病原诱导的中间偃麦草ERF转录因子基因的克隆及其表达特性

应用RT-PCR、RACE技术，从病原诱导的中间偃麦草叶片cDNA中，分离出1个编码ERF基因的全长cDNA序列，该基因暂命名为TiERF1a，编码由292个氨基酸组成的蛋白质，具有ERF转录因子典型的结构，即保守的AP2/ERF DNA 结合域、核定位位点和酸性激活区。TiERF1a的氨基酸序列与一个水稻ERF蛋白OsBIERF3具有66%的同源性，与拟南芥AtERF1同源性仅39.7%，为植物ERF转录因子家族B3亚群的一个新成员。表达分析结果表明，纹枯病菌、赤霉病菌侵染可诱导TiERF1a基因的上调表达，与防卫相关的激素乙烯、茉莉酸也可诱导该基因上调表达，且TiERF1a对外源乙烯、茉莉酸的响应时期早于对纹枯病菌、赤霉病菌响应时期，说明TiERF1a可能通过乙烯、茉莉酸信号途径参与寄主调控对纹枯病菌、赤霉病菌的防御反应。     

ERF转录因子对生物胁迫的反应及对棉花抗性改良的意义

大量研究表明植物中ERF(ethylene responsive factor)类转录因子广泛参与外界环境胁迫应答基因表达的调控.它编码的蛋白能够特异结合GCC-box元件,从而调控启动子含有GCC-box的病程相关蛋白基因的表达,在植物抗病反应中发挥重要的调控作用.棉花中ERF基因表达受生物和非生物胁迫诱导,并在乙烯、茉莉酸和水杨酸信号传导途径中发挥一定的作用.一些ERF基因在转基因植物中的超表达表现出了一定的广谱抗性,因而在棉花分子育种中具有较为广阔的应用前景.本文主要论述了棉花等植物中ERF的结构与功能特征,当前相关研究进展,及其对棉花抗病性分子遗传改良的意义.     

利用大麦基因芯片筛选簇毛麦抗白粉病相关基因及其抗病机制的初步研究

利用大麦基因芯片筛选差异表达基因并结合RT-PCR分析技术，对簇毛麦的抗白粉病机制进行了初步研究。基因芯片杂交试验获得了抗病簇毛麦非诱导叶片、抗病簇毛麦和感病突变体经白粉菌（Blumeria graminis f. sp. tritici）诱导叶片的基因表达谱。抗病簇毛麦经白粉菌诱导前后的表达谱及RT-PCR分析结果表明，抗病簇毛麦中乙烯和水杨酸信号途径的部分基因被白粉菌诱导增强表达，参与了白粉病的抗性过程。另外，通过比较诱导的抗病簇毛麦与诱导的簇毛麦感病突变体的表达谱并结合RT-PCR分析，发现感病突变体中乙烯和茉莉酸途径的部分基因被白粉菌诱导表达参与防卫反应，未观察到水杨酸信号途径参与防卫反应的证据。同时对抗、感簇毛麦经白粉菌诱导不同时间的叶片进行了内源水杨酸含量的测定，结果表明抗病簇毛麦经白粉菌诱导后水杨酸含量明显上升，而感病突变体中水杨酸含量始终处于较低水平。由于乙烯信号途径是抗、感簇毛麦中共同的信号途径，而水杨酸途径只在抗病簇毛麦中参与抗病反应，所以在簇毛麦的抗病过程中，水杨酸途径是一种最有效的信号传导途径。还筛选出一批与簇毛麦抗白粉病相关的基因，包括病程相关蛋白基因、防卫反应基因、转录因子、信号传导因子和抗病基因类似物等。     

AP2/ERF转录因子对植物非生物胁迫的应答机制研究进展

AP2/ERF (APETALA2/ethylene responsive factor)家族转录因子是植物中的一大类转录因子,最早从拟南芥(Arabidopsis thaliana)中分离出。该家族转录因子一般包含两个AP2/ERF结构域,该结构域大约由60～70个氨基酸组成与DNA结合有关。AP2/ERF家族参与非生物胁迫的反应,比如干旱胁迫、高盐胁迫和低温胁迫等。这些胁迫会激活植物激素ABA、茉莉酸(jasmonate, JA)、乙烯(ethylene, ET)、水杨酸(salicylic acid, SA)等信号途径,同时激活AP2/ERF转录因子家族基因的表达,进而调控其下游功能基因的表达。本研究综述了近年来有关植物AP2/ERF转录因子响应非生物胁迫应答机制的新进展,探讨了该家族转录因子的结构特点、分布,及其对植物发育和次级代谢的影响以及对非生物胁迫的应答。     

