黄肉苹果转色期前后类胡萝卜素合成相关差异表达基因鉴定

为了了解黄肉苹果种质成熟时富集类胡萝卜素的分子机制,以黄肉种质‘东北黄海棠’为材料,选择转色期前后(盛花后90 d和115 d)两个发育时期的果实进行RNA-seq测序,分析两个样本差异表达基因,并利用实时荧光定量PCR技术验证获得的类胡萝卜素合成相关差异表达基因的表达水平。结果获得两个样本显著差异表达基因共3 056个,与盛花后90 d果实比较,成熟期果实中有1 270个基因上调表达,1 786个基因下调表达。对这些表达差异基因进行了Pathway富集分析,结果显示差异表达基因涉及类胡萝卜素、苯丙氨酸以及类黄酮等代谢途径,其中类胡萝卜素代谢途径基因有3个上调,4个下调。通过对这7个差异表达基因进一步的qRT-PCR及聚类分析,发现一个新的候选基因MdCCD4b,该基因与菊花以及桃等CCD4聚为一类,与其他作物功能已知的CCD4一样,其表达水平与黄肉种质中类胡萝卜素积累呈负相关,推测黄肉种质果实成熟后类胡萝卜素的富集与MdCCD4b基因显著下调有关。     

‘砀山酥梨’褐皮芽变木质素含量及相关酶活性与CCoAOMT 表达量分析

 为了探讨‘砀山酥梨’芽变品系‘锈酥’果皮褐色形成机理,采用分光光度法测定盛花后25、50、75、100、125、150和175 d果皮中木质素含量和相关酶活性变化;从构建的‘锈酥’正向SSH-cDNA文库中筛选出与木质素生物合成密切相关的CCoAOMT-EST,通过实时荧光定量PCR测定了‘砀山酥梨’和‘锈酥’果皮中CCoAOMT的相对表达量。结果表明：‘锈酥’果皮发育前期木质素增量较大,且木质素增量累计比‘砀山酥梨’高12.2%;‘砀山酥梨’和‘锈酥’果皮中PAL、4CL、CAD酶活性均在花后75 d达到最大值,而POD酶活性则在花后125 d出现高峰;二者果皮中4种酶活性变化趋势基本一致,但‘锈酥’果皮中均相对较高;‘锈酥’果皮中的PAL和4CL酶活性与木质素增量变化均呈显著正相关,而‘砀山酥梨’则未呈现出此规律;在果实生长发育各个时期,‘锈酥’果皮中CCoAOMT相对表达量均高于‘砀山酥梨’。因此推测,‘锈酥’果皮褐色形成与果皮中木质素积累及相关酶活性提高有关,果皮中CCoAOMT的增量表达是‘锈酥’果实褐皮形成的重要原因之一。     

茄子单性结实差异表达序列鉴定及分析

利用实时荧光定量PCR技术,研究茄子单性结实SSH-c DNA文库中的96条EST序列在单性结实自交系和非单性结实自交系果实发育过程中的表达模式。分析表明,在低温条件下,相对表达量有显著差异的EST序列有31条,总体上调表达的EST序列有17条,总体下调表达的EST序列有14条。通过NCBI对EST序列进行Blastx比对,得到与其同源性高的序列信息。其中5条EST与抵御低温相关,6条EST与植物激素代谢相关,多条EST与植物代谢过程中的蛋白质和碳水化合物合成相关,1条EST无比对结果,可能为新基因。差异表达序列可作为研究茄子单性结实和耐低温的候选基因。     

