银杏锰型超氧化物歧化酶GbMnSOD基因的克隆与表达

利用RACE技术首次从银杏中克隆到锰型超氧化物歧化酶基因(GbMnSOD ) 的cDNA全长。GbMnSOD的cDNA全长965 bp (GenBank accession number: EF633506) 。生物信息学分析GbMnSOD cDNA序列含有一个681 bp最大读码框, 编码一个226氨基酸多肽链, 通过软件预测分子量为2515 kD, 等电点为8197。三维结构预测结果显示, GbMnSOD 含有12个α螺旋和3 个β折叠构成一个篮子状的活性中心。GbMnSOD氨基酸序列与其它植物MnSOD具有很高的相似性。进化树分析结果表明GbMnSOD和其他物种的MnSOD源自于相同的祖先。Southern杂交显示, GbMnSOD属于一个小的多基因家族。Northern 杂交表明GbMnSOD在银杏的根、茎、叶和果中都有表达, 在叶中的表达量最高, GbMnSOD 的转录受到ABA、IAA、蔗糖、甘露醇、NaCl 和低温的诱导。     

IBA对芍药扦插生根的影响及生根过程中相关酶活性的变化

以芍药‘大富贵’品种为材料, 用1 000、2 000和3 000 mg·L^-1 3个浓度的IBA处理进行扦插对比试验, 研究其生根过程中过氧化物酶( POD) 、多酚氧化酶( PPO) 、吲哚乙酸氧化酶( IAAO) 活性的动态变化。结果表明: IBA处理极显著地提高了插穗的生根率、根数和总根长, 其中以2 000 mg·L ^-1为IBA最佳生根浓度, 将生根周期缩短18 d, 并明显提高了生根质量。在生根过程中, 插穗中POD、PPO活性分别呈现“缓慢上升→下降→急剧上升→下降”和“降→升→降”的变化趋势, 但与对照组相比, 处理组的变化更为剧烈, 峰值出现提前了18 d。IAAO活性在对照和处理中呈现不同的变化趋势: 前者为“升→降→升”, 而后者为“降→降→升”。此外, 高活性的POD、PPO有利于不定根的形成, 低活性的IAAO则促进生根。     

气相色谱-嗅觉测量法鉴定蒌蒿中的香气化合物

 采用顶空固相微萃取法提取了蒌蒿样品中的挥发性成分, 利用气相色谱-嗅觉测量法(GC-O)并结合气相色谱质谱联用分析(GC - MS) , 鉴定了萎蒿中的香气化合物。共检出21个呈香化合物, 基于其嗅感强度, 反式- 2 - 己烯醛、水芹烯、顺式- 罗勒烯、3 - 崖柏酮、樟脑, 黄瓜醛、( - ) - 龙脑和( - ) - 乙酸龙脑酯被认定为形成蒌蒿香气的重要化合物。     

南亚热带李新品种‘华蜜大蜜李’

 ‘华蜜大蜜李’是从‘三华李’中选育出来的优良新品种, 果实近圆形, 平均单果质量51.5 g, 可溶性固形物含量9%～11% , 酸甜味浓, 肉质软溶, 丰产稳产, 是‘三华李’中的大果品种, 适合于南亚热带山区栽培。在广东6月中下旬果实红熟。     

利用DAD1反义片段转化创建菜薹可调控雄性不育材料

 菜薹因为没有好的雄性不育材料或自交不亲和系,至今尚无一代杂种用于生产,根据拟南芥及白菜型油菜的花药不开裂基因DAD1的保守序列设计引物,扩增菜薹的DAD1基因片段（DAD1F）,构建反义DAD1F植物表达载体,用农杆菌介导法转化菜薹,对转基因植株进行分子检测,鉴定其雄性不育性并进行育性恢复试验.克隆得到的菜薹的DAD1基因片段大小为678 bp,命名为BrcpDAD1F,其序列与拟南芥和白菜型油菜的DAD1高度同源,同源率分别为88％和99％;共得到了12株转基因植株,有6株在转录水平上得到表达,表现为雄性不育,花器官畸形,花粉活力低,萌发率不到10％,且开花后不能结角果或结空角果,或者得到极少种子但种子不萌发;用对照的花粉给转基因植株授粉可使其正常结实.以500 μmol·L^-1茉莉酸甲酯处理可使其雄性不育得到恢复,花粉可以在柱头和培养基上萌发,具有受精能力。T₁代可育株与不育株的比例都呈1:3分离, T₂代不同株系的育性分离比例不同,有些株系继续呈1:3的分离,有些株系全是可育株或全是不育株,说明反义抑制呈单基因稳定遗传。     