遗传发育所揭示生长素介导乙烯反应的信号转导过程

正植物激素生长素和乙烯协同调控植物根的生长,乙烯促进了生长素的合成与运输,生长素受体TIR1/AFB2感受到生长素后,结合并泛素化转录抑制子Aux/IAA蛋白,使其通过26S蛋白酶体途径降解,从而将转录因子ARF释放出来调控下游基因的表达。目前介导乙烯反应的生长素信号过程并不清楚。     

番茄ACC氧化酶基因cDNA的克隆及三种植物表达载体构建

乙烯在植物生长发育过程中发挥着重要的作用.ACC氧化酶是乙烯生物合成途径的限速酶.通过反义基因工程调控ACC氧化酶基因的表达,进而起到调控乙烯的生成是乙烯研究的主要途径.本研究采用RT-PCR方法获得番茄ACC氧化酶基因cDNA序列1 018 bp,构建该基因的正义、反义、RNAi表达载体,通过电转化法导入农杆菌LBA4404中.     

调控植物生物碱生物合成的转录因子研究进展

生物碱是植物中广泛存在的一种次生代谢产物,在抵御生物胁迫及非生物胁迫中发挥着重要作用。转录因子是调控基因转录翻译,控制基因表达量的一种蛋白质。转录因子通过诱导或抑制生物碱合成相关基因的表达来控制植物中生物碱的含量。目前有关调控SGA、烟碱、TIAs和BIAs生物合成的转录因子已有报道。本研究综述了参与SGA、烟碱、TIAs和BIAs生物合成的转录因子家族类型,转录因子在生物碱生物合成中的调控机制以及生物碱合成涉及的信号传导途径,以期为调控农作物中生物碱含量提供理论依据,培育出抗逆性强、安全和优质的农作物品种。     

板栗种子淀粉积累过程中ABA响应的AP2/ERF转录因子家族分析

板栗(Castanea mollissima)是中国重要的经济林树种之一,素有木本粮食之称,主要组成成分是淀粉,其含量对坚果品质具有重要影响。采用双波长紫外分光光度计法,测定ABA处理后板栗坚果不同发育时期的直链淀粉、支链淀粉和总淀粉含量;选择成熟期坚果的转录组数据,对AP2/ERF转录因子家族开展生物信息学和表达分析。结果表明,外施一定量ABA后,坚果仁中支链淀粉含量提高。转录组数据共检测出7 603个差异表达基因,检测出转录因子344个,AP2/ERF转录因子家族占比最多为18.31%。从板栗基因组中检测出AP2/ERF转录因子155个;在ABA处理后83 d的坚果中,AP2/ERF家族中有63个基因响应。AP2/ERF家族转录因子在坚果发育过程中通过响应ABA信号,调控糖和淀粉类物质的积累。本试验分析了淀粉积累过程中响应ABA的AP2/ERF转录因子家族,可为进一步实践验证板栗该基因家族功能和调控表达提供一定参考。     

棉花抗枯萎病相关转录因子基因GhERFB101的克隆与表达分析

为了探究ERF转录因子家族与棉花枯萎病抗性之间的关系,也为海岛抗枯萎病品种的选育工作提供新的基因资源,从Solexa高通量测序技术建立的棉花基因表达谱中筛选探针序列,通过电子克隆结合RT-PCR技术从高抗枯萎病的棉花品种中棉所12中克隆到一个新的ERF-B1亚组转录因子基因,命名为Gh ERFB101(Gen Bank:KF850521)。序列分析表明,该基因开放阅读框738 bp,编码245个氨基酸,含有一个保守的AP2/ERF结构域,在进化上与拟南芥At ERF11的亲缘关系最近。实时荧光定量PCR分析显示,枯萎病菌诱导后,Gh ERFB101基因在抗病品种根中的表达量呈现下降趋势;而在感病品种中,该基因呈上调表达,随着病菌处理后时间延长,其表达量呈现先增加后降低的变化趋势,在病菌处理后12 h表达量达到最大,在48 h下降到最低。乙烯和茉莉酸诱导后,该基因表达量均呈现明显的先增加后降低的变化趋势,在乙烯处理后2 h基因的表达量达到最大;茉莉酸则在处理后1 h基因的表达量达到最大;而水杨酸诱导后基因的变化幅度不大;推测该基因可能通过茉莉酸、乙烯信号传导途径参与对枯萎病菌的防御反应。     