梨两个休眠相关MADS-box 基因特征及在其休眠过程中的表达分析

 PpMADS1 和PpMADS2 是从‘酥梨’（Pyrus pyrifolia white pear group‘Suli’）休眠芽转录组 文库筛选的两个与休眠相关的MADS-box 基因序列。为了解序列的特征,对其进行了相关生物信息学分 析,并以‘翠冠’和‘圆黄’梨为试材,用实时定量PCR 技术分析其花芽休眠不同阶段的表达变化。结 果表明：两个基因都具有MADS-box 家族的特征序列MIKC-基序,PpMADS1 和PpMADS2 分别与日本梨 （P. pyrifolia‘Kosui’）休眠相关的两个MADS-box 基因PpMADS13-1 和PpMADS13-2 聚在一起,且单独 聚为一支,与李属植物休眠相关的MADS-box 基因关系最近。在两个品种的休眠过程中,PpMADS1 和 PpMADS2 的表达呈现相似的变化趋势,都有一个表达高峰,但‘翠冠’梨PpMADS1 和PpMADS2 的表 达高峰均出现在11 月15 日,而‘圆黄’梨PpMADS1 的表达高峰延后到12 月30 日,PpMADS2 的表达 高峰出现在12 月15 日。两个品种PpMADS1 和PpMADS2 的表达高峰都出现在内休眠解除之前,随内休 眠解除其表达量下调,在生态休眠阶段表达量维持在较低的水平。据此推测PpMADS1 和PpMADS2 的表 达对梨芽内休眠的解除具有调控作用     

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’.     

金枝黄花柳回交子代枝条颜色与转录组研究

为了探究枝条颜色变异的原因，以金枝黄花柳回交子代及其亲本为材料，分析子代枝条性状遗传变异规律及性状与基因表达的关系。结果显示，金枝型回交子代枝条表现为黄色到橙色，普通型回交子代枝条表现为墨绿色至棕色，不同单株个体之间存在颜色差异。枝条转录组共检测到差异表达基因2 499个，其中上调基因984个，下调基因1 515个。功能注释和富集分析发现，卟啉与叶绿素代谢通路(KEGG&ko00860)中与色素合成有关的基因表达量存在差异，其中叶绿素/细菌叶绿素a合成酶(2.5.1.133;2.5.1.62)与叶绿素/细菌叶绿素a还原酶(1.3.1.111)基因表达上调，叶绿素酶(3.1.1.14)基因表达下调。基因差异表达会影响代谢通路，进而影响枝条中叶绿素的含量，导致枝条颜色存在差异。     

仁用杏花芽3个发育时期数字基因表达谱分析

以仁用杏‘优一’为试材,运用转录组、数字基因表达谱技术,研究了仁用杏花芽萌动期前后相关基因的表达规律。结果发现,仁用杏花芽在花芽萌动期前后进行着各种旺盛的生物合成和代谢活动,且4个开花调控途径（光周期途径、春化途径、自主途径、GA调控途径）均已启动,光周期途径在花芽萌动时发挥重要作用,涉及到了74条基因,其中有38条差异基因上调表达,29条下调表达;GA途径在花芽萌动期对花芽的生长发育调控表现为抑制作用,涉及到了60条基因,其中有13条差异基因上调,9条差异基因下调;其他两条途径于花芽萌动前作用,其中春化途径涉及到了23个基因,其中有4条差异基因上调,15条差异基因下调;自主途径涉及到了24条基因,其中有3条差异基因上调,11条差异基因下调。本研究通过数字基因表达谱分析,初步了解仁用杏花芽萌动前后的网络途径,为后期对仁用杏花芽相关开花基因的研究、探明仁用杏早花的分子机理及通过分子育种方法培育仁用杏晚花品种提供坚实的理论依据。     

几种果实不同组织总RNA提取及质量分析

以菊水梨为试验材料,通过2种改良CTAB法对果实不同成熟阶段提取的总RNA质量和产量的变化的影响,研究果实不同成熟阶段果皮和果肉总RNA提取的质量和产量,并针对梨果实不同成熟阶段采用不同的改良CTAB法,以较低的成本从果实中提取完整性好、质量高的总RNA。同时提取近成熟的红富士葡萄、富士苹果和丰香草莓3种果实总RNA。总RNA的质量和产量分析结果表明,方法Ⅰ和Ⅱ分别适用于成熟度较低和较高梨果实总RNA的提取,方法Ⅰ也适用于其它3种近成熟果实总RNA提取。并通过RT-PCR验证,所提取的总RNA可以满足基因克隆和表达等分子生物学实验的要求。     

