奉节脐橙果实苯丙氨酸解氨酶活性及其基因表达与果皮褐变的关系

柑橘果皮褐变严重影响果实的商品价值和耐贮性．以奉节脐橙（Citrus sinensis Osbcck）果实为材料,通过采后常温、涂蜡、低温、机械损伤等处理研究了果实果皮褐变率、苯丙氨酸解氨酶（PAL）活性及PAL上基因在果皮褐变过程中表达水平的变化。结果表明,涂蜡、损伤处理均极显著地提高果实的果皮褐变率,而低温贮藏可显著降低其发生率;贮藏期问各处理的PAL活性均呈上升趋势,损伤处理PAL活性显著高于对照。果实发生褐变或受到机械损伤后,PAL2、PAL6基因的表达均比对照明显增强。结果首次表明脐橙果实PAL活性变化以及PAL2基因的表达与果皮褐变具有非常密切的关系．     

几种培养基及光照对蝴蝶兰叶片外植体褐变的影响

蝴蝶兰叶片外植体在MS纸桥培养基上培养10 d发生褐变率较低,1/2 MS次之,MS、B₅和N₆培养基外植体褐变率最高。MS培养基中铁盐加倍或微量元素缺乏造成外植体严重褐变,培养基积累很多褐色物质;减少琼脂含量,外植体褐变加重;添加柠檬酸可减轻外植体褐变;而添加活性炭、抗坏血酸或PVP对减轻外植体褐变无显著作用。光照强度增加外植体褐变严重。     

多酚氧化酶抑制剂对蝴蝶兰叶外植体褐变的影响

将多酚氧化酶(PPO)抑制剂添加到酶反应液中,抗坏血酸和半胱氨酸在0.5mmol/L就完全抑制蝴蝶兰PPO活性。300mg/L柠檬酸和100~200mg/L亚硫酸氢钠分别添加到培养基中,可使蝴蝶兰外植体褐变程度降低;采用抑制剂浸泡处理外植体,对外植体褐变抑制效果最好的为50mg/L抗坏血酸,外植体在褐变发生前PPO活性低于对照。     

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理.方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系.结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低.结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合.     

蝴蝶兰组织培养研究进展(综述)

本文从蝴蝶兰外植体的选择．不同基本培养基、激素及添加物对其增殖与分化的影响,外植体褐变的防治以及生根壮苗方法等,概述蝴蝶兰组培快繁方面的研究进展,为其组培技术研究提供参考。     

蝴蝶兰组培快繁及热激处理抑制褐变的研究

以9个蝴蝶兰品种的花梗和无菌苗叶片为试材,研究了不同品种间不定芽诱导率及增殖系数的差异,并选出增殖系数高、中、低的3个代表性品种‘内山姑娘’、‘梦幻兄弟’和‘万花筒’,对不定芽增殖条件进行优化,并对培养过程中褐变严重的品种‘内山姑娘’进行了热激处理抑制褐变的研究。结果表明:(1)相同条件下,不同蝴蝶兰品种间不定芽诱导率和增殖(分化)系数存在显著差异,且花梗芽诱导率和繁殖系数高的品种,其叶片的不定芽诱导率和分化系数也较高。(2)6-BA是影响蝴蝶兰不定芽增殖的主要因素,椰汁次之。培养基1/2MS+7.0mg.L-16-BA+0.2mg.L-1 NAA+100mL.L-1椰汁适于‘内山姑娘’和‘梦幻兄弟’品种的不定芽增殖,1/2MS+5.0mg.L-1 6-BA+0.2mg.L-1 NAA+50mL.L-1椰汁适合‘万花筒’品种的不定芽增殖。(3)40℃热激处理9min恢复48h后再切割接种能够显著减轻蝴蝶兰‘内山姑娘’叶片的褐变程度,热激处理后叶片组培过程中的褐变指数、总酚含量以及苯丙氨酸解氨酶(PAL)活性均显著低于对照,且三者的两两之间均具有极显著正相关关系,证明短时间的热激处理可降低酚合成酶PAL的活性,减少酚类化合物的积累,从而减轻组培过程中的褐变伤害。     