AP2基因家族的起源和棉花AP2转录因子在抗病中的作用

植物中庞大的AP2基因家族成员因其广泛参与植物响应外界环境胁迫、生长发育相关的转录调控而备受重视。AP2基因曾被认为植物所特有,但最近在蓝藻、线虫和病毒中发现了具有AP2结构域和位点特异核酸内切酶的蛋白。所以有人认为当今植物中的AP2基因起源于细菌或者病毒的基因的横向转移,AP2结构域可能来自后来进化为叶绿体的原始蓝细菌的内共生。ERF是AP2大家族中的一个亚族,它编码的蛋白能特异结合含有GCC盒的病程相关基因的表达,参与植物抗病反应。ERF基因的表达受到疾病相关刺激以及环境胁迫的诱导,并且在乙烯、茉莉酸和水杨酸信号传导途径中发挥一定的作用。同时,某些ERF基因在转基因植物中的超表达表现了一定的广谱抗性,因而在分子育种中具有一定的应用前景。棉花上AP2基因家族的基因克隆与分析最近才得以进行,介绍了我们在棉花上相关的研究工作并讨论了它们在植物抗病反应中的作用。     

桃果实中ACC合酶基因克隆及基因沉默载体构建

摘 要：植物中乙烯是一种具有促进果实成熟和衰老的内源激素。ACC合酶是植物乙烯生物合成途径中一个重要的限速酶,沉默ACC合酶基因的表达能减少植物性内源性乙烯的产生。本研究以中华寿桃为研究材料,采用RT-PCR 技术,克隆获得ACC合酶基因。将该基因酶切回收后连接到pTRV-RNA2载体上,转化DH5α,筛选阳性克隆,进行酶切鉴定。测序后与已知序列进行同源性比较,其同源性达到99.6%,表明将ACC合酶基因成功连接到pTRV-RNA2基因沉默载体上。     

1—MCP对红星苹果贮藏期间伤乙烯合成的影响

以红星苹果果实为试材，设机械损伤、机械损伤＋1-MCP、对照3种处理，进行贮藏期间伤诱导乙烯的生物合成过程中ACC合成酶（ACS）活性、ACC积累水平、ACC氧化酶（ACO）活性和乙烯释放速率变化的试验研究。试验结果表明，机械伤刺激了果实ACS和ACO活性，促进了果实乙烯释放，加速了果实衰老；而1-MCP则抑制了受伤果实中ACS和ACO活性，提高了受伤果实贮藏后期ACC积累水平，显著地减少了受伤果实乙烯的释放，改善了受伤果实的贮藏品质。     

Ethylene-regulated hastening of perianth senescence after pollination in Dendrobium flowers is not due to an increase in perianth ethylene production

Flowers of Dendrobium cv. Kenny were hand-pollinated using pollinia from cv. Sakura. This resulted in a large increase in flower ethylene emission and rapid perianth (tepal) senescence. The increase in flower ethylene emission was correlated in time with an increase in ethylene emitted by the column (the fused stigma, style and stamens) plus the ovary. No ethylene emission was observed from perianth parts that were isolated at various periods after pollination. The increased ethylene emission by the column plus ovary was correlated with an increase in ACC synthase and ACC oxidase activity in these flower parts. The perianth parts, in contrast, only showed an increase in ACC oxidase activity, following pollination. The data show that pollination-induced early perianth senescence in Dendrobium is mediated by increased ethylene biosynthesis by the column + ovary, and not due to increased ethylene biosynthesis in the perianth parts. Apparently, ethylene synthesized in the gynoecium diffuses to the perianth parts where it induces senescence. The data are very similar to those found previously in pollinated Phaleanopsis orchids and in emasculated Cymbidium orchids, with the exception that ethylene was emitted from the tepals of these two orchids and not from Dendrobium.     

