柑橘采后品质变化及保鲜技术研究进展

柑橘Citrus spp.是中国南方重要的热带、亚热带果树,其果实具有较高营养价值。然而,柑橘果实采后容易发生腐烂、失水、营养损失和病害,导致果实品质劣变,商品价值降低。本文就柑橘果实采后外观色泽,果实水分,果肉糖、酸、维生素C含量变化,果实病害青霉病、褐斑病、枯水病、油斑病发生,以及低温贮藏、热处理、短波紫外线(UV-C)照射、化学保鲜剂、生物技术保鲜等保鲜技术的研究进展进行综述,为提高柑橘保鲜效果、保持柑橘果实采后品质、延长果实保鲜期提供科学依据与生产实践指导。     

应用基因芯片检测酸味抗病甜瓜果实基因表达

利用Melon cDNA array ver1.0检测新疆厚皮甜瓜(Cucumis melo var.ameri)果实基因表达的可行性,并检测了经60Coγ射线辐射诱变后的新疆厚皮甜瓜酸味抗病变异株成熟果实基因的表达.结果显示:该芯片平均能够检测新疆厚皮甜瓜基因2 008个,检测出的基因占该芯片基因探针总数的65.4%;检测酸味抗病变异株上调表达的基因251个,占检出基因总数的12.5%;下调表达的基因224个,占检出基因总数的11.16%.利用RT-PCR验证芯片结果的可靠性,结果表明,用Melon cDNA array ver 1.0检测新疆厚皮甜瓜成熟果实基因表达水平是可行的.     

金柚贮藏真菌病害及保鲜技术研究

1998年5月-2000年10月间调查了梅州柚果贮藏期的真菌病害,它们是:①柚果绿霉病(Penicilliumdigitatum),②柚果炭疽病(Collettrichumgloeosporioides),③柚果黑腐病(Alternariacitri),④柚果焦腐病(Diplodianatalensis)和⑤柚果酸腐病(Oosporacitri-aurantii)等5种.从几种安全性高的保鲜剂中筛选出1个混用配方,该配方对柚果贮藏防腐保鲜效果明显,贮藏5个月无病斑果达100%,6个月也只有20%的柚果出现少量病斑,且柚果几项质量指标较好.     

Induction of a Citrus gene highly homologous to plant and yeast thi genes involved in thiamine biosynthesis during natural and ethylene-induced fruit maturation

Maturing citrus fruit undergo pigment changes which can be enhanced by exogenous ethylene. In order to identify genes induced by ethylene in citrus fruit peel, we cloned the gene c-thi1. mRNA corresponding to c-thi1 increased gradually in the peel during natural fruit maturation and in response to ethylene. GA₃ pretreatment reduced the inductive effect of ethylene. Levels of c-thi1 increased also in juice sacs but the effect of ethylene was much less prominent. c-thi1 is homologous to yeast and plant genes encoding for an enzyme belonging to the pathway of thiamine biosynthesis. The data suggest that thiamine is involved in citrus fruit maturation.     

利用功能分类基因芯片研究不同品种猪脂肪中特定基因的发育性变化

利用Oligo功能分类基因芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在1、2、3、4和5月龄间背部皮下脂肪中脂肪沉积代谢和细胞生长调控相关基因的动态表达变化。差异表达分析结果显示1~5月龄的品种间分别有10、6、11、8和19个基因的表达差异倍数大于2倍, 且长白猪有25个基因在不同月龄间的表达差异达显著水平(P<0.05)。其中血管生成素样蛋白4 (ANGPTL4)、组织蛋白酶K (CTSK) 、异柠檬酸脱氢酶2(NADP+) (IDH2)、脂蛋白脂酶 (LPL)、苹果酸酶1 (ME1)、 硬酯酰辅酶A去饱和酶 (SCD)和解藕联蛋白2 (UCP2)这7个基因不仅在同月龄的品种间和品种内的不同月龄间差异表达, 主成分分析结果也显示其表达模式明显偏离其他基因, 提示受到了特殊的调控。聚类分析结果显示1~5月龄间长白猪中正调控脂肪酸代谢基因的表达量逐渐上调, 太湖猪中参与细胞生长调控基因的表达量平缓波动且变化幅度相对较小。另外, 5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势。结果成功筛选出了对猪胴体和肉质性状可能具有重要影响并值得深入研究的一些候选基因, 初步揭示了相关基因的表达变化规律, 为了解生长发育过程中脂肪酸合成与水解的动态平衡过程提供了基础数据。     

