盐地碱蓬甲硫氨酸合成酶基因(SsMS)的克隆与表达分析

从盐地碱蓬 (Suaedasalsa)中克隆了甲硫氨酸合成酶基因的cDNA片段 .通过Southern杂交和Northern杂交对其表达特性进行分析 ,结果表明 ,同一盐浓度不同时间处理下、双氧水及冷害刺激条件下甲硫氨酸合成酶在碱蓬叶中的表达量先呈现减少然后增加的趋势 ;聚乙二醇 (PEG)透导下呈现下降趋势 .由此可见该酶在盐、氧化和低温胁迫条件下产生一定的反应 .     

NaCl诱导表达的盐藻果糖-1,6-二磷酸醛缩酶基因克隆及原核表达

通过RACE技术克隆到盐藻（Dunaliella salina Teod.)果糖－1，6－二磷酸醛缩酶（DsALDP)的全长cDNA,对其序列分析及在GeneBank中进行同源搜索，发现DsALDP基因属于果糖－1，6－二磷酸醛酶基因家族，其与植物叶绿体果糖－1，6－二磷酸酯缩酶（AldP)基因的一致性为73％－66％。Northern杂交结果及醛缩酶活性分析均表明它角为在盐诱导下特异表达。将DsALDP cDNA构建到pET32a载体上，转入E.coli BL21进行表达分析。IPTG诱导后可以检测到－62ku基因产物大量表达。经不同浓度NaCl处理，表达DsALDP基因产物的细菌耐盐性明显高于对照组。     

盐藻磷酸甘油脱氢酶基因cDNA文库的构建

采用Ｌａｍｂｄａ ｇｔ１０载体构建了盐藻ｃＤＮＡ文库，文库大小为１０＾６／ｍＬ插入片段平均长度大于１ｋｂ。根据果蝇，兔，鼠编码其３－磷酸甘油脱氢酶基因的辅酶ＮＡＤ的结合功能区保守序列设计一对引物，大小分别为２１ｂｐ和１８ｂｐ。ＰＣＲ扩增得到与果蝇相同的２９０ｂｐ特异扩增片段，以该片段为探针，采用缺口平移系统标记原位杂交，从盐藻ｃＤＮＡ文库中获得３６个３－磷酸甘油脱氢酶基因的阳性克隆。     

宝山堇菜Pb耐性相关基因筛选及表达特性

Cd超富集植物宝山堇菜Viola baoshannensis也有一定Pb耐性/富集能力,为了解宝山堇菜对Pb的耐性/富集分子机制,采用抑制消减杂交技术(suppression subtractive hybridization,SSH),筛选了宝山堇菜根在Pb诱导下差异表达的基因片段,进而对与Pb耐性与富集相关的基因克隆片段(expressed sequence tags,ESTs)进行半定量RT-PCR和Northorn杂交验证。利用SSH方法,从宝山堇菜根部获得27个在Pb胁迫下特异表达的无重复的有意义EST克隆片段。半定量RT-PCR和Northern杂交进一步证实和Lycopersicon esculentum木葡聚糖内源转糖基酶(xyloglucan endotransglycosylase,XTH)基因、Arabidopsis thaliana GTP结合蛋白基因(GTP binding mRNA)基因相似的2个克隆片段在宝山堇菜受Pb胁迫时,为上调表达基因。可初步假设,Pb胁迫下,伴随有细胞壁的变化,GTP结合蛋白可能参与宝山堇菜体内Pb胁迫信号的传导。     

盐地碱蓬甲硫氨酸合成酶基因（MsMS）的克隆与表达分析

从盐地碱蓬(Suaeda salsa)中克隆了甲硫氨酸合成酶基因的cDNA片段．通过Southem杂交和Notthem杂交对其表达特性进行分析，结果表明，同一盐浓度不同时间处理下、双氧水及冷害刺激条件下甲硫氨酸合成酶在碱蓬叶中的表达量先呈现减少然后增加的趋势；聚乙二醇(PEG)透导下呈现下降趋势．由此可见该酶在盐、氧化和低温胁迫条件下产生一定的反应．     

