杨梅酸性转化酶基因cDNA分离及表达分析

杨梅果实富含蔗糖,酸性转化酶是蔗糖代谢关键酶,根据植物酸性转化酶基因保守区序列设计引物,提取杨梅叶片RNA,逆转录获得cDNA,以此为模板通过PCR技术扩增到长度为516bp的基因片段,克隆入pMD18-T载体中,命名为MrIVR1(GenBank:DQ339699)。测序及同源性检索表明,该基因推导氨基酸序列与君子兰、葡萄、草莓、胡萝卜等酸性转化酶基因氨基酸序列同源性为60%～69%。运用ClustalX软件对植物转化酶基因进行了系统树分析,结果显示,MrIVR1编码的蛋白质属于细胞壁酸性转化酶。半定量RT-PCR表达分析显示,MrIVR1基因在杨梅果实发育早期表达量最高,随着果实的发育表达量下降,在成熟果实中表达水平较低。     

Molecular cloning and expression analysis of the gene encoding sedoheptulose-1, 7-bisphosphatase from Cucumis sativus

Sedoheptulose-1, 7-bisphosphatase (SBPase; EC 3.1.3.37) is a key enzyme in the photosynthetic carbon reduction cycle (Calvin cycle). In the present study, a full-length cDNA encoding SBPase (designated as CsSBP, GenBank accession no. FJ911553) was isolated from cucumber (Cucumis sativus L. cv. ‘Jinyou 3’) grown in solar-greenhouse by RT-PCR and RACE. The cDNA contained 1452 nucleotides with a complete open reading frame (ORF) of 1167 nucleotides, which was deduced for encoding a peptide of 388 amino acids whose molecular mass was inferred to be 42 kDa with its isoelectric point at 6.17. Sequence comparison analysis showed that the deduced amino acid of SBPase from cucumber leaves had high homology to that from other species. Expression analysis by real-time PCR and Western blot revealed that CsSBP mRNA and protein was most abundant in leaves, detectable in stems and fruits, but invisible in roots. A similar difference was found in SBPase activity among leaves, stems, fruits and roots. CsSBP expression at the mRNA and protein level was the highest in mid-position leaves (4th–8th apical expanded leaves) of plants, medium in base position leaves (12th), and the lowest in upper position leaves (1st). However, SBPase activity decreased successively as leaf position declined. The net photosynthetic rate (Pn) of mid-position leaves was also found to be the highest, but that of base-position leaves was lower than upper-position leaves. The diurnal variations of gene expression and SBPase activity in optimal functional leaf (6th leaf) was a single-peak pattern curve, which was similar to that of Pn, and the peak appeared at 12:00. These data indicate that CsSBP expresses only in green tissues of cucumber plants and there are close positive correlations between SBPase activity and gene expression at the mRNA and protein levels, and between SBPase activity and Pn in leaves of cucumber plants.     

几个早熟苹果品种果实糖酸组分及风味品质的评价

采用高效毛细管电泳仪分离测定了6个早熟苹果品种果实的糖酸组分,并对早丰甜、贝拉、辽伏3个品种可溶性糖、可滴定酸、糖酸比及主要香气成分含量与风味品质的关系进行了研究.结果表明,(1)所有参试品种均以果糖含量最高,品种间变异系数最小,仅为14.59%,但蔗糖和葡萄糖含量与果糖明显不同,在不同品种间存在较大差异,品种间变异系数分别为67.58%和29.94%,果糖和蔗糖含量是影响早熟苹果品种糖类总含量的主要因素.有机酸中苹果酸含量在不同早熟苹果品种也存在较大差异,最高为6.61 mg/g,最低仅为1.55 mg/g;(2)果实可溶性糖、可滴定酸、糖酸比及3种主要香气成分测定和官能鉴评结论在早丰甜、贝拉和辽伏3个品种间存在显著差异,官能鉴评的这种差异性与糖、酸及香气成分等成分测定结果基本一致,因此,在试验条件下,3个早熟苹果品种风味品质评价指标应包括可溶性糖含量、可滴定酸含量、糖酸比及1-丁醇、(E)-2-己烯醛和乙酸丁酯等3种主要香气成分的总含量.     

翠冠梨PG基因家族两成员的克隆及其表达与货架期果实软化的关系

以早熟砂梨翠冠果实为材料,克隆了多聚半乳糖醛酸酶（PG）基因家族中的两成员功PGj和PpPG2。PpPGI cDNA序列全长1793bp,开放阅读框为287-1666bp,DNA序列长2757bp,包括8个外显子和7个内含子,编码一个含有459个氨基酸残基的蛋白,预测的等电点（pI）为8．47,估计的相对分子质量为49...     

