ipt基因促进矮牵牛遗传转化效率及热激启动子驱动基因删除

本研究主要探讨ipt基因对矮牵牛遗传转化不定芽诱导影响及拟南芥热激启动子hsp18.2驱动重组酶基因flp的热诱导外源基因删除表达载体在矮牵牛中的基因删除效果.本研究中将植物表达载体pBin-hsp18.2:flp-35S:ipt及对照载体pBin-hsp18.2:flp遗传转化矮牵牛,以获得转基因植株,分析比较不定芽的诱导和转基因植株进行的热激基因删除.研究结果表明,ipt基因可促进矮牵牛遗传转化过程中不定芽的诱导,其不定芽诱导率为21.5%,显著高于对照的8.7%.在44℃,6 h,热激6次的条件下,转基因矮牵牛植株表型恢复正常,经 GUS蛋白表达分析及PCR、RT-PCR检测,证明外源基因已经被删除.转基因矮牵牛基因删除效率最高可达43.8%.本研究为ipt基因在一些遗传转化困难植物转基因中的应用奠定了基础.     

ocs/mas|一个受损伤和植物激素诱导的嵌合启动子的构建与功能分析

选择适宜的转录调控序列以提高启动子的转录效率,增强外源基因在转基因植株中的表达,对改良作物的抗病虫性具有重要意义。将甘露碱合成酶基因(mas)启动子和章鱼碱合成酶基因(ocs)增强子杂合而成的嵌合启动子ocs/mas与GUS报告基因连接,构建了植物表达载体pOMS-GUS。对照载体pMAS-GUS仅携带mas启动子驱动的GUS基因。利用根癌农杆菌介导法,将以上植物表达载体分别转化烟草。应用半定量RT-PCR和GUS荧光定量分析法分别检测不同胁迫条件下启动子驱动的GUS基因表达量的变化。结果显示,未诱导处理的转基因植株GUS基因仅有微弱表达。伤害处理1h后,mas启动子驱动的GUS活性是未诱导处理的1.8倍,而嵌合启动子ocs/mas的诱导表达活性是未处理的5.7倍。植物激素水杨酸(SA)和茉莉酸甲酯(MJ)处理也诱导了较高水平的ocs/mas嵌合启动子活性;而且SA和MJ联合作用时呈现叠加效应,转基因烟草的GUS活性明显高于伤害处理后的GUS表达水平。以上结果表明,ocs/mas嵌合启动子是一种强诱导型启动子,可以接受多种刺激因子的诱导,从而为更有效地改良作物抗病虫的能力提供新的候选高效启动子元件。     

利用热诱导的位点专一性重组系统在烟草中控制基因表达

采用热激启动子Gmhsp17．5C控制Cre定位重组酶介导的DNA删除系统。在这个系统中,在热激启动子控制下的Cre重组酶的表达导致两侧带有相同方向loxp位点的CaMV35S—GUS片段从转基因烟草(Nicotiana tabacum L.cv.W38)的基因组中删除。通过定量PCR的方法鉴定这个转基因系统,显示了这个系统的重组效率。结果显示在两个小时热激处理后转基因烟草中有41％的CaMV35S—Gus片段被删除。由于热激诱导的定点重组系统有容易操作、对热敏感和无背景表达等优点,因此有利于采用这个系统在转基因植物中进行可诱导的基因操作。     

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。     

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 (Arabidopsis 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。     

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.     

玉米核基质结合区在烟草中对外源转基因表达水平的影响

为研究核基质结合区 (matrixattachmentregion ,MAR)在转基因植物中的功能 ,将来自玉米基因组的MAR序列构建在植物表达载体T_DNA中 ,并将报告基因β_葡糖醛酸酶 (β_glucuronidase ,GUS)基因 (uidA)插入两段MARs序列之间。将此载体与不包含MARs序列的植物表达载体分别转化烟草 (NicotianatabacumL .)。GUS活性检测表明 ,MARs可以显著提高外源基因uidA在转基因烟草中的表达水平 ,平均表达水平提高 2倍 ,最高单株活性可达 10倍。并且转基因植株GUS活性高低与稳定mRNA的量成正比 ,表明MARs在转录水平提高基因表达。     

DGP1, 一个受干旱诱导的保卫细胞特异性启动子的构建与功能分析

植物气孔运动的分子调控离不开高效的基因开关系统, 保卫细胞特异性表达的启动子近几年备受关注. 将保卫细胞特异性表达元件和干旱应答元件进行改造, 构建成一个新的受干旱诱导的保卫细胞特异性的启动子DGP1. 转基因烟草的组织化学定位表明, DGP1驱动的gus基因在受到干旱诱导的情况下, 在保卫细胞中特异性表达, 而在未经干旱处理植株的保卫细胞和干旱处理的根、茎和花中均不表达. 对GUS活性进行定量测定, 结果显示GUS活性明显地受干旱诱导, 并且随着处理时间的延长而增强, 其中干旱诱导8 h后GUS的活性是诱导前的179倍. 而根、茎和叶肉组织在干旱诱导后GUS活性虽然有所提高, 但变化不大. 这些结果表明, 植物在遭遇干旱胁迫时, DGP1启动子可以驱动目的基因在保卫细胞专一性表达, 这就为通过基因工程的途径调控气孔开关运动奠定了基础.     