K+/H+逆向转运体基因PpeKEA在桃开花期的表达及对钾肥施用的响应

从‘霞晖8号’桃花中克隆并鉴定了6个K+/H+逆向转运体基因PpeKEA,在转录水平分析其在桃花发育不同时期的表达模式及对钾肥施用的响应,明确关键基因并验证其功能。结果表明,钾肥施用显著增强盛花期桃花的鲜样质量,提高花朵钾素富集水平。PpeKEA1 ~ PpeKEA6在桃花开放不同时期的表达水平存在差异,均在盛花期达到最高,其转录水平对施用钾肥的响应不同;PpeKEA1在整个桃花开放不同时期的表达量较高,钾肥处理显著增强其在花蕾膨胀期和初开期的表达并抑制其在败落期的表达量;表达载体pDR196-shortKEA1能赋予酵母菌感受态细胞AXT3突变体适应高钾环境而正常生长,说明PpeKEA1的C端区域具有调节桃花组织内K+转运和平衡的功能。本研究表明PpeKEA1是一个桃花开放过程中调节K+平衡的K+/H+逆向转运体,并可能在桃树钾素营养的动态平衡中起着重要作用。     

Physiological and Technical Aspects of Cactus Pear [Opuntia ficus-indica (L.) Mill.] Double Rellowering and Out-of-Season Winter Fruit Cropping

Abstract

A commercial cactus pear plantation in Sicily, Italy was manipulated to induce late cropping. The spring flush of flowers and cladodes were removed as was the second induced bloom of flowers and cladodes. The third induced bloom was harvested for a late out-of-season crop of cactus pear (Opuntia ficus-indica Mill.). The double removal induced a third flush of flowers and cladodes during late August with a fruit production that ripened the following winter (to March). The number of flowers per fertile cladode was halved after the double removal and the length of the fruit development period increased from 100-120 days to 160-190 days for the out-of-season crop. Polyethylene covering reduced sunlight but was essential for establishing optimal temperatures for cladode photosynthetic activity and fruit growth and ripening. Out-of-season fruits were regular in size and percent flesh with only a slight reduction in total soluble solid content.     

Effect of soil water stress on the development of stone cells in pear (Pyrus pyrifolia cv. ‘Niitaka’) flesh

This study was carried out to investigate the cause of stone cell formation in pear (Pyrus pyrifolia cv. ‘Niitaka’) flesh. Potted plants grown in a glass house were subjected to water stress conditions without irrigation for 30 days from 30 days before full bloom (BFB treatment), full bloom (FB treatment) and 30 days after full bloom (AFB treatment). Control plants were drip-irrigated daily maintaining a soil matrix potential around −40 ± 5 kPa. The formation of stone cells in pear flesh increased in the FB treatment and AFB treatment plants and this tendency was sustained until the harvest season. Root activity was investigated 60 days after full bloom (DAFB) and the triphenyltetrazolium chloride (TTC) reduction potential, the formazan content and leaf water potential were investigated 30, 45, and 60 DAFB. Root activity decreased progressively due to the effect of water stress. Also, the Ca content in leaf and flesh was lower. The peroxidase activity was high in the flesh at the early stages of fruit growth and decreased at the late stages of fruit growth, and then a higher increase of peroxidase activity was observed in water-stressed fruit. The reduction in calcium content of leaf and fruit in plants under water stress may be related to the reduction of root activity and leaf water potential. The increase in peroxidase activity under water stress may be due to limited calcium absorption. Higher peroxidase activity may induce the accumulation of lignin in the cell wall and promote the formation of stone cells in pear flesh. We conclude that water stress condition during the early stages of fruit growth is one of several factors that determine the formation of stone cells in pear flesh.     

辣椒N 基因介导抗根结线虫作用早期表达基因的抑制性消减杂交SSH分析

 以含N 基因的辣椒(Capsicum annuum L. ) 为试验材料, 接种南方根结线虫(Meloidogyne incognita) 12、24、36 h的根尖材料作为测验方( tester) , 相应的未接种根尖材料作为驱动方( driver) , 构建一个南方根结线虫诱导N 基因表达早期的正向抑制消减杂交cDNA文库, 并结合文库高密度点阵膜杂交差异筛选, 获得了237条表达序列标签( EST) 。在GenBank上进行BLASTn与BLASTx分析, 得到148条功能已知的EST序列, 获得已知的上调抗性相关EST 68个。分离出了具有NBS2LRR结构的抗线虫蛋白和类LRR抗性蛋白的基因, 防御作用相关的类萌芽素(GLP) 、HSR203J 蛋白、蜜腺蛋白、蛇毒素肽等基因,抗性相关的WRKY、ERFBP等转录因子基因, 以及G蛋白、142323蛋白等多种信号蛋白基因。通过GeneOntology分析, N 基因介导的早期表达抗病基因涉及病原物的识别、抗性信号传导、过敏性坏死、系统获得性抗性以及植物细胞保护机制等多个方面, 并有许多功能未知的基因有待于进一步的研究。     

