of mRNA expression in two groups.The genes were identified using real time quantitative RT- PCR.RESULTS: 19 down-regulated genes were determined by microarray,which were classified as chaperones,transport,growth factors,signal transduction,nuclear factor and lipoprotein.The result was confirmed by the method of real time quantitative RT- PCR.It was found that the Slc7a8 gene was up-regulated 9.3 fold in SHR.CONCLUSION: Slc7a8 gene may relate to hypertension.Further study on the Slc7a8 gene and its function would help us wholly understand the mechanism of hypertension and provide new clue to hypertension causes.     

黄皮洋葱新品种连葱7号的选育

 连葱7号是以连引洋葱1号为母本、富永中生为父本,配组杂交后经多代系统选育而成的黄皮洋葱新品种。中熟,生育期250d(天)左右;植株直立,生长势旺盛,株高60～70cm;鳞茎圆球形,球形指数0.85以上,外皮金黄色,有光泽,辛辣味淡;分球率低,耐抽薹,单球质量290g左右,每667m2产量6000kg左右,适宜黄淮地区栽培。     

早秋大白菜新品种淄白7号的选育

淄白7号属早秋品种,从播种到收获60 d(天)。母本96-9是从台湾耐热白菜杂交后代中经多代定向选育而成的自交不亲和系,父本9542-2选自石特变异株。该品种外叶浅绿色,球叶叠抱,倒卵圆形,球顶圆,结球紧实。较抗病毒病、霜霉病和软腐病。平均单球质量2.5 kg,一般每667 m~2净菜产量3 600～4 000 kg。     

High-efficient genetic transfection of CD41+,UT7, U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadher in-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41⁺ cells and cell culture: Cells expressing CD34⁺ from cord blood were isolated. The inducement of cells expressing CD41 from CD34⁺ cells was performed by using TPO and cells CD41⁺ were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41⁺, UT7, U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1×10⁷) and EC1-4 pMSCV retroviruses (1.0×10⁸). With 8-folds dilution retroviruses, 60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells, 72.56% in U937 cells and 30.57% in CD41⁺ cells, respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41⁺ and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION:The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41⁺,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.     

辣椒新品种长辣7号的选育

长辣7号是以903A为母本,恢复系7122为父本配制而成的三系辣椒新品种。中熟,生长势中等,果实细长羊角形,顺直少皱,青果深绿色,红果色泽鲜艳,果长20 cm左右,果肩宽1.2～1.4 cm,肉厚0.22 cm,单果质量15 g左右,商品性好|田间对炭疽病的抗性优于对照湘辣4号,对病毒病和疫病的抗性与对照相当,耐旱|一般每667 m2产鲜椒2 400 kg。适宜湖南、贵州、河南及其相似生态区域栽培。     

马铃薯新品种郑薯7号的选育

郑薯7号是由Favorita×豫马铃薯1号的杂交后代选育而成。早熟,生育期68d(天)左右,休眠期短,45d(天)左右。薯块椭圆形,黄皮黄肉,芽眼浅而稀,表皮光滑,平均商品薯率95.0%左右。淀粉含量为12.2%,VC含量为158.0mg·kg-1,粗蛋白含量为2.48%,还原糖含量为0.81%。对病毒病、早疫病、晚疫病的抗性均强于郑薯5号,春季栽培每667m2产量2000kg左右,高产可达2500kg以上。适宜河南省各地种植,种植面积已达8400hm2。     

FGFR2-IIIb D3 extracellular domain inhibits lipid synthesis in keratinocytes

AIM:To investigate the inhibitory effect of extracellular domain of fibroblast growth factor receptor 2 (FGFR2)-Ⅲb D3 on lipid synthesis in HaCaT keratinocyteas. METHODS:The bioactivity of FGFR2-Ⅲb D3 extracellular domain was detected by isothermal titration calorimetry (ITC) and CCK-8 assay. The inhibitory effect of FGFR2-Ⅲb D3 extracellular domain on lipid synthesis in HaCaT cells was analyzed by real-time PCR, Western blot, flow cytometry and oil red O staining. RESULTS:The results of ITC, real-time PCR and CCK-8 assay showed that FGFR2-Ⅲb was highly expressed in HaCaT cells, and FGFR2-Ⅲb D3 extracellular domain specifically bound to FGF7 to inhibit HaCaT cell viability. The results of real-time PCR and Western blot showed that FGF7 significantly up-regulated the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA synthase (ACS), stearoyl-CoA desaturase (SCD) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and the protein levels of p-FGFR, p-AKT and SREBP-1 in HaCaT cells, and these effects were inhibited after the treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, which was significantly different from the FGF7 group. and The results of oil red O staining and flow cytometry showed that the lipid content of HaCaT cells was increased after FGF7 induction. After treatment with FGFR2-Ⅲb D3 extracellular domain and AKT inhibitor, the lipid content was decreased (P<0.01). CONCLUSION:FGFR2-Ⅲb D3 extracellular domain inhibits the autophosphorylation of FGFR2-Ⅲb by its competitive binding to FGF7, and then blocks downstream PI3K-AKT signaling pathway, thus resulting in the down-regulation of SREBP-1c and downstream lipid synthases, and inhibiting lipid synthesis in keratinocytes.     

Overexpression of Smad7 inhibits TGF-β-induced Smad2 mRNA and protein expression in peritoneal mesothelial cells

AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-β₁ (TGF-β₁) in rat peritoneal mesothelial cells (PMCs).METHODS: Rat PMCs were cultured at different doses of TGF-β₁ (0，1.25，2.5，10 μg/L) for different time (0，5，15，30，60，120 min).PCDNA3-Smad7 was then transfected into cultured rat PMCs by lipofectamine, and the cells were stimulated like the above.Endogenous Smad2 and Smad7 expression was evaluated by RT-PCR and Western blotting.RESULTS: TGF-β₁ induced increase in Smad2 mRNA and protein expression at 5 min, peaked at 30 min, and declined to baseline levels at 120 min, which was in a time-dependent manner.TGF-β₁ also induced Smad7 mRNA expression at 5 min, and then declined, down to the lowest at 30 min, but at 60 min it increased again.Smad2, Smad7 mRNA and protein expression induced by TGF-β₁ were also dose-dependent.After transfection, overexpressions of Smad7 mRNA and protein in rat PMCs were observed, which did not decline with time.The expression of Smad2 mRNA significantly decreased by 33%, 56%, 67%, 71%, 63% and 57% (P<0.05), the expression of Smad2 protein declined by 78%，89%，89%，88% and 76% (P<0.05) respectively at 0, 5, 15, 30, 60 and 120 min.CONCLUSION: Overexpression of Smad7 inhibits Smad2 gene and protein expression in peritoneal mesothelial cells.Smad7 may be a negative regulator of TGF-β₁ signaling.