玉米核基质结合区在烟草中对外源转基因表达水平的影响

为研究核基质结合区(matrix attachment region, MAR)在转基因植物中的功能,将来自玉米基因组的MAR序列构建在植物表达载体T-DNA中, 并将报告基因β-葡糖醛酸酶(β-glucuronidase, GUS)基因(uidA)插入两段MARs序列之间.将此载体与不包含MARs序列的植物表达载体分别转化烟草(Nicotiana tabacum L.).GUS活性检测表明,MARs可以显著提高外源基因uidA在转基因烟草中的表达水平,平均表达水平提高2倍,最高单株活性可达10倍.并且转基因植株GUS活性高低与稳定mRNA的量成正比,表明MARs在转录水平提高基因表达.     

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pCAMBIA-YS3GUS,利用农杆菌介导转化拟南芥,获得pYS3::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pYS3::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方法od3 gmaigensuan有重要意义。     

Tight regulation of expression of two Arabidopsis cytosolic Hsp90 genes during embryo development

The spatial and temporal distribution of expression of two cytosolic members of the AtHsp90 gene family was assessed during early development. In stressed transgenic plants bearing the AtHsp90-3 promoter, beta-glucuronidase (GUS) activity was strong in meristematic tissues. Expression was also detected in vascular tissues, leaf veins, siliques, and in pollen sacs. The promoter induced gene expression after heat shock in a time-course dependent manner. AtHsp90-1 promoter activity was low throughout the early stages of embryo development but high just before embryo maturation, with expression most prominent in cotyledons. AtHsp90-3 promoter activity was almost constant and restricted to the root and the cotyledon tips of the embryo. This highly specific spatial distribution of GUS activity changed when the tissues were heat-stressed. Both promoters were also active in unstressed mature pollen grains and during pollen germination. The results shown here indicate that different regulatory and developmental mechanisms control and differentiate the expression of the two cytosolic members of the Arabidopsis AtHsp90 gene family under normal conditions. The developmental and restricted pattern of expression of the AtHsp90-1 and -3 gene promoters in unstressed transgenic plants suggest prominent and distinctive roles of these two genes during different developmental processes.     

Ca2+ and AtCaM3 are involved in the expression of heat shock protein gene in Arabidopsis

The involvement of calcium and different calmodulin isoforms (Ca²⁺-CaM) in heat shock (HS) signal transduction in Arabidopsis ( Arabidopsis thaliana ) was investigated. Using transgenic Arabidopsis plants which have the AtHsp18.2 promoter/GUS fusion gene, it was found that the level of β -glucuronidase (GUS) activity was up-regulated by the addition of CaCl₂ and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl₃ and verapamil, or the CaM antagonists N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), chlorpromazine (CPZ) and trifluoperazine (TFP). CaCl₂ not only increased the GUS activity after HS, but also up-regulated the GUS activity under non-HS conditions. These results provide additional support for the involvement of the Ca²⁺-CaM signalling system in HSP gene expression. The expression of nine CaM genes (AtCaM1–9) from Arabidopsis was differentially regulated by HS at 37 °C. The expression of AtCaM3 and AtCaM7 genes increased during HS. The temporal expression of the AtCaM3, AtCaM7 and hsp18.2 genes demonstrated that up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or hsp18.2 .     

Short Communication Involvement of Calcium-calmodulin in the Expression of hsp26 Gene in Wheat

The treatment of wheat ( Triticum aestivum L.) seedlings with CaCl2 increased mRNA levels of hsp26 gene at 37 ℃ for 1 h, while that with Ca2+ chelator EGTA decreased the expression of the hsp26 gene. The expression of the wheat calmodulin gene CaM1-2 is rapidly up-regulated in wheat seedlings by heat shock at 37 ℃. The heat-induced up-regulation of CaM1-2 occurred earlier than that of the hsp26 gene did. Calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamid (W7) inhibited the expression of the hsp26 gene in wheat seedlings at 37 ℃ for 1 h. These results implied that the calcium-calmodulin is probably involved in the signal transduction of the hsp26 gene expression induced by heat shock.     