利用功能分类基因芯片研究不同品种猪脂肪中特定基因的发育性变化

利用Oligo功能分类基因芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在1、2、3、4和5月龄间背部皮下脂肪中脂肪沉积代谢和细胞生长调控相关基因的动态表达变化。差异表达分析结果显示1~5月龄的品种间分别有10、6、11、8和19个基因的表达差异倍数大于2倍, 且长白猪有25个基因在不同月龄间的表达差异达显著水平(P<0.05)。其中血管生成素样蛋白4 (ANGPTL4)、组织蛋白酶K (CTSK) 、异柠檬酸脱氢酶2(NADP+) (IDH2)、脂蛋白脂酶 (LPL)、苹果酸酶1 (ME1)、 硬酯酰辅酶A去饱和酶 (SCD)和解藕联蛋白2 (UCP2)这7个基因不仅在同月龄的品种间和品种内的不同月龄间差异表达, 主成分分析结果也显示其表达模式明显偏离其他基因, 提示受到了特殊的调控。聚类分析结果显示1~5月龄间长白猪中正调控脂肪酸代谢基因的表达量逐渐上调, 太湖猪中参与细胞生长调控基因的表达量平缓波动且变化幅度相对较小。另外, 5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势。结果成功筛选出了对猪胴体和肉质性状可能具有重要影响并值得深入研究的一些候选基因, 初步揭示了相关基因的表达变化规律, 为了解生长发育过程中脂肪酸合成与水解的动态平衡过程提供了基础数据。     

甲型肝炎病毒衣壳蛋白融合基因植物表达载体的构建 与遗传转化柑桔的研究

基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系。以含甲型肝炎病毒结构基因cDNA的克隆载体pCDNAⅡA16为模板,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增,得到全长2.2kb衣壳蛋白融合基因序列。经测序鉴定后正向克隆于植物表达载体pBI121中,衣壳蛋白融合基因位于pBI121质粒T-DNA左右边界区间内,处于CaMV35S启动子控制之下。经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒pBI121-A导入根癌农杆菌LBA4404。以锦橙 (Citrus. Sinensis Osbeck) 上胚轴为转化材料,通过根癌农杆菌介导法将衣壳蛋白融合基因转 化到植物基因组中。120株转化外植体经卡那霉素50 mg/L筛选,其中13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养。PCR分析证明,13株拟转化植株中有5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因,转化率为4.1%。此研究是对遗传转化柑桔表达外源蛋白的初步探讨,为进一步研究食用疫苗开辟了新途径。Abstract: The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.     

DNA芯片技术中利用内标对数据归一化后检测基因的表达变化

在DNA芯片技术中 ,通过反转录反应 ,由mRNA合成带有荧光标记物的cDNA的过程中 ,往往要参入已知质量的poly(A) + RNA ,以对DNA芯片的检测灵敏度进行归一化处理 .通过体外转录的方法 ,以真核生物的cDNA克隆中的DNA片段为模板合成poly(A) +RNA ,对之定量后 ,以不同的质量比参入到样品的反转录体系中 ,代表不同的RNA拷贝丰度 ,从而对DNA芯片检测的灵敏度进行了定量 ,并得到DNA芯片上杂交点的荧光信号强度与基因表达的RNA拷贝数成正相关的关系 .利用含有内标的DNA芯片检测了热击反应后酵母细胞的基因表达变化 ,结果与Northern印迹方法检测结果是相符的     

cDNA Microarray Analysis of Changes in Gene Expression Associated with MPP+ Toxicity in SH-SY5Y Cells

cDNA microarray analysis of 1-methyl-4-phenyl-pyridinium (MPP⁺) toxicity (1 mM, 72 h) in undifferentiated SH-SY5Y cells identified 48 genes that displayed a signal intensity greater than the mean of all differentially expressed genes and a two-fold or greater difference in normalized expression. RT-PCR analysis of a subset of genes showed that c-Myc and RNA-binding protein 3 (RMB3) expression decreased by 50% after 72 h of exposure to MPP⁺ (1 mM) but did not change after 72 h of exposure to 6-hydroxydopamine (25 M), rotenone (50 nM), and hydrogen peroxide (600 M). Exposure of retinoic acid (RA)-differentiated SH-SY5Y cells to MPP⁺ (1 mM, 72 h) also resulted in a decrease in RMB3 expression and an increase in GADD153 expression. In contrast, c-Myc expression was slightly increased in RA-differentiated cells. Collectively, these data provide new insights into the molecular mechanisms of MPP⁺ toxicity and show that MPP⁺ can elicit distinct patterns of gene expression in undifferentiated and RA-differentiated SH-SY5Y cells.     