Rapid induction of a novel ACC synthase gene in deepwater rice seedlings upon complete submergence

Elongation growth is a typical feature of deepwater rice plants in response to submergence. The growth phenomenon is known to be induced by hypoxia, which results in the expression of genes implicated in ethylene biosynthesis. Ethylene is considered to trigger the growth response as it accumulates in the submerged tissues and submergence enhances the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase. However, ACC concentration in rice plants increases much faster after submergence than the activity of the ACC synthase genes studied previously. Here, we studied the expression characteristics of the fifth member of this gene family, OS-ACS5, and show that submergence induces the messenger concentration of this gene before the accumulation of ACC could be observed. OS-ACS5 may play a fundamental role in the growth-promoting increase in ethylene biosynthesis during the first hours of submergence in deepwater rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of high temperature stress on ethylene biosynthesis, respiration and ripening of ‘Hayward’ kiwifruit

Temperatures up to 35°C have been shown to increase ethylene production and ripening of propylene-treated kiwifruit (Stavroulakis, G., Sfakiotakis, E.M., 1993. We attempted to study the regulation by high stress temperature of the propylene induced ethylene biosynthesis and ripening in ‘Hayward’ kiwifruit. ‘Hayward’ kiwifruit were treated with 130 μl/l propylene at temperatures from 30 to 45°C up to 120 h. Ethylene biosynthesis pathway and fruit ripening were investigated. Propylene induced normal ripening of kiwifruit at 30–34°C. Fruit failed to ripe normally at 38°C and above 40°C ripening was inhibited. Propylene induced autocatalytic ethylene production after a lag period of 24 h at 30–34°C. Ethylene production was drastically reduced at 38°C and almost nil at 40°C. The 1-aminocyclopropane-1-carboxylic acid (ACC) content was similar at 30–38°C and was very low at 40°C. The 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) activities decreased with a temperature increase above 30°C, but ACC oxidase decreased at a faster rate than ACC synthase. Fruit not treated with propylene showed no ripening response or ethylene production. However, kiwifruit respiration rate increased with temperature up to 45°C, reaching the respiration peak in 10 h. At temperatures up to 38°C, propylene treatment enhanced the respiration rate. After 48 h at 45°C, fruit showed injury symptoms and a larger decrease in CO₂. The results suggest that high temperature stress inhibits ripening by inhibiting ethylene production and sensitivity while respiration proceeds until the breakdown of tissues.     

Mode of action of abscisic acid in triggering ethylene biosynthesis and softening during ripening in mango fruit

The role of abscisic acid (ABA) in triggering ethylene biosynthesis and ripening of mango fruit was investigated by applying ABA [S-(+)-cis,trans-abscisic acid] and an inhibitor of its biosynthesis [nordihydroguaiaretic acid (NDGA)]. Application of 1 mM ABA accelerated ethylene biosynthesis through promoting the activities of ethylene biosynthesis enzymes (1-aminocyclopropane-1-carboxylic acid synthase, ACS; 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), enhanced fruit softening and activity of endo-polygalacturonase and reduced pectin esterase activity in the pulp. The activities of ethylene biosynthesis and softening enzymes were significantly delayed and/or suppressed in the pulp of NDGA-treated fruit. The ABA-treated fruit had higher total sugars and sucrose as well as degradation of total organic acids, and citric and fumaric acids compared with NDGA treatment. These results suggest that ABA is involved in regulating mango fruit ripening and its effects are, at least in part, mediated by changes in ethylene production.     

Ca2+参与NO对切花月季瓶插期间乙烯合成的调控

分别用0.1 mmol?L-1 SNP（NO供体）、0.1 mmol?L-1 SNP+0.3 mmol?L-1的TFP（CaM）、0.1 mmol?L-1 SNP+10 mmol?L-1的TFP（Ca2+螯合剂）、6 mmol?L-1 Ca2+、6 mmol?L-1 Ca2++0.05 mmol?L-1的PTIO（NO清除剂）处理切花月季‘Kardinal’,研究切花瓶插期间内源乙烯的生物合成变化以及Ca2+在NO对切花月季瓶插期间乙烯合成调控中的作用。结果表明：Ca2+处理能提高月季瓶插前期花瓣中的NOS活性,保持了花瓣中的NO的较高水平,减缓切花瓶插后期NOS活性的升高,进一步研究表明,Ca2+螯合剂EGTA和CaM的抑制剂TFP处理却可使花瓣中的ACS和ACO活性升高,ACC的含量增加,从而加速了乙烯的生物合成;同时,NO的清除剂PTIO处理也可以抑制由于Ca2+处理导致的ACS和ACO的活性降低以及乙烯合成底物ACC的含量下降。因此,Ca2+和CaM可能参与了NO对切花瓶插期间乙烯的合成调控及其信号转导。     