鲤NADH泛醌氧化还原酶亚基3基因的克隆及低温适应相关性分析

根据鲤低温下特异表达的基因（片段）序列设计引物,采用2轮菌落PCR方法对鲤脑全长cDNA文库中的克隆进行筛选,确定了基因序列152在文库中的位置,经生物信息学比对分析确定其为NADH泛醌氧化还原酶亚基3基因.这一基因在低温下的特异表达,对于低温下鱼类机体的供能,起着重要作用.结果还表明,NADH泛醌氧化还原酶亚基3基因在鲤氧化呼吸链电子传递系统中与低温适应性状在一定程度上存在相关性.     

盐生盐藻肌动蛋白基因及其5′上游片段的克隆

在盐生盐藻 c DNA文库和基因组文库中对肌动蛋白基因进行了筛选 ,结果得到了一个c DNA克隆和两个基因克隆 ,并进一步从基因克隆中克隆出该基因的 5′上游片段 .检索结果表明 ,所得到的 c DNA和基因属于盐生盐藻的肌动蛋白基因 .通过将盐生盐藻肌动蛋白基因序列同其c DNA序列及其他生物的肌动蛋白序列相比较 ,给出了一个此基因 5′上游区大概的结构     

盐生盐藻肌动蛋白基因及其5‘上游片段的克隆

在盐生盐藻cDNA文库和基因组文库中对肌动蛋白基因进行了筛选,结果得到了一个cDNA克隆和两个基因克隆,并进一步从基因克隆中克隆出该基因的5'上游片段,检索结果表明,所得到的cDNA和基因属于盐生盐藻的肌动蛋白基因。通过将盐生盐藻肌动蛋白基因序列同其cDNA序列及其他生物的肌动蛋白序列相比较,给出了一个此基因5'上游区大概的结构。     

棉花类耐盐锌指蛋白基因的克隆与结构分析

从棉花花瓣cDNA文库中随机挑选部分克隆，经测序发现一个与拟南芥耐盐锌指蛋白基因同源的cDNA(CSTZ),CSTZ序列全长1012bp，开放阅读框共编码272个氨基酸，含典型的植物双锌指（Cys2/His2)结构区，Northern杂交证实，CSTZ的表达随棉花幼苗钠盐处理浓度的升高而增强，在棉花花龄期，CSTZ基因在叶片，根，花瓣和花药组织中大量表达，在柱头组织中表达相对较弱。     

盐地碱蓬Δ~1-二氢吡咯-5-羧酸合成酶基因(SsP5CS)的克隆及表达特性

从盐地碱蓬 (Suaedasalsa)中克隆了Δ1 -二氢吡咯 -5 -羧酸合成酶基因的cDNA片段 (S .salsaΔ1 -pyrroline -5 -carboxylatesynthasegene,Ssp5CS) .Southern杂交表明SsP5CS基因在盐地碱蓬基因组中只有一个拷贝 ;Northern杂交结果表明 ,不同盐处理 (4 0 0mmol/LNaCl)时间下SsP5CS在盐地碱蓬叶中的表达量明显增加 ;同时盐胁迫条件下盐地碱蓬叶中的脯氨酸含量与对照相比显著的增加 ,并且其渗透调节能力也逐渐增强 .推测SsP5CS基因可能在保护盐地碱蓬免受盐胁迫损伤的过程中起到重要作用     

Identification of genes associated with cotyledon senescence in upland cotton

Leaf senescence in plants is an essential develop- mental phase, and an understanding of senescence is important not only for pure scientific reasons, but also for practical purposes. During the last decade, a number of senescence-associated genes (SAGs) …     

Selective activation of human beta-but not gamma-globin gene in human fibroblast x mouse erythroleukaemia cell hybrids.

The human alpha- and beta-globin genes have been activated in MEL X human fibroblast cell hybrids. However, even though the human gamma- and beta-globin genes are closely linked and were shown in these hybrid clones to be present in approximately equal numbers, no human gamma-globin mRNA was produced. Thus, the human beta- and gamma-globin genes in these cells are differentially regulated apparently by a positive regulatory factor(s) specific for individual globin genes.     

