茶树醇脱氢酶基因的表达特征及番茄遗传转化分析

根据茶树醇脱氢酶基因(CsiADH1)的cDNA序列设计引物,采用RT-PCR方法从茶树品种‘龙井43’中克隆了CsiADH1序列,分析了CsiADH1在生物和非生物胁迫下的诱导表达情况并转化番茄。结果表明:CsiADH1包含一个1 044bp的最大开放阅读框,编码347个氨基酸。qRT-PCR分析显示,CsiADH1的表达受到茶尺蠖取食、机械损伤、茉莉酸和水杨酸的诱导;将CsiADH1基因ORF区域克隆进pCAMBIA1301载体中,构建了由CaMV35S启动子驱动的CsiADH1基因植物表达载体pCAMBIA-ADH,并以农杆菌介导的方法侵染番茄‘中蔬四号’子叶,经PCR鉴定,获得了8个转CsiADH1基因阳性植株。该结果为进一步揭示CsiADH1基因在植物诱导防御反应中的分子机理研究奠定了基础。     

花生白藜芦醇合酶基因cDNA的克隆及植物表达载体的构建

为得到含有白藜芦醇合酶cDNA(RS cDNA)并能表达白藜芦醇(Res)的转基因植物,以花生为材料,从其根部提取基因组DNA,并以其为模板,利用引物悬挂延伸PCR法扩增得到大小约1200bp的片段,将这个片段连接到克隆载体PBS-T上进行测序,测序结果证明,此片段就是花生的RS cDNA。将此片段再正向插入到植物表达载体PBI121的CaMV35s启动子和NOS终止子之间,对重组子进行筛选与鉴定,证明花生的RS cDNA已经正确插入PBI121中,成功的构建了植物表达载体PBI121L。     

康乃馨ACC氧化酶cDNA的克隆及其反义植物表达载体的构建

以康乃馨（Dianthus caryophyllus L.)花瓣为材料,用改进的异硫氰酸胍一步法提取总RNA,根据已报道的康乃馨ACC氧化酶（1-aminocyclopropane-1-carboxylic acid oxidase,CO)基因的序列设计产合成一对引物,通过RT-PCR方法获得一约1.2kb特异片段,把该片段连接pGEM^(R)-Teasy vector上进行测序,其全长共1156bp,编码区915bp。共编码304个氨基酸残基,序列分析结果表明该序列与GenBankL35152中的康乃馨ACC氧化酶基因的cDNA序列完全相符,推断该基因在康乃馨种内可能是完全或高度保守的,随 后将此片段反向插入植物表达载体pBI121的35S启动子和NOS终止子之间,构建了一反义植物表达载体pBO;又把花特异表达启动子PchsA插入pBI121的HindⅢ Xbal位点构建中间载体pGHB,再把康乃馨ACC氧化酶基因反向插入中间载体pCHB的XbaI Satl位点构建成另一反义植物表达载体pCBO。     

番茄交替氧化酶基因的克隆和表达

利用简并PCR扩增产物做探针筛选番茄cDNA基因文库获得一个全长交替氧化酶cDNA基因LeAoxlau.经序列分析得出,该基因全长1 418bp,编码区序列长1 077 bp,编码约40 kD的前体蛋白.该蛋白在转运到线粒体时被加工成32kD的成熟蛋白.Southern印迹杂交分析结果显示该基因以单拷贝形式存在于番茄的基因组中RT-PCR显示,该基因在在番茄植株的根、茎、叶和子叶中表达.重组表达实验表明该基因能在大肠杆菌中表达.     

木薯SBEI基因片段克隆及块根特异性反义表达载体的构建

为了抑制木薯支链淀粉的合成,提高直链淀粉含量,改变木薯淀粉结构,培育工业用燃料酒精型木薯品种,本研究利用RT-PCR方法,用木薯块根cDNA克隆获得支链淀粉合成的关键酶基因SBEI的部分片段,对其进行序列分析表明与GenBank序列高度同源,同源性为99.22%;并以pBI121为基础,构建了以块根特异性表达启动子Sporamin驱动的SBEI基因反义结构的植物表达载体.     