单性结实和自花结实柑橘果实发育过程中IAA、ZR和GA3含量的变化

对单性结实的龟井温州蜜柑(以下简称龟井)和自花结实的鄂柑1号橘(以下简称鄂柑1号)果实发育过程中不同部位的IAA、ZR和GA3的含量变化进行了测定。结果表明：(1)两品种果皮IAA含量呈相似的变化趋势,均于果实增大期间出现明显高峰,但鄂柑1号的IAA高峰值显著较高;二者果皮ZR含量动态却相反,增大期间龟井果皮ZR的含量相对较高且趋上升,而鄂柑1号却趋下降;二者果皮GA3含量均于增大期间出现类似的上升过程,之后均趋下降。(2)龟井果肉IAA、ZR和GA3含量前期均较高,此后均明显下降并居较低水平;而鄂柑1号果肉IAA和GA3均于增大期间出现明显的上升且含量明显高于龟井,而ZR含量与龟井一样呈下降趋势,但ZR的含量更低。(3)鄂柑1号种子的IAA、ZR和GA3在花后72d均相对较高,之后急剧回落至相对较低的稳定水平。     

Organic Acid Secretion and Phosphate Solubilizing Efficiency of Pseudomonas sp. PSB12: Effects of Phosphorus Forms and Carbon Sources

Secretion of organic acids is an important mechanism for phosphate solubilizing bacteria (PSB) to dissolve insoluble phosphorus in soil. However, the composition of organic acids produced by PSB in the presence of different phosphorus compounds is poorly known, and little is known about the ability of PSB to degrade pollutants in sediment. In this study, we isolated a strain Pseudomonas sp. PSB12 from the sediment of the Qihe River. PSB12 had maximum phosphate solubilization index (SI) of 3.86 on Pikovskaya's agar medium. The phosphate solubilizing activity was associated with the release of organic acids produced from glucose, while the composition of organic acids produced by PSB12 was dependent on the phosphorus forms. When initial soluble phosphorus was insufficient (in MP1 and MP2 media), gluconic acid was the predominant organic acid. In contrast, formic acid, butyrate, and propanedioic acid were the main organic acids produced when only soluble phosphorus (MP3) was supplied. RT-PCR indicated that the expression of glucose dehydrogenase gene (gcd) of PSB12 was two- to four-fold higher in MP1 than in MP3. PSB12 also possessed the phenol hydroxylase gene (phe) suggesting that phenol could be used as the carbon source to dissolve insoluble phosphorus. PSB12 is a potential candidate for in situ bioremediation and for promoting plant growth in soil contaminated by phenol with low levels of soluble phosphorus.     

绿色荧光蛋白标记的D-氨基酸氧化酶基因在人宫颈癌细胞中的表达研究

为了探讨绿色荧光蛋白标记的红色酵母D 氨基酸氧化酶 (DAAO)基因在人宫颈癌细胞 (HeLa细胞 )中的表达及其功能 ,采用基因重组技术构建了含有CMV启动子和EGFP、DAAO基因开放阅读框 (ORF)的真核表达载体 pIRES DAAO。脂质体法转染HeLa细胞 ,荧光显微镜下观察转染细胞中绿色荧光蛋白的表达 ,流式细胞术分析转染效率并筛选荧光阳性细胞 ,命名为HeLa D。以不同浓度的前药D Ala处理HeLa D细胞 ,MTT法检测细胞存活率。结果显示 ,荧光显微镜下可见绿色荧光蛋白在HeLa D细胞中表达 ,流式细胞术成功筛选出HeLa D细胞。前药D Ala能明显杀伤HeLa D细胞。结果表明 ,EGFP可作为报告基因快速筛选DAAO表达载体转染的细胞 ,DAAO/D Ala自杀基因系统可进一步用于肿瘤的基因治疗研究     