Identification of auxin responsive genes in Arabidopsis by cDNA array

The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．     

大规模原位杂交筛选爪蟾发育调控基因

应用半自动化的系统表达筛选,研究了2400个爪蟾EST克隆在胚胎发育中的表达模式,从筛选出的624个分化表达克隆中,发现了协同表达组的新成员及调控爪蟾体节形成的新基因.     

用扣除杂交法分离藏红花苷合成相关基因的克隆

对藏红花苷合成的相关基因进行了初步分离与克隆实验研究,旨在分离与藏红花苷合成相关的特异性表达的cDNA克隆,为进一步从分子水平上研究藏红花素的合成机理奠定基础。实验采用经添加诱导子和合成前体、处于藏红花苷合成状态下的藏红花细胞和未添加诱导子和合成前体、处于生长状态的对照藏红花细胞为材料,采用扣除杂交法成功地分离到6条与藏红花苷合成相关的特异性表达的cDNA克隆。结果表明:其中一条与胡杨中由高盐环境引起的差异表达的一条mRNA的序列有较高的同源性。     

盐胁迫下拟南芥去乙酰化酶调控下游基因表达的转录组分析

胁迫是指植物持续地暴露在环境的刺激下,有能力建立保护和适应的机制。组蛋白的去乙酰化作为一种表观遗传现象,在植物适应环境中发挥了重要的作用。为了探究高盐胁迫下,组蛋白的去乙酰化酶在植物抵御和适应高盐胁迫中的作用,本研究以拟南芥野生型WT和四突（h2tq,HD2四个基因的突变体）为材料,采用RNA-seq技术和生物信息学方法,对生长10d的幼苗在高盐（150 mM NaCl）处理前和后进行比较转录组分析,并结合实时荧光定量PCR验证转录组数据的可靠性。结果显示：在高盐处理前WT和hd2q共有25个差异基因,其中上调基因2个,下调基因23个。在高盐处理后,WT和hd2q共有1407个差异基因,其中上调基因772个,下调基因635个。GO和KEGG分析显示,对照的差异基因主要富集在胞外区域和响应脱落酸上,盐处理的差异基因主要富集在胞外区域和细胞壁上。植物响应盐胁迫是一个涉及不同交叉途径的复杂过程,这些结果可为研究表观遗传在盐胁迫逆境下的刺激响应提供一定参考意义。     

Analysis of genes differentially expressed during initial cellular dedifferentiation in cotton

The early phase of phytohormone induction is a vital stage of somatic embryogenesis. This phase includes a key process for acquiring cellular totipotency through cellular dedifferentiation. To unravel the molecular mechanism of cellular dedifferentiation in cotton, we constructed a cDNA library using the suppression subtractive hybridization method. A total of 286 differential cDNA clones were sequenced and identified. Among these clones, 112 unique ESTs were significantly up-regulated during the early phase of phytohormone induction, and 40.2% of the ESTs were first identified. GST was highly ex- pressed from 6 to 24 h after induction with phytohormone treatment. PRPs were predominantly ex- pressed and exhibited distinct expression patterns in different treatments, suggesting that they are closely related to cellular dedifferentiation in cotton. Putative GhSAMS, GhSAMDC, GhSAHH and GhAC03 involvement in SAM metabolism was identified in this library. The analysis of qRT-PCR showed that two remarkable increased expressions of the four SAM-related genes happened during the early phase of phytohormone induction, and that a highly positive correlation existed between GhSAMS and GhSAHHo The highest expression level of GhSAMS might be associated with its reentry into the cell cycle. The histological observations further showed that some cells accomplished cellular dedifferentiation and division within 72 h in 2,4-D treatment, and that cellular dedifferentiation might be regulated through two alterations in SAM-dependent transmethylation activity in cotton. In addition, the expression patterns of differential genes in different treatments disclosed the complicated interaction between 2, 4-D and kinetin.