Functional genomics of maize submergence tolerance and cloning of the related gene <Emphasis Type="Italic">Sicyp51</Emphasis>

In this study, SSH (Suppression Subtractive Hybridization) and cDNA microarray were used to identify genes associated with waterlogging response of maize roots. Mo17 and Hz32 are two maize inbred lines with differential tolerance to hypoxia. Seedlings of the inbred lines with two leaves were submerged in hypoxia buffer. SSH libraries were constructed with cDNA samples from roots. Both forward and reverse subtractions were performed for each inbred line, and 105 positive clones induced by hypoxia were selected by differential screening. The treated and control message RNA were hybridized with the cDNA microarray of Mo17, sequentially, 57 of 3-fold differentially expressed clones were obtained. A total of 162 positive clones were all sequenced. Bioinformatics analysis showed these positive clones represent 85 TUGs, including genes involved in several biochemistry pathways, such as glycolysis, protection, signal transduction, cell construction and energy metabolism and 41 EST with unknown function. Comparison between Mo17 and Hz32 indicates that genes related to hypoxia tolerance have different expression patterns in submerged roots. Several positive clones' expression patterns were revealed by Northern or RT-PCR, and a new gene (Sicyp51), which may contribute to hypoxia tolerance, was identified.     

Functional genomics of maize submergence tolerance and cloning of the related gene Sicyp51

In the middle and lower Yangtze River area, the major corn-growing region of South China, seasonal rainfall greatly affects maize plantation. Maize seed-lings meet with excessive precipitation and low tem-perature in spring, and when they grow up to begin flowering, they usually encounter Mei-yu storm ac-companied by hot days. At the same time, bad irriga-tion system and a higher level of underground water cause waterlogging, which further leads to yield losses. In order to reveal the molecu…     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

条锈菌诱导的小麦抗病与感病近等基因系SSH文库构建及分析

为分析条锈菌诱导下的小麦抗病与感病近等基因系之间差异表达的基因,以接种小麦条锈菌CY26小种的抗病近等基因系Yr4/6×Taichung 29幼苗叶片cDNA作为实验方,接种CY26的感病亲本Taichung 29幼苗叶片cDNA为驱动方,利用抑制消减杂交(SSH)技术构建了一个包含1 300余克隆的消减文库。对文库中600个克隆进行了反向Northern点杂交筛选,对获得的阳性克隆进一步进行了Northern杂交验证,获得显著差异的克隆12个。经测序和BlastX分析,其中6个差异表达序列的推测产物分别为亮氨酸重复序列蛋白、过氧化氢酶、硫氧还蛋白、RNA结合蛋白、抗坏血酸过氧化物酶和热激蛋白。除亮氨酸重复序列为信号传导类蛋白外、其他几个均为抗病防御类蛋白。     

无核荔枝果实形成差异表达基因cDNA的克隆

采用抑制差减杂交技术(Suppression Subtractive Hybridization,SSH)分离与海南无核荔枝果实形成相关的差异表达基因的cDNA片段,为克隆相关基因提供研究基础。分别以无核荔枝的有核幼果为driver;无核幼果为tester,建立差减cDNA文库。经Reverse Northern Dot-Blot筛选该文库,共获得61个阳性克隆,随机选取17个克隆进行测序,共获得10条非重复序列,对其中较长的7个序列进行同源分析,结果表明:有6个序列在荔枝中为首次报道。     

利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因

以晚疫病病原菌混合小种接种处理48h的马铃薯水平抗性材料(R-gene-free)叶片为目的材料,以未处理材料作为对照,用抑制差减杂交技术构建了一个富集晚疫病抗性相关基因的差减文库。应用反向Northern技术对840个克隆进行斑点杂交筛选,筛选出150个病原诱导后信号明显增强的克隆。26个片段测序结果表明：部分片段基因功能与抗病性明显相关。7个差异表达片段与GenBank EST数据库中已有晚疫病原诱导马铃薯叶片得到的EST有很高同源性(达95％～100％);部分片段核苷酸或氨基酸序列分别与番茄、烟草、拟南芥等的EST序列或氨基酸序列有较高同源性;另有4个基因片段在GenBank EST数据库中未找到明显的同源序列,可能为新发现的基因片段。     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2 transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways.The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (A1) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to A1 stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg^2 transportation, and other functions. Under A1 stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to A1 stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

血管紧张素Ⅱ诱导心肌成纤维细胞表达上调基因的分离和鉴定

为了对成年大鼠心肌成纤维细胞（cardiac fibroblasts,CF）受血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）刺激后上调基因表达谱进行筛选及分析,以受AngⅡ刺激CF为实验方,未刺激CF为驱动方,进行抑制消减杂交（suppression subtractive hybridization,SSH）,建立消减cDNA文库。经斑点杂交筛选文库后将表达变化显著的部分阳性克隆测序及同源性分析,共获得19个上调表达的基因,分别与细胞外基质、细胞周期、胞内信号转导、细胞骨架及细胞代谢等功能相关,并克隆到7个新的基因表达序列标签（expressed sequence tags,EST）。我们的数据证实了SSH可以有效地克隆成年大鼠CF受AngⅡ刺激后上调表达基因,对这些基因的研究将有助于阐明心肌重塑的分子机制。     

小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因

应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。