二氢黄酮还原酶基因的克隆与转基因烟草的获得

以蒺藜苜蓿(Medicagotrunctulacv.5160)幼果总RNA为模板,采用RT-PCR技术克隆到二氢黄酮还原酶(DFR)基因的cDNA序列,所获得的cDNA序列全长1018 bp,具有完整的ORF,编码337个氨基酸。Blast分析表明,该片段与GenBank中注册的DFR基因同源性为99.80%。以植物表达载体pBI121为基础,构建了Ca MV35S启动子驱动的DFR基因植物表达载体pBIDFR;采用直接转化法将pBIDFR导入根癌农杆菌EHA105,用该菌株对普通烟草进行遗传转化获得6株转基因植株。     

番茄叶片GDP-甘露糖焦磷酸化酶基因表达载体的构建与转化

GDP-甘露糖焦磷酸化酶(GMPase,EC 2.7.7.22)是维生素C合成途径的第一步关键酶。通过RT-PCR扩增到1498 bp的GMPase全长序列,GenBank登录号为DQ449030。利用克隆到的基因分别构建得到正义及反义植物表达载体。并将其克隆至PBI121真核表达载体,转化农杆菌LBA4404,利用农杆菌介导法转化烟草植株,经基因组PCR及琼脂糖凝胶电泳检测,结果表明GMPase真核表达载体构建成功,并成功获得转基因植株。     

斑马鱼TK1基因的克隆表达

目的:克隆斑马鱼TK1基因的cDNA序列,并在大肠杆菌中诱导表达,对其产物进行生物学活性鉴定。方法:采用RT-PCR和RACE方法,克隆TK1的cDNA全长序列。表达载体在大肠杆菌BL21(DE3)中进行诱导表达。表达蛋白利用镍离子柱纯化。结果:获得TK1基因的cDNA全长序列,编码一个分子量为26kD的蛋白。结论:TK1融合蛋白在28℃条件表现出比较高的生物学活性,达到0.45 U/mg。     

巴西橡胶树HbWRKY1基因的克隆及转化烟草的初步研究

利用RACE结合RT-PCR技术,从巴西橡胶树(Hevea brasiliensis)总RNA中扩增得到长度为1234 bp的WRKY基因cDNA全长编码序列。通过氨基酸同源性比对,该序列推导的氨基酸序列与蓖麻、白杨的WRKY同源性分别为79%和73%,表明分离的cDNA序列为橡胶树WRKY基因,命名为HbWRKY1。通过构建pCAMBIA1304-HbWRKY1植物表达载体,经农杆菌GV3101介导,将HbWRKY1基因导入烟草(Nicotiana tabacum)中,对所获得的潮霉素抗性烟草株系进行PCR鉴定。结果表明,HbWRKY1基因已整合到65株转基因植株中。干旱胁迫试验表明,HbWRKY1的过量表达可以明显提高转基因烟草对干旱胁迫的耐受能力。这说明WRKY基因与橡胶树抗旱能力之间存在一定的关系。     

逆境诱导型启动子rd29A的克隆及植物表达载体的构建

根据文献上发表的逆境诱导型启动子rd29A 序列设计并合成了一对引物, 通过PCR 的方法从拟南芥基因组中扩增到rd29A 的启动子序列。根据GenBank 中已发表的转录因子DREB1A基因的cDNA 序列设计并合成了一对引物, 通过RT-PCR 的方法从低温处理的拟南芥总RNA 中扩增出DREB1A 基因的全长cDNA 片段。以植物表达载体pBch 为基础, 构建了由rd29A 调控的DREB1A 基因的植物表达载体pBDR29A, 为利用DREB1A 基因改良植物抗逆性奠定了物质基础。     

Expression analysis of LeNHX1 gene in mycorrhizal tomato under salt stress

The plant growth, stem sap flow, Na⁺ and Cl^? content, and the expression of vacuolar Na⁺/H⁺ antiporter gene (LeNHX1) in the leaves and roots of tomato under different NaCl stresses (0.5% and 1%) were studied to analyze the effect of arbuscular mycorrhizal fungi (AMF) on Na⁺ and Cl^? accumulation and ion exchange. The results showed that arbuscular mycorrhizal (AM) plant growth and stem sap flow increased and salt tolerance improved, whereas Na⁺ and Cl^? accumulated. Na⁺ significantly decreased, and no significant decline was detected in Cl^? content after AMF inoculation compared with the non-AM plants. The LeNHX1 gene expression was induced in the AM and non-AM plants by NaCl stress. However, AMF did not improve the LeNHX1 level, and low expression was observed in the AM tomato. Hence, the mechanism that reduced the Na⁺ damage to tomato induced by AMF has little relation to LeNHX1, which can export Na⁺ from the cytosol to the vacuole across the tonoplast.     

A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants

In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.     