nm23-H1基因转染L9981肺癌细胞前后基因表达谱的变化

应用基因芯片检测L9981细胞转染nm23-H1基因前后细胞基因表达谱的改变.提取L9981细胞转染nm23-H1基因前后细胞的总RNA,纯化为mRNA后再转录为cDNA.cDNA经限制性内切酶Sau3AI消化后,cDNA片段分别用cy3和cy5标记,与定制的包含14000个基因芯片杂交.杂交结果经扫描和软件分析,nm23-H1基因转染L9981细胞后发现1156(8.26%,1156/14000)个基因表达上调,而642(4.59%,642/14000)个基因表达下调.涉及基因包括信号传导、癌基因与抑癌基因、转移相关基因、细胞周期与凋亡、细胞外基质与细胞骨架相关基因,以及细胞因子和转录因子等.nm23-H1基因是通过对转移相关基因的调节来发挥其抑制肺癌细胞株L9981侵袭和转移作用的.     

Influence of environmental factors on the emissions of gaseous formic and acetic acids from orange (Citrus sinensis L.) foliage

Gaseous acids can be emitted as well as taken up by plant foliage, but little is known about the influence of environmental factors on the exchange process. In a laboratory study we investigated the short-term effects of temperature and light on the exchange of acetic and formic acids between orange foliage and the atmosphere. The results were compared with diurnal exchange cycles obtained with the same species under field conditions. In the field, the exchange of volatile acids showed pronounced diurnal variations with maximum emissions of about 0.15 nmol m^-2 projected leaf area s^-1 for acetic acid and 0.3 nmol m^-2 s^-1 for formic acid during noon and afternoon and with acid deposition of up to 0.1 nmol m^-2 s^-1 for both acids during early morning and night. Under laboratory conditions no significant acid deposition (<0.01 nmol m^-2 s^-1) could be observed, and emission rates were lower than in the field and ranged around 0.035 nmol m^-2 s^-1 (SD = 0.013) for acetic acid and about 0.08 nmol m^-2 s^-1 for formic acid (SD = 0.033) at 30 °C and 1000 mol m^-2 s^-1 PAR. A clear positive response of acid release to light could be observed. Emissions were very low in darkness and strongly increased with light up to 1000 mol m^-2 s^-1 PAR. The emission response to light occurred within our hour and was also observed with other acids emitting plant species investigated. Response of emission to temperature was more variable, though, on the whole, the effect was also positive. Finally, we observed a diurnal rhythm in acid release which was not related to other measured climatic or physiological parameters. We conclude that in the short-term the exchange of volatile acids between leaves and air under natural conditions is stimulated by light and, somewhat, by temperature, while a leaf-internal process promotes acid release in the morning and higher acid concentrations in ambient air promote acid deposition.     

三价铁螯合物还原酶在香橙和枳中的表达

用耐缺铁的香橙 (CitrusjunosSieb .exTanaka)和极不耐缺铁的枳 (Poncirustrifoliata (L .)Raf.) ,在铁胁迫条件下对根的三价铁螯合物还原酶活性变化和酶基因的表达情况进行了研究。离体根的酶活性测定表明 ,在铁胁迫 4周时 ,香橙根的酶活性增强约 2 0倍 ,枳仅增强约 3倍。用拟南芥的三价铁螯合物还原酶基因作探针进行组织印迹的Northern杂交检测香橙和枳三价铁螯合物还原酶的mRNA ,在铁胁迫 2周时 ,香橙吸收根、幼茎和新叶中均检测到强烈的表达信号 ,而枳相同器官的表达信号则极其微弱。实验结果表明 ,三价铁螯合物还原酶活性在缺铁胁迫下被诱导强烈增加是香橙耐缺铁的重要原因 ,该酶活性的调控发生在转录水平上 ,而且该酶基因在诱导条件下在根、茎和叶中均有表达。     

三价铁螯合物还原酶在香橙和枳中的表达

用耐缺铁的香橙(Citrus junos Sieb. ex Tanaka)和极不耐缺铁的枳(Poncirus trifoliata (L.) Raf.),在铁胁迫条件下对根的三价铁螯合物还原酶活性变化和酶基因的表达情况进行了研究.离体根的酶活性测定表明,在铁胁迫4周时,香橙根的酶活性增强约20倍,枳仅增强约3倍.用拟南芥的三价铁螯合物还原酶基因作探针进行组织印迹的Northern杂交检测香橙和枳三价铁螯合物还原酶的mRNA,在铁胁迫2周时,香橙吸收根、幼茎和新叶中均检测到强烈的表达信号,而枳相同器官的表达信号则极其微弱.实验结果表明,三价铁螯合物还原酶活性在缺铁胁迫下被诱导强烈增加是香橙耐缺铁的重要原因,该酶活性的调控发生在转录水平上,而且该酶基因在诱导条件下在根、茎和叶中均有表达.     