Mode of action of nitric oxide in inhibiting ethylene biosynthesis and fruit softening during ripening and cool storage of ‘Kensington Pride’ mango

The mode of action of nitric oxide (NO) in inhibiting ethylene biosynthesis and fruit softening during ripening and cool storage of mango fruit was investigated. Hard mature green mango (Mangifera indica L. cv. ‘Kensington Pride’) fruit were fumigated with 20 μL L⁻¹ NO for 2 h at 21 °C and allowed to ripen at 21 ± 1 °C for 10 d, or stored at 13 ± 1 °C for 21 d. During ripening and cool storage, ethylene production and respiration rate from whole fruit were determined daily. The 1-aminocyclopropane-1-carboxylic acid (ACC) content, activities of ACC synthase (ACS), ACC oxidase (ACO), and fruit softening enzymes such as pectin esterase (PE), endo-1,4-β-d-glucanase (EGase), exo- and endo-polygalacturonase (exo-PG, endo-PG) as well as firmness and rheological properties of pulp were determined at two- and seven-day intervals during ripening and cool storage, respectively. NO fumigation inhibited ethylene biosynthesis and respiration rate, and maintained higher pulp firmness, springiness, cohesiveness, chewiness, adhesiveness, and stiffness. NO-fumigated fruit during cool storage and ripening had lower ACC contents through inhibiting the activities of both ACS and ACO in the fruit pulp. NO-fumigated fruit showed decreased activities of exo-PG, endo-PG, EGase, but maintained higher PE activity in pulp tissues during ripening and cool storage. In conclusion, NO fumigation inhibited ethylene biosynthesis through inhibition of ACS and ACO activities leading to reduced ACC content in the fruit pulp which consequently, reduced the activities of fruit softening enzymes during ripening and cool storage.     

Effects of Ethylene on Cotton Adaption to Waterlogging Stress and the Underlying Mechanism

[Objective] Waterlogging adversely affects cotton growth and development, and continuous waterlogging may further result in considerable yield loss or crop failure. Enhancing the ability of cotton to adapt to waterlogging stress to preserve yield and quality is therefore critical. Ethylene is an important signal molecule, which plays a vital role in the process of plant stress resistance. However, the mechanism of ethylene in mitigating cotton waterlogging damage is still unclear. [Method] In this study, we setup an experiment with a cotton (Gossypium hirsutum L.) variety K638 in an electric rain shelter at the experimental station of the Shandong Cotton Research Center at Linqing, Shandong. We treated the cotton plants by waterlogging for 10 d during the flowering stage and used a non-waterlogged treatment as the control. During the waterlogging stress treatment, cotton plants were treated with an ethylene signal transduction inhibitor (1-MCP) or ethylene synthesis precursor (ACC) to detect the effects of ethylene content on cotton waterlogging injury and its physiological mechanism. [Result] The results revealed that a foliar spray of 1-MCP significantly inhibited ethylene synthesis in the stressed cotton plants, the content of ethylene and malondialdehyde (MDA) decreased by 5.3% and 39.2%, and the activities of alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lactate dehydrogenase (LDH) decreased by 37.8%, 20.5%, and 8.2%, respectively. The photosynthetic rate, dry weight of the whole plant, and seed cotton yield increased by 13.5%, 3.3%, and 4.6%, respectively. The effect of ACC on the plants was the opposite because spraying ACC promoted ethylene accumulation in the waterlogged cotton. The ethylene and MDA content increased by 8.0% and 19.5%, respectively. The activities of ADH, PDC, and LDH increased by 17.5%, 11.2%, and 8.0%, respectively, while the photosynthetic rate, dry weight of the whole plant, and seed cotton yield decreased by 6.0%, 7.7%, and 8.0%, respectively. [Conclusion] In summary, reducing ethylene content in waterlogged cotton plants can significantly alleviate hypoxia damage caused by waterlogging stress and subsequently promote cotton growth and development by restoring physiological metabolism.