基于EST数据库的葡萄APETALA2基因cDNA克隆及其表达分析

APETALA2(AP2)是拟南芥生长发育特别是花器官发育过程中重要的调控基因。利用生物信息学方法以拟南芥APETALA2基因cDNA序列作为模板,对葡萄EST数据库进行同源检索筛选,电子克隆出葡萄AP2基因(Vv-AP2)cDNA序列。以香悦葡萄花cDNA为模板,根据电子克隆的葡萄AP2基因cDNA序列设计特异引物,分别利用RACE技术和特异PCR技术获得该基因3’末端和5’末端,序列拼接后获得葡萄的APETALA2基因的cDNA全长。该cDNA的全长为2 208 bp,命名为Vv-AP2。Vv-AP2核苷酸序列有一个1 536 bp完整的开放读码框(ORF),其5’与3’末端非翻译区分别为268 bp和376 bp。其中,3’末端含有28 bp的Ploy+(A)。该基因在GenBank基因数据库的注册号为FJ809943。氨基酸推导结果表明该cDNA共编码了511个氨基酸,具备完全保守的核定位信号序列(KKSR)以及2个高度保守的重复序列即AP2结构域。分别采用半定量RT-PCR和SYBR Green I实时定量RT-PCR初步分析了Vv-AP2在葡萄叶、茎、花序、花等不同器官中的表达水平。其中,Vv-AP2在花序和花中的表达量明显高于叶和茎。     

Identification of organic acid-related genes and their expression profiles in two pear (Pyrus pyrifolia) cultivars with difference in predominant acid type at fruit ripening stage

‘Yandangxueli’ is a pear cultivar with predominant citric acid in the ripe fruit, different from most of pear cultivars such as ‘Gengtouqing’ in which malic acid is the predominant acid type. It was found that ‘Yandangxueli’ accumulated citric acid for three times against that in ‘Gengtouqing’ at fruit ripening stage. To investigate the mechanism of citric acid accumulation in ‘Yandangxueli’, organic acids content, gene expression and enzyme activity were studied in both cultivars. Five genes, Pp:mtCs, Pp:cyAco, Pp:cyIdh, Pp:mtMdh and Pp:cyMe which encoded citric synthase (CS), cytosolic aconitase (cyACO), NADP-dependent isocitrate dehydrogenase (NADP-IDH), NAD-dependent malate dehydrogenase (NAD-MDH) and NADP-dependent malic enzyme (NADP-ME) respectively, were identified from pear fruit. Their expression profiles and the corresponding enzyme activities were determined throughout fruit development in both cultivars. Results from these enzymes indicated that there were no strict relationship between gene expression, enzyme activity and citric acid accumulation. Expression analysis for two Py:vVAtp genes encoding vacuolar H⁺-ATPase A subunit and one Py:vVpp gene encoding Vacuolar H⁺-pyrophosphatase showed that they were all with up-regulated expression at the later development stage of ‘Yandangxueli’ but with down-regulated expression in ‘Gengtouqing’. Therefore, it is concluded that the different ability in citric acid transportation and storage might be involved in the high citric acid content in ‘Yandangxueli’.     

Physico-chemical and antioxidant properties of six apple cultivars (Malus domestica Borkh) grown in southern Brazil

The objective of this study was to compare the physico-chemical properties and antioxidant activity of six apple cultivars grown in southern Brazil. Apple peel color, dry matter, total soluble solids, pH, total sugars, titratable acidity, total phenolics, total monomeric anthocyanin and total antioxidant activity were measured in the apple cultivars Imperatriz, Daiane, Fred Hough, Fuji Suprema, Galaxy and Baronesa. The results showed great quantitative differences in the composition of the apple cultivars. Of all the cultivars, the peel of Galaxy was slightly more red-colored and that of Fred Hough was the least red-colored. The dry matter varied from 15.24% (Galaxy) to 19.55% (Fuji Suprema), the soluble solids content was between 11.8 (Fred Hough) and 14.0 (Daiane) °Brix, pH values varied from 3.90 (Imperatriz) to 4.27 (Fred Hough), the total sugar content (g 100 g⁻¹) ranged from 11.54 (Imperatriz) to 14.78 (Fuji Suprema) and the titratable acidity content (g 100 g⁻¹) varied from 0.20 (Baronesa) to 0.36 (Imperatriz). The total phenolic content (GAE 100 g⁻¹ fresh matter) observed in the apple cultivars was between 105.4 (Baronesa) and 269.7 mg (Imperatriz). The values of the total anthocyanin content (mg 100 g⁻¹ FM) ranged from 4.79 (Fred Hough) to 41.96 (Galaxy). The highest total antioxidant activity was observed in Imperatriz (739 μmol TEAC 100 g⁻¹ FM), while the lowest value was found in Fuji Suprema (335 μmol TEAC 100 g⁻¹ FM). There was a strong correlation between total monomeric anthocyanin content and all peel color measurements and between antioxidant activity and total phenolic content. The results suggested that genotype is the main factor that determines the composition of bioactive compounds in apples and this provides important information on how to make the best use of the apple cultivars investigated.     

Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

香蕉WAT1基因的鉴定及表达分析

【目的】基于香蕉基因组数据筛选Walls are thin 1(WAT1)基因,分析它们的序列及表达特性。【方法】以拟南芥WAT1为参考序列,通过本地Blast筛选获得香蕉WAT1基因,分析其核苷酸、启动子及编码蛋白特性,并利用实时定量PCR技术研究其在不同组织部位、不同激素和逆境胁迫处理下的表达情况。【结果】筛选获得5个香蕉WAT1基因(命名为MaWAT1-1~5)。蛋白亚细胞定位预测结果显示,MaWAT1-1、MaWAT1-2、MaWAT1-4主要定位在液泡和细胞膜上,MaWAT1-3主要定位在细胞质和细胞膜,MaWAT1-5定位在细胞膜和叶绿体。基因结构分析和系统进化树分析均将MaWAT1s分为两组,MaWAT1-1、MaWAT1-2、MaWAT1-4聚为一组(含6个外显子和5个内含子),MaWAT1-3和MaWAT1-5归为一组(含7个外显子和6个内含子)。启动子顺式作用元件分析结果显示:MaWAT1s启动子含有大量激素和胁迫响应相关元件。实时定量PCR结果显示,MaWAT1-4在叶片中表达量最高,MaWAT1-1在根和假茎中表达量最高,其余均在根中表达量最高;大多数MaWAT1s的表达受GA3、SA、盐胁迫和干旱等显著诱导,受高温显著抑制,同时部分成员的表达受IAA、ABA、JA、低温、机械损伤和枯萎病影响显著。【结论】MaWAT1s的表达受多种激素和逆境影响显著,可能在香蕉生长发育和抗逆防御反应中发挥着重要作用。     

全基因组基因表达分析技术及其在果树上的应用

基因表达分析对于揭示基因功能、了解基因间相互关系、阐明植物生长发育过程中的生理生化调控机制起着至关重要的作用。综述了mRNA差异显示、cDNA-AFLP、基因芯片、EST数据分析、基因表达系列分析(SAGE)等全基因组基因表达分析技术的原理特点及在果树上的应用情况,并介绍了利用全基因组基因表达分析技术开展果树研究的思路。     

北京昌平苹果园土壤养分、pH与果实矿质营养的多元分析

2008-2009年,通过对北京昌平34个成龄苹果园土壤养分、pH和果实矿质元素含量的调查和多元统计分析,探讨了果实矿质元素含量最佳的土壤养分含量和pH优化方案.结果表明,土壤有机质与土壤各种养分均呈正相关,土壤养分与果实矿质元素含量的相关性非常弱,单因子相关分析不能揭示土壤养分与果实矿质元素间的关系;典型相关分析表明...     

渭北3种不同类型苹果园冠层特征及光照特性

渭北果区正在推广的旨在提高果园通风透光条件的“大改形”技术已进行几年,为对其效果进行观察,应用WinsCanopy2004a冠层分析仪对陕西渭北3种不同类型苹果园冠层及光照的有关参数进行了测定。结果表明,3类果园的平均冠下总辐射偏大(透光率高),每666.7m2留枝量偏低,说明改形对改善树体的光照条件效果明显,但是改形偏重;中冠园果树的透光率最高,小冠园最低;大冠园果树的叶面积指数最大,当年生枝量与主枝数的比例最大。     

Cloning,molecular analysis,and developmental expression of 3 oleosin cDNA isoforms in coconut (Cocos nucifera L.)

Coconut stores its lipid reserves in the endosperm, specifically in oil bodies that contain proteins called oleosins which stabilise their structure. This study reports the complete cDNA sequences of 3 genes for 3 isoforms of oleosin termed CnOLE500a, CnOLE500c, and CnOLE300a with 396, 375, and 381 nucleotides, respectively. Their predicted amino acid sequences were 131, 124, and 126 residues in length, respectively, with molecular weights of 13,787, 12,982, and 12,988 Da, respectively. The complete CnOLE500a cDNA sequence had 83.1% similarity with that of CnOLE500c, while CnOLE300a cDNA had only 50.7% and 46.5% similarity with CnOLE500a and CnOLE500c, respectively. None of the 3 coconut oleosins had the 18 amino acid insertion characteristics of H-class oleosins, thus they belonged to the L-class of oleosins. However, phylogenetic analysis showed that CnOLE300a was more related to H-class oleosins from dicots. All 3 isoforms were highly expressed at all stages of coconut endosperm development. CnOLE500c exhibited 6% higher expression than CnOLE500a and 15% higher than CnOLE300a at all stages of coconut endosperm development. However, oleosin proteins were barely detectable in the solid endosperm of coconut at 5–6 months, but this increased 20-fold at 6–7 months, and increased by a further 2- and 12-fold, at 7–8 and 8–9 months, respectively.