联合调控对中国红豆杉细胞关键酶基因表达的影响

红豆杉悬浮培养细胞可以持续提供抗癌药物紫杉醇及一些紫杉烷类。在中国红豆杉悬浮培养细胞中,云南紫杉烷C(Tc)是主要的紫杉烷。为了更理性地调控紫杉醇或有用紫杉烷的生产,有必要深入了解其生物合成过程。采用实时定量PCR(Real-time Quantitative PCR,即RQPCR)技术考察经调控后紫杉醇及紫杉烷代谢中关键酶基因—TASY,T5αH,TDAT,T10βH,TαH,T14βH表达水平的变化。在细胞培养的第7天和12天,分别以100μmol/L2,3-二羟丙基茉莉酸(DHPJA)诱导,同时在细胞培养第7天进行20g/L蔗糖饲喂、100g/LXAD-7HP的原位吸附。该联合调控处理使得细胞培养第30天时,Tc产量高达1517±37mg/L,是对照处理的11.1倍,是DHPJA重复诱导联合蔗糖饲喂处理的1.7倍。RQ-PCR结果显示:DHPJA的加入可使6个基因表达水平显著提高,但在12小时后快速下降,需补充DHPJA以再次提高基因表达水平。吸附剂同时引入会延缓基因表达水平的提高速度,但却能维持基因表达处于一个较高的水平,表现为在细胞培养中后期,基因表达水平将显著高于无吸附剂的调控体系。与13α-羟化相对应的TαH基因有所不同,吸附剂的存在更显著地抑制其表达,但仍有维持表达的功能。     

蝴蝶兰离体培养及其相关生物技术研究进展

对蝴蝶兰(Phalaenopsis)的离体培养、褐化及控制、物理化学诱变、转基因研究进展进行了综述,包括蝴蝶兰离体培养中的外植体选择、体细胞胚胎及类原球茎诱导、培养基及培养条件、培养方式、褐化控制等关键因素,并概括了生物反应器、转基因、诱变育种等相关生物技术研究进展。成体植株的花梗、茎尖和种子以及试管苗的叶片、茎尖、根尖等均可作为外植体;影响外植体褐化的主要因素有培养基的种类、pH值、温度、外植体的生理状况等;蝴蝶兰诱变育种及转基因研究尚处于初步阶段。     

丝瓜苯丙氨酸解氨酶基因PAL克隆及表达分析

苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase)是苯丙烷途径代谢的关键酶,在果蔬褐变中发挥着重要作用。为了探究丝瓜中PAL基因的功能,本研究以丝瓜果实为材料,采用RACE和RT-PCR技术从丝瓜果肉中分离获得丝瓜PAL基因,命名为Lc PAL,Gen Bank登录号为:KP341758。该基因全长2406 bp,具有完整的开放阅读框(ORF),共2145个碱基;预测编码715个氨基酸,理论分子量(Mw)为77.69 k D,等电点(p I)为6.11,编码的蛋白与黄瓜和香瓜的同源蛋白相似性均在93%以上,显示其高度的保守性。系统进化树分析显示,丝瓜与同为葫芦科的黄瓜、香瓜的PAL蛋白的亲缘关系较近,聚为同一类。Motif Scan分析显示,Lc PAL编码的蛋白包含有PAL-HAL(60~525位)、PLN02457(8~715位)及phe_aml_yase(24~705位)3个保守结构域和1个酶活性中心序列(197~212位),属于典型的Lyase_I_Like超家族。Wolf Psort预测其亚细胞定位于叶绿体或内质网中。实时荧光定量PCR分析显示,Lc PAL基因在普通丝瓜果实、花、茎、叶和根中均有表达。Lc PAL在所选的8个丝瓜品种中表达存在一定差异,在普通丝瓜中的表达量均高于有棱丝瓜;在普通丝瓜品种闽丝3号鲜切及采后储藏褐变过程中,Lc PAL初期表达上调,后期表达量受到抑制,且与其酶活性的变化趋势基本一致。普通丝瓜Lc PAL表达与果肉褐变的发生过程高度相关,表明Lc PAL在普通丝瓜果肉褐变中发挥着作用。     