生物信息学对番茄LeNHX1基因的研究

应用生物信息学的方法和工具对番茄LeNHX1蛋白质的理化性质、跨膜区域、疏水性/亲水性、二级结构、结构功能域、功能分类和同源性进行分析.结果表明此蛋白为疏水性稳定蛋白,包含一个保守的氨氯吡嗪咪结合位点LFFIYVLPPI区域,相对分子量为59.0 kD,等电点为6.60,存在10个跨膜区域,蛋白质二级结构中的主要构成元件是α-螺旋和不规则卷曲,功能分类和蛋白质同源性分析表明番茄LeNHX1属于液泡膜Na+/H+反向转运蛋白.     

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.     

Improved yield,fruit quality,and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes

The K⁺, Na⁺/H⁺ antiporter LeNHX2 and the regulatory kinase SlSOS2 are important determinants of salt tolerance in tomato plants and their fruit production ability. In this work, we have analyzed the effects of LeNHX2 and SlSOS2 co-overexpression on fruit production, quality in tomato plants (Solanum lycopersicum L. cv. MicroTom), and analyzed physiological parameters related to salt tolerance. Plants overexpressing LeNHX2, SlSOS2 or both were grown in greenhouse. They were treated with 125 mM NaCl or left untreated and their salt tolerance was analyzed in terms of plant biomass and fruit yield. Under NaCl cultivation conditions, transgenic tomato plants overexpressing either SlSOS2 or LeNHX2 or both grew better and showed a higher biomass compared to their wild-type plants. Proline, glucose and protein content in leaves as well as pH and total soluble solid (TSS) in fruits were analyzed. Our results indicate that salinity tolerance of transgenic lines is associated with an increased proline, glucose and protein content in leaves of plants grown either with or without NaCl. Salt treatment significantly reduced yield, pH and TSS in fruits of WT plants but increased yield, pH and TSS in fruits of transgenic plants, especially those overexpressing both LeNHX2 and SlSOS2. All these results indicate that the co-overexpression of LeNHX2 and SlSOS2 improve yield and fruit quality of tomato grown under saline conditions.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

一个新的油菜NHX基因电子克隆及序列分析

基于电子克隆的方法,从甘蓝型油菜中获得一个新的反向转运蛋白基因cDNA序列,暂被命名为BnNHX6。BnNHX6包含一个完整的长为1593bp的开放阅读框架,编码530个氨基酸。BnNHX6蛋白属于跨膜蛋白,有9个跨膜区,含有信号肽,预测在质膜上。通过同源比对和进化分析发现,BnNHX6的氨基酸序列与拟南芥AtNHX5和AtNHX6、西红柿LeNHX2、毛白杨PtNHX2基因所编码的氨基酸序列高度同源,同源性分别为78.4%、92.6%、77.1%、76.9%,亲缘关系较近;但与已报道的油菜BnNHX1同源性仅为24.9%,亲缘关系很远,表明BnNHX6是一个新的油菜反向转运蛋白基因。     

Genetic analysis of Na+ and K+ concentrations in leaf and stem as physiological components of salt tolerance in Tomato

The sodium and potassium concentrations in leaf and stem have been genetically studied as physiological components of the vegetative and reproductive development in two populations of F₈ lines, derived from a salt sensitive genotype of Solanum lycopersicum cv. Cerasiforme, as female parent, and two salt tolerant lines, as male parents, from S. pimpinellifolium, the P population (142 lines), and S. cheesmaniae, the C population (116 lines). Genetic parameters of ten traits under salinity and five of them under control conditions were studied by ANOVA, correlation, principal component and QTL analysis to understand the global response of the plant. Two linkage maps including some tomato flowering time and salt tolerance candidate genes encoding for SlSOS1, SlSOS2, SlSOS3, LeNHX1, LeNHX3, were used for the QTL detection. Thirteen and 20 QTLs were detected under salinity in the P and C populations, respectively, and four under control conditions. Highly significant and contributing QTLs (over 40%) for the concentrations of Na⁺ and K⁺ in stems and leaves have been detected on chromosome 7 in both the populations. This is the only genomic position where the concentration QTLs for both the cations locate together. The proportion of QTLs significantly affected by salinity was larger in the P population (64.3%, including all QTLs detected under control) than in the C population (21.4%), where the estimated genetic component of variance was larger for most traits. A highly significant association between the leaf area and fruit yield under salinity was found only in the C population, which is supported by the location of QTLs for these traits in a common region of chromososome C1. As far as breeding for salt tolerance is concerned, only two sodium QTLs (lnc1.1 and lnc8.1) map in genomic regions of C1 and C8 where fruit yield QTLs are also located but in both the cases the profitable allele corresponds to the salt sensitive, cultivated species. One of those QTLs, lnc1.1 might involve LeNHX3.