温度胁迫下棉花粉蚧热激蛋白基因的表达分析

【目的】为了研究热激蛋白 Hsp70, Hsp70-4和Hsp90在棉花粉蚧Phenacoccus solenopsis抵抗逆温中的作用。【方法】在测序棉花粉蚧转录组的基础上,分析了该虫热激蛋白Hsp70基因家族的2个序列［Pshsp70（GenBank登录号为KJ909505）和Pshsp70-4（GenBank登录号为KJ909506）］和Hsp90基因家族的1个序列,［Pshsp90(GenBank登录号为KJ909507)］,采用实时荧光定量 PCR（RT-qPCR）检测了在不同温度（18和32℃恒温, 37, 39, 41, 43和45℃热激1 h 后26℃恢复1 h)下棉花粉蚧不同发育阶段（2龄若虫、3龄若虫、雌成虫）3种热激蛋白基因的表达量。【结果】Pshsp70 cDNA序列包含1 923 bp的开放阅读框,编码641个氨基酸,理论分子量和等电点分别为70.9 kDa和5.65; Pshsp70-4 cDNA序列包含1 962 bp的开放阅读框,编码654个氨基酸,理论分子量和等电点分别为71.8 kDa和5.38;Pshsp90 cDNA序列包含2 172 bp的开放阅读框,编码724个氨基酸,理论分子量和等电点分别为83.5 kDa和4.93。Pshsp70 和Pshsp70-4均含有Hsp70基因家族高度保守的基序,Pshsp70编码的氨基酸序列与烟粉虱Bemisia tabaci和家蚕Bombyx mori等昆虫的Hsp70 的氨基酸序列一致性为 85%;Pshsp70-4编码的氨基酸序列与白蜡蚧Ericerus pela和点蜂缘蝽Riptortus pedestris等昆虫的Hsp70的氨基酸序列一致性高达95%;Pshsp90也含有Hsp90基因家族高度保守的基序,Pshsp90编码的氨基酸序列与赤拟谷盗Tribolium castaneum和东亚小花蝽Orius sauteri等昆虫的Hsp90 的氨基酸序列一致性为 87%。热激蛋白基因表达量分析结果表明,在18℃恒温条件下,粉蚧2龄若虫的3个PsHsps基因的mRNA相对表达量均比对照(26℃)低,在32℃恒温条件下,各龄期的Hsp70基因的相对表达量均显著高于对照。在37~45℃下热激1 h并在26℃下恢复1 h,棉花粉蚧3个龄期的3个热激蛋白PsHsps基因的相对表达量随温度的升高总体呈增加趋势,相关性分析表明,除Pshsp70-4在雌成虫中的表达量与热胁迫温度的相关系数为0.225外,各龄期中3个基因的表达量与温度的相关系数均大于0.6,显著相关;43℃和45℃胁迫下,各龄期的3个热激蛋白基因相对表达量均显著高于对照组（P<0.05）。【结论】棉花粉蚧热激蛋白基因的表达与温度呈正相关,在该虫应对高温中起着重要作用。     

Production of medium-chain-length hydroxyalkanoic acids from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in pH stat

The production of β-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36–42°C for 2 h then recovery at 27°C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42°C heat shock and a recovery phase at 27°C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl β-D-glucuronic acid (MU) min⁻¹ mg⁻¹ protein at 24 h of recovery. When tissues were continuously heated at 42°C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.     

温度对中华稻蝗小分子热休克蛋白基因表达的影响

【目的】小分子热休克蛋白(s HSPs)能够被环境胁迫所诱导,不同温度可诱导中华稻蝗体内sHSPs基因表达。【方法】本文采用实时荧光定量PCR(RT-q PCR)技术,分析不同温度处理后中华稻蝗Oxya chinensis体内6个Ocs HSPs基因的表达。【结果】结果表明,OcHSP19.1、OcHSP19.8、OcHSP20.4和OcHSP20.7基因的表达水平在27、32和37℃相同处理时间没有显著变化,但其表达量均在42℃显著升高,尤其是在42℃处理后2 h表达量升高明显。OcHSP21.1和OcHSP23.8基因的表达水平在不同温度处理后无显著变化。多因素方差分析结果表明,基因与处理温度间具有显著的交互作用。【结论】由此可见,温度能够对中华稻蝗s HSPs基因的表达产生影响,但是,不同温度处理下各基因的表达模式存在差异。     

Transient expression of GUS and anthocyanin constructs in intact maize immature embryos following electroporation

Electroporation was used for the delivery and subsequent expression of GUS and anthocyanin reporter genes into intact maize immature embryos. The optimal conditions consisted of culturing immature embryos for 4 days on N6 1-100-25-Ag medium prior to electroporation (375 V/cm; 960 µF capacitance) in EPR buffer containing DNA and 0.07 M sodium glutamate at room temperature (22°C) after a 10 min heat shock at 37°C. Under these conditions, over 40 spots of GUS transient activity were observed per immature embryo. Transient gene expression after electroporation was further demonstrated using an anthocyanin construct, which is specific for expression in plant cells.