月季RcHSP17.8 基因表达提高大肠杆菌对非生物胁迫的耐性

 月季RcHSP1718为编码小分子热激蛋白基因, Southern检测其在月季基因组中为多拷贝存在。38 ℃/3 h热激处理后, 该基因在耐热月季品种‘曼海姆宫殿’ ( Schloss Mannieim) 、‘彩虹’ (Radio) 、‘赌城’ (Las Vegas) 、‘梅郎随想曲’ (Caprice de Meiland) 、‘小女孩’ ( Girl) 中强表达, 而在不耐热品种‘新十全’ (Kordes’Perfecta) 、‘绯扇’ (Hiogi) 、‘莱茵黄金’ ( Pfalzer Gold) 中弱表达或不表达, 表明该基因与月季耐热性关系密切。为确定月季RcHSP1718基因功能, 将其转化E.coli BL21, 结果表明重组菌株能正常诱导表达包含RcHSP17.8的融合蛋白, 并因此提高了重组菌株对高温、低温、高盐、高pH、重金属、氧化等非生物胁迫的耐性, 表明RcHSP17.8参与了上述非生物胁迫的响应。     

黄金树花器官发生及发育的形态观察

利用普通光学显微镜和扫描电镜技术对黄金树花器官的发生及发育过程进行了观察.结果显示:(1)黄金树花器官的形态发生及发育过程集中于3月下旬-5月上旬;(2)花原基分化形成花的整个过程符合一般的分化顺序:花萼原基-花冠原基-雄蕊原基-雌蕊原基,且各原基在分化顺序上存在交叉;(3)花药及胚珠的发育与花器官的形态发生之间有明显的连续性,当花蕾直径为3.0 mm左右时花粉母细胞及完整的花粉囊壁形成,直径达到3.5 mm左右时胚珠中出现孢原细胞的分化,它直接起大孢子母细胞的功能.     

Nontypeable haemophilus influenzae induces IL-8 expression in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner

AIM:To investigate the key molecular mechanism of inflammatory response in alveolar epithelial cells induced by nontypeable haemophilus influenzae (NTHi). METHODS: A549 cells were co-cultured with NTHi (multiplicity of infection, MOI: 10) and harvested 15 min and 30 min after stimulation. The phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) in A549 cells was detected by Western blotting. The intracellular expression of nuclear factor-κB (NF-κB) p65 was examined by flow cytometry 4 h after stimulation. A549 cells were preincubated with p38 inhibitor (SB203580) or NF-κB inhibitor (PDTC) for 1 h and then stimulated with NTHi for 24 h. The level of interleukin 8 (IL-8) in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The phosphorylation of p38 MAPK was rapidly induced by NTHi stimulation. The expression of NF-κB p65 in A549 cells after NTHi stimulation was significantly up-regulated compared with control group (P<0.05). The level of IL-8 in the supernatants was increased 24 h after bacterial stimulation compared with control group (P<0.05). Blockage of p38 MAPK or NF-κB remarkably decreased IL-8 secretion in A549 cells (P<0.05). CONCLUSION:NTHi induces inflammatory response in alveolar epithelial cells in a p38 MAPK and NF-κB dependent manner.     

Relationship between zinc and zinc transporter expression in human lung cancer cells

AIM: To study the changes of zinc transporter gene expression in A-549 cell line exposed to ZnCl2 and N,N,N’,N’-tetrakis 2-pyridylmethyl ethylenediamine (TPEN).METHODS: Human lung cancer cell line A-549 was exposed to different concentrations of ZnCl2 (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15, 20 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT (methyl thiazolyl tetrazolium) and levels of zinc transporter mRNA was detected by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations.RESULTS: A-549 cell viability rate was significantly decreased when exposed to ZnCl2 at concentrations of 150 and 200 μmol/L, and to TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl2 and decreased when exposed to TPEN. Zinc transporter (ZnT-1) mRNA level was increased along with the increase in the concentration of ZnCl2 but decreased when exposed to TPEN. The expressions of ZIP-1 and ZIP-10 (Zrt-and Irt-like protein) were increased along with the increase in the concentration of TPEN but decreased when exposed to ZnCl2.CONCLUSION: ZnT-1 expression is induced by zinc supplement. ZIP-1 and ZIP-10 expressions are induced by zinc deficiency and repressed by zinc supplement.     

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

AIM: To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells (MSCs) on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS: MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X-gal staining. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry (factor Ⅷ and ADM) and the DAPI-labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type I and III was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type I and III were detected. RESULTS: With X-gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose-effect relationship with multiplicities of infection (MOI). When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time-dependent manner. A maximum production of ADM was observed at 7 d after infection ［(26.53±1.42) ng/L vs (1.34±0.08) ng/L, P<0.05］, and ADM secretion reduced to normal level at 15 d ［(2.20±1.44) ng/L vs (1.52±0.33) ng/L, P>0.05］. DAPI-labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad-ADM or MSCs alone. MSCs transplantation decreased the ratio of collagen type I and III, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type I and III, and significantly improved the left ventricular functions. CONCLUSION: Ad-ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type I and III through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.