病毒感染相关基因微阵列的制备及其在HBV感染应答基因筛选中的应用

为筛选乙型肝炎(乙肝)病毒(HBV)感染应答基因,探讨HBV感染分子机理,采用生物信息学分析、筛选宿主细胞中与乙肝病毒、丙型肝炎(丙肝)病毒、流行性感冒(流感)病毒等感染密切相关的基因,设计并合成寡核苷酸探针,制备了含231种病毒感染相关基因的寡核苷酸微阵列.利用此微阵列比较HepG2细胞、HepG2.2.15细胞之间的基因表达谱差异,筛选乙肝病毒感染候选应答基因,从分子水平对乙肝病毒感染作用机理进行初步研究.制备的病毒感染相关基因表达谱微阵列的监测结果显示,阳性对照和看家基因探针出现较强信号,空白点样液和阴性对照探针未出信号,大部分基因探针信号强度在可分析范围内,上矩阵和下矩阵反映的基因表达情况一致,证明微阵列的特异性、敏感性、重复性都较好.HepG2.2.15与HepG2细胞基因表达谱比较结果显示,28个宿主基因在HepG2.2.15细胞中高表达,包括ASGR1、AFP、Fibronectin、APOC等基因;4个基因低表达,包括RRM1、ICSBP等基因.初步筛选获得HBV感染候选应答基因.此结果表明,制备的微阵列敏感性、特异性、重复性好,可为研究病毒宿主相互作用关系提供技术平台,应用此微阵列筛选获得的HBV候选应答基因可为揭示HBV感染的分子致病机理提供新的信息,为抗HBV药物研究提供潜在的作用靶点.     

Early and Late Gene Changes in MPTP Mice Model of Parkinson's Disease Employing cDNA Microarray

Recently, we reported specific brain gene expression changes in the chronic MPTP model in the late stage of degeneration, employing cDNA expression array, which indicate a domino cascade of events involved in neuronal cell death. In an attempt to elucidate early gene expression profile in the region of the substantia nigra (SN) and the striatum of acute MPTP-treated mice (3–24 h), we elected a restricted number of genes affected by the long-term MPTP treatment, and their expression was examined. Specifically, we detected alterations in the expression of genes implicated in oxidative-stress, inflammatory processes, signal transduction and glutamate toxicity. These pro-toxic genes appear to be compensated by the elevated expression in trophic factors and antioxidant defenses, which are also activated by short exposure to MPTP. The time course of these gene expression changes indicates the importance of investigating the early gene cascade of events occurring prior to late nigrostriatal dopamine neuronal cell death.     

单性结实和自花结实柑橘果实发育过程中IAA、ZR和GA3含量的变化

对单性结实的龟井温州蜜柑(以下简称龟井)和自花结实的鄂柑1号橘(以下简称鄂柑1号)果实发育过程中不同部位的IAA、ZR和GA₃的含量变化进行了测定。结果表明:(1)两品种果皮IAA含量呈相似的变化趋势,均于果实增大期间出现明显高峰,但鄂柑1号的IAA高峰值显著较高;二者果皮ZR含量动态却相反,增大期间龟井果皮ZR的含量相对较高且趋上升,而鄂柑1号却趋下降;二者果皮GA₃含量均于增大期间出现类似的上升过程,之后均趋下降。(2)龟井果肉IAA、ZR和GA₃含量前期均较高,此后均明显下降并居较低水平;而鄂柑1号果肉IAA和GA₃均于增大期间出现明显的上升且含量明显高于龟井,而ZR含量与龟井一样呈下降趋势,但ZR的含量更低。(3)鄂柑1号种子的IAA、ZR和GA₃在花后72d均相对较高,之后急剧回落至相对较低的稳定水平。