苹果6-磷酸山梨醇脱氢酶基因启动子逆境诱导表达特性研究

为研究6-磷酸山梨醇脱氢酶(sorbitol-6-phosphate dehydrogenase,S6PDH)基因启动子(S6PDHp)的逆境诱导表达特性,利用Gateway技术构建了S6PDH基因启动子区5'端系列缺失体与GUS基因的融合表达载体,并通过农杆菌介导法转化拟南芥。对转基因拟南芥进行低温和外源ABA处理,通过GUS蛋白活性变化分析S6PDHp的逆境诱导表达特性。研究结果发现,通过Gateway技术构建了4个S6PDHp 5'端系列缺失体与β-葡萄糖苷酸酶(GUS)基因的融合表达载体(pGWB433-S6PDHp1、pGWB433-S6PDHp2、pGWB433-S6PDHp3和p GWB433-S6PDHp4)并获得了相应的转基因拟南芥。对转基因植株进行低温处理后发现,p GWB433-S6PDHp3转基因植株中的GUS活性增幅最大,达到显著水平,而其他转基因植株中的GUS活性基本保持不变。外源ABA处理后发现,除p GWB433-S6PDHp4外,其余启动子缺失体转基因拟南芥中GUS活性显著升高。以上结果表明,低温和外源ABA能够诱导S6PDHp的表达,但不同的缺失体响应程度不同,意味着在S6PDHp序列(-2 396bp至-236bp)中可能存在着响应逆境胁迫的正负调控顺式作用元件。     

拟南芥AtNCED2基因启动子区域序列克隆及其活性分析

目的:克隆拟南芥AtNCED2基因启动子区域序列,并分析其组织器官特异性及对外界刺激的响应.方法:通过PCR从拟南芥基因组中克隆AtNCED2基因5'侧翼2295bp启动子区域序列(AtNCED2p),并进行生物信息学分析.构建AtNCED2p驱动GUS的植物双元表达载体pAtNCED2p::GUS,通过根癌农杆菌介导法将其转化野生型拟南芥,检测转基因植侏中GUS表达的组织器官特异性.结果:该启动子序列中存在TATA-box、CAAT-box、根器官特异性元件、ABA响应元件、低温响应元件、昼夜节律响应元件等顺式作用元件.GUS活性主要集中在转基因拟南芥根尖及侧根发生部位.外源ABA处理的转基因植株根中GUS活性为174.8nmol 4-MU min-1 mg-1蛋白,明显高于对照值91.7nmol 4-MU min-1mg-1蛋白.结论:AtNCED2基因可能在根的生长和发育中起作用,且外源ABA处理增强其在根中的表达.     

拟南芥AHAl基因启动子的表达特性分析

从拟南芥中分离到编码质膜H^＋-ATPase的AHA1基因启动子序列813bp，并构建了此种启动子与GUS嵌合的重组载体，通过农杆菌介导转化拟南芥，得到转基因拟南芥。用组织化学方法分析AHA1基因启动子驱动GUS转基因拟南芥的结果表明，GUS基因在转基因的拟南芥根、茎、叶、花和荚的维管组织中均有表达，且不同发育时间内GUS基因表达也不同。以上研究表明拟南芥AHA1基因可能参与植物的生长发育与抗逆胁迫反应。     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

人工合成启动子SAR在拟南芥中的表达分析

利用植物防御基因中的病原诱导响应元件和最小35S启动子(-62～+1),人工合成了启动子SAP,并以GUS基因为报告基因,在转基因拟南芥中分析了合成启动子的表达特性.通过对转基因拟南芥GUS组织染色的分析表明:SAR启动子在子叶、毛刺、根茎交接处和根系中优势表达,在老叶中的表达量高于幼叶,说明SAR启动子具有组织和发育表达特异性.     

病程相关基因启动子片段与GUS的融合基因在拟南芥中的表达

PR1是拟南芥(Arabidopsisis thaliana L.)系统获得抗性的一个标志基因.利用PCR技术,从拟南芥中扩增并克隆了PR1基因的启动子片段.将该启动子片段与GUS报告基因拼接,构建成含有PR1-GUS融合基因的重组表达质粒.经根癌农杆菌介导转化,得到了转基因的拟南芥植株.用已知的系统获得抗性激活剂处理转基因植物,检测到GUS活性.因此,这一转基因体系可以作为一种简便、灵敏的实验体系以筛选激活植物系统获得抗性的化合物.     

拟南芥钙调素结合蛋白基因启动子的克隆及表达

采用PCR技术从拟南芥中克隆了SCBP60g基因的启动子,并与GUS报告基因融合构建重组表达载体,转化野生型拟南芥,对获得的转基因株系进行GUS组织染色,从基因调控水平上探讨其在功能方面的差异。结果显示:SCBP60g基因的启动子能指导GUS报告基因在拟南芥的根、茎、叶和花中表达,并且在这些部位的维管束表达较强。这种表达方式与LCBP60g基因的启动子指导的GUS基因组织化学染色有差异,表明这个启动子的表达调控具有一定的特异性。     