蝴蝶兰胚性愈伤组织诱导的氧化还原动态

离体培养蝴蝶兰（Phalaenopsis spp.）类原球茎切块诱导胚性愈伤组织,在培养第11～18 d时出现胚性细胞,第25～32 d观察到幼嫩的球形胚。在培养第0～11 d,外植体.OH相对含量波动不大,O2.生成速率持续提高并达到高峰,H2O2水平明显提高,抗氧化能力在低位波动,推测外植体可能经受较强的氧化胁迫,以利细胞脱分化和获得胚性潜能。此外,此期间SOD活性持续提高,POD和CAT活性达到一个高峰。在愈伤组织诱导全过程中,总抗氧化能力、DPPH自由基清除率、H2O2水平基本上都持续提高,与SOD活性均极显著正相关,SOD是影响培养物清除自由基能力和提高抗氧化水平的主要因素。综合分析实验结果表明,在蝴蝶兰胚性愈伤组织诱导过程中,SOD、O2.和H2O2起到关键的作用。     

蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析

本研究从蝴蝶兰叶片中克隆了茎部特异基因Ph TSJT1的全长序列(GenBank登录号为MF797883),并分析了其在不同组织及低温胁迫下的表达特性。结果表明,Ph TSJT1基因全长994 bp,编码239个氨基酸,属于Class-Ⅱ谷氨酰胺酰胺基转移酶超家族成员;同源性分析表明,该蛋白与多种植物的茎部特异蛋白和铝诱导蛋白有较高的同源性,进化上与小兰屿蝴蝶兰的茎部特异蛋白亲缘关系最近;该基因在营养器官中表达水平较高,在花器官中表达水平较低;13℃/8℃(昼/夜)的低温胁迫抑制PhTSJT1基因的转录表达,并随着低温胁迫时间的延长,Ph TSJT1基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃冷胁迫低温条件下,PhTSJT1基因在处理1 h时,表达水平升高,处理8 h时表达水平最高,16 h后表达水平逐渐降低。由此推测,PhTSJT1参与4℃冷胁迫的分子调控。本研究不但有助于理解热带亚热带植物的耐冷机制,也为蝴蝶兰新品种的遗传改良提供帮助。     

Enhancement of direct somatic embryogenesis and plantlet growth from leaf explants of <Emphasis Type="Italic">Phalaenopsis</Emphasis> by adjusting culture period and explant length

Two Phalaenopsis orchids, Phalaenopsis amabilis and Phalaenopsis ‘Nebula’, were used to test the effects of induction period (30, 45 and 60 days), subculture period (30, 45 and 60 days), and explant length (1, 1.5 and 2 cm) on direct somatic embryogenesis from different regions (leaf tip, adaxial side, abaxial side and cut end) of leaf explants from in vitro grown seedlings. The results showed that the cut end had a highest competence to form embryos than the other regions of the leaf explants from both orchids. In addition, the suitable culture conditions were 60 days for induction period in darkness, 45 days for subculture period in light and 1 cm for explant length. Besides, the combinations of N⁶-benzyladenine (BA) and naphthaleneacetic acid were tested on their effects on plantlet conversion and further development of leaf-derived embryo. It was found that 0.5 mg/l of BA showed the highest response on plantlet conversion rate and the lowest browning rate of explants. In this communication, the embryo structures and development were proved by scanning electron microscopy.     

Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids

The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light, direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher embryogenic competence than the leaf tip segment.     

植物组织培养外植体褐变的研究进展

外植体的褐变是组织培养过程中的主要障碍。目前一般认为影响褐变的因素主要有外植体材料、培养基和培养条件。为了防止褐变的发生,主要可以从外植体和培养条件、进行细胞筛选和材料的预处理、抑制剂或吸附剂的加入等方面入手。酚、酚酶的区域性分布假说、自由基伤害假说、保护酶系统假说是褐变机理方面较为成熟的3种假说。     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.