不同中间砧对苹果果实苹果酸代谢关键酶活性及其相关基因表达的影响

为深入探究中间砧影响苹果果实苹果酸代谢的机理,以SH40实生后代（代号53、111和236）为中间砧嫁接的‘天红2号’苹果树为试材,测定果实发育过程中苹果酸含量、相关代谢酶活性及基因相对表达量。结果表明：果实成熟时,以53号为中间砧嫁接的‘天红2号’果实苹果酸含量显著高于以111号为中间砧的。盛花后30、40、100 ~ 160 d,以53号为中间砧的果实中苹果酸脱氢酶（NAD-MDH）活性显著高于以111号为中间砧的;盛花后30、40和130 d,53号处理的磷酸烯醇式丙酮酸羧化酶（PEPC）活性显著高于111号处理的;盛花后100 d,以111号为中间砧的果实中苹果酸酶（NADP-ME）活性显著高于以53号为中间砧的。基因表达结果显示,盛花后100和160 d,以53号为中间砧的NAD-MDH基因相对表达量显著高于以111号为中间砧的,同时NADP-ME基因表达量也显著高于以111号为中间砧的;盛花后30 和160 d,以53号为中间砧的PEPC基因相对表达量显著高于以111号为中间砧的。     

葡萄活性赤霉素合成关键基因VvGA3ox家族的克隆和表达分析

在植物赤霉素合成代谢中,GA3ox可将几种非活性赤霉素转化为具生理活性的赤霉素。以葡萄有核品种‘黑比诺’和无核品种‘无核白’为材料,克隆葡萄的VvGA3ox基因家族成员,分析基因结构、进行染色体定位和系统发育树构建,检测其组织器官表达特性和在胚珠（幼种子）发育过程的表达变化。结果表明,葡萄基因组VvGA3ox家族有3个成员,cDNA长度分别为1 313、1 225和1 480 bp,都包含2个外显子和1个内含子结构。组织器官特异性表达分析表明,VvGA3ox1在胚珠中的表达很低,在根中高表达;VvGA3ox2和VvGA3ox3在花和胚珠中的表达较高。在受精后胚珠（幼种子）发育过程中,VvGA3ox家族基因的表达趋势存在差异,其中VvGA3ox1在‘黑比诺’与‘无核白’中均是在盛花后30 d时上调之后下降;VvGA3ox2在‘黑比诺’中20 ~ 30 d极速上调之后下降,相应地‘无核白’中是先缓慢下降之后上升;VvGA3ox3在‘黑比诺’中逐渐下降,对应地‘无核白’中是在10 ~ 15 d先上升后在20 ~ 45 d呈下降趋势并逐渐与‘黑比诺’中的表达持平。VvGA3ox在有核和无核葡萄品种中表现较大差异表达,与葡萄无核性状有一定关系。     

Screening of the differentially expressed genes in lymphocytes of patients with unstable angina with suppression subtractive hybridization

AIM: To screen and identify the differentially expressed genes in lymphocytes of patients with unstable angina in order to find the molecular mechanism of unstable angina . METHODS: Suppression subtractive hybridizations (SSH) and dot blot hybridizations were performed to screen the relatively differentially expressed genes in lymphocyte RNA between the patients with unstable angina pectoris and stable angina pectoris. The obtained expressed sequence tags (ESTs) were used as probes to perform Reverse Northern blot with forward and reverse suppression products. And the positive ESTs were performed RNA slot hybridization with unstable and stable angina group. The obtained ESTs were sequenced and analyzed using BLAST (nr) at NCBI. RESULTS: Three up-regulated ESTs in the unstable angina group, and one down-regulated EST in the stable angina group were obtained. All of them are sequences of known genes. CONCLUSION: All these ESTs may be associated with the unstablization of plaque of coronary artery in patients with unstable angina.     

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.