芥菜型油菜BjuA09DFR基因及其启动子的克隆与转化和表达

该研究利用实时荧光定量(qRT-PCR)检测了BjuA09 DFR基因的时空表达特异性,并通过克隆BjuA09 DFR基因启动子片段,构建该基因的启动子GUS融合表达载体,利用农杆菌介导法将重组质粒转入野生型拟南芥,最后对拟南芥转基因材料不同发育时期的不同组织部位进行GUS组织化学染色,分析BjuA09 DFR基因启动子的表达模式,为BjuA09 DFR基因启动子功能的进一步研究提供理论依据。结果表明:(1)BjuA09 DFR基因在芥菜型油菜的多个组织部位都有表达,尤其是在叶、花、角果和授粉后15d种子中表达量较高。(2)成功构建了BjuA09 DFR基因启动子和GUS基因融合表达载体(pBjuA09 DFR∷GUS),采用农杆菌介导法将重组质粒转入野生型拟南芥,经卡那霉素筛选和PCR检测抗性苗,获得转基因拟南芥阳性苗。(3)GUS组织化学分析结果显示,转基因拟南芥材料的GUS活性具有明显的时空特异性,在叶、花、角果和种子中的染色较深,具有很强的GUS活性。     

玉米脱氧麦根酸分泌通道蛋白基因YS3启动子的克隆与启动活性分析

缺铁是世界范围内农业生产面临的严重问题,玉米通过分泌脱氧麦根酸(2’-deoxymugineic acid,DMA)吸收利用土壤中的难溶性铁。为探明玉米DMA分泌通道蛋白基因YS3的表达和调控机制,本文通过克隆获得长为2813 bp的YS3基因启动子,该序列含有大量TATA-box、CAAT-box等启动子基本元件,以及光响应、激素调控等多个顺式调控元件;构建YS3启动子驱动GUS基因的植物表达重组载体p CAMBIA-YS3GUS,利用农杆菌介导转化拟南芥,获得p YS3::GUS转基因植株,对转基因植株进行GUS组织化学染色,并通过石蜡切片技术对转基因植株进行组织观察,分析p YS3::GUS转基因植株中YS3基因启动子的活性。结果表明,YS3启动子主要驱动GUS基因在拟南芥根部表达,且主要集中在根部表皮细胞,机械损伤可激发YS3启动子活性,驱动GUS基因在损伤临近部位表达。本研究对于理解玉米DMA分泌的分子调控机理具有重要意义。     

厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析

为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants

Emerging evidence suggests that plant cell-wall-modifying enzymes induced by root-parasitic nematodes play important roles in feeding cell formation. We previously identified a tobacco endo-β-1,4-glucanase (cellulase) gene, NtCel7 , that was strongly induced in both root-knot and cyst nematode feeding cells. To characterize further the developmental and nematode-responsive regulation of NtCel7 , we isolated the NtCel7 promoter and analysed its expression over a time course of nematode infection and in response to auxin, gibberellin, ethylene and sucrose in soybean and tomato hairy roots and in Arabidopsis containing the NtCel7 promoter fused to the β-glucuronidase (GUS) reporter gene. Histochemical analyses of transgenic plant materials revealed that the NtCel7 promoter exhibited a unique organ-specific expression pattern during plant development suggestive of important roles for NtCel7 in both vegetative and reproductive growth. In all plant species tested, strong GUS expression was observed in root tips and lateral root primordia of uninfected roots with weaker expression in the root vasculature. Further analyses of transgenic Arabidopsis plants revealed expression in shoot and root meristems and the vasculature of most organs during plant development. We also determined that the NtCel7 promoter was induced by auxin, but not gibberellin, ethylene or sucrose. Moreover, strong GUS activity was observed in both cyst and root-knot nematode-induced feeding sites in transgenic roots of soybean, tomato and Arabidopsis. The conserved developmental and nematode-responsive expression of the NtCel7 promoter in heterologous plants indicates that motifs of this regulatory element play a fundamental role in regulating NtCel7 gene expression within nematode feeding sites and that this regulation may be mediated by auxin.     

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T₀). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T₁) produced the highest GUS activity when treated with 150 μM Cu²⁺ compared to the control (without Cu²⁺). However, Zn²⁺ and Fe²⁺ treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T₁ seedlings of tomato when subjected to Cu²⁺ ions.     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers

The Arabidopsis thaliana etr1-1 allele, capable of conferring ethylene insensitivity in a heterologous host, was introduced into transgenic carnation plants. This gene was expressed under control of either its own promoter, the constitutive CaMV 35S promoter or the flower-specific petunia FBP1 promoter. In about half of the transgenic plants obtained flower senescence was delayed by at least 6 days relative to control flowers, with a maximum delay of 16 days, a 3-fold increase in vase life. These flowers did not show the petal inrolling phenotype typical of ethylene-dependent carnation flower senescence. Instead, petals remained firm and finally started to rot and decolorize.In transgenic plants with delayed flower senescence, expression of the Arabidopsis etr1-1 gene was detectable and the expression pattern followed the activity of the upstream promoter. In these flowers expression of the ACO1 gene, encoding the final enzyme in the ethylene biosynthesis pathway, ACC oxidase, was down-regulated. This indicates that the autocatalytic induction of ethylene biosynthesis, required to initiate and regulate the flower senescence process, is absent in etr1-1 transgenic plants due to dominant ethylene insensitivity.The delay in senescence observed in transgenic etr1-1 flowers was longer than in flowers pretreated with chemicals that inhibit either ethylene biosynthesis (amino-oxyacetic acid) or the ethylene response (silver thiosulfate). This may have important implications for post-harvest management of carnation flowers.