毛尖紫萼藓总RNA的提取方法研究

介绍一种提取苔藓植物毛尖紫萼藓(Grimmia pilifera P.Beauv)总RNA的方法。以新鲜的紫萼藓为材料,采用改良的SDS的方法,以氯化锂为沉淀试剂,成功地提取了该植物的总RNA。并与其他方法作了对比,结果发现该方法获得的RNA条带清晰、完整性好、纯度高、DNA污染小。可满足反转录及RT-PCR等实验要求。     

不同微生境中水光温变化对毛尖紫萼藓叶绿素荧光特性的影响

毛尖紫萼藓（Grimmia pilifera）多生长在裸岩表面且具有多种微生境,其生长过程受到水分、光照和温度等环境因素的交互影响,但其光合生理特征如何响应这种变化的环境条件尚不清楚。开展原位（荫蔽和向阳裸岩2种微生境）和室内模拟实验,分析了不同水分（模拟降水量和降水频次）、光照、温度及其复合梯度处理对毛尖紫萼藓叶绿素荧光特性的影响。原位实验结果显示荫蔽生境原位生长毛尖紫萼藓光化学效率显著高于向阳生境。在室内相同培养条件下,脱水过程中来自荫蔽生境植株有效光合效率保持时间比向阳生境明显缩短,表现出较低的脱水耐受性。水-光-温复合模拟实验显示,降水频次和光-温变化对毛尖紫萼藓光化学效率均有极显著影响且存在一定的交互作用,而降水量的影响相对较弱;弱光低温及1次/（2 d）的降水频率条件下毛尖紫萼藓具有最高的光合活性。总体来看,荫蔽生境、弱光低温条件及中等频次降水有利于毛尖紫萼藓生长,但向阳生境毛尖紫萼藓则具有更强的环境耐受性。     

毛尖紫萼藓干旱胁迫cDNA文库的构建

干旱胁迫是影响植物生长发育的主要环境因素,严重影响农作物的产量。解决这个问题的有效途径是培育和利用优良的抗旱品种。应用比较功能基因组学方法筛选抗旱相关基因,并通过基因工程培育抗旱品种已成为植物遗传资源与品种改良研究的重要内容。毛尖紫萼藓（Grimmia pilifera）是典型旱生藓类,生长在向阳的裸岩上,具有很强的抗旱能力,是很好的抗旱基因资源。本研究采用SMART技术构建毛尖紫萼藓干旱cDNA文库,文库滴度为2.8×10⁵ pfu·mL^-1,重组率为91.7%,插入片段大小为500~2 000 bp,平均为800 bp。通过测序我们获得了1 045条ESTs,其中高质量的996条,通过拼接获得875个Unigenes,为进一步筛选抗旱相关基因奠定了基础。     

毛尖紫萼藓GpUCH基因克隆及表达分析

从毛尖紫萼藓干旱cDNA文库中筛选了一条与抗旱相关的泛素羧基末端水解酶基因的EST序列,采用RACE技术从毛尖紫萼藓中克隆出了该基因的cDNA全长序列,命名为白UCH。对该基因的序列特征、进化关系和表达谱进行了分析,为全面研究其功能奠定了基础。该基因cDNA全长951bp,开放阅读框711bp,共编码236个氨基酸。生物信息学分析显示,印UCH基因相对分子质量为25．7kD,等电点为4．67,为不稳定蛋白,属于跨膜蛋白但不存在信号肽。系统进化树分析表明,GpUCH与小立碗藓UCH蛋白一致性较高。实时荧光定量PCR结果显示,GpUCH基因在复水和干旱的条件下均能表达,但是在不同的条件下表达情况差异显著。表明该基因可能在毛尖紫萼藓干旱过程中发挥一定的作用。     

毛尖紫萼藓抗旱相关基因Gp-LEA的克隆与表达分析

以毛尖紫萼藓干旱cDNA文库中获得的一段与LEA基因同源性较高的EST序列为基础,采用RACE技术分离该基因cDNA全长序列,命名为Gp-LEA。Gp-LEA基因的cDNA全长814bp,开放阅读框456bp,编码含151个氨基酸蛋白质。生物信息学分析结果显示,Gp-LEA蛋白为稳定蛋白,分子质量为16.612kD,理论等电点（pI）为5.06,含有LEA₂功能结构域,不属于跨膜蛋白且不存在信号肽。系统发生分析表明,Gp-LEA基因编码蛋白与花旗松LEA蛋白亲缘关系最近。荧光定量PCR分析显示,Gp-LEA基因在复水和快速干旱模式下均能表达。推测Gp-LEA基因在毛尖紫萼藓的复水和干旱过程中起着重要作用。     

毛尖紫萼藓超氧化物歧化酶基因GpSOD的克隆及表达分析

超氧化物歧化酶(SOD)在植物中普遍存在,具有清除自由基、维持活性氧代谢平衡的功能,在植物抗逆中发挥着重要的作用。本研究以耐旱能力极强的毛尖紫萼藓(Grimmia pilifera)为试材,采用RACE技术克隆获得1个超氧化物歧化酶基因SOD的cDNA序列,命名为GpSOD(GenBank登录号:GU989312)。该基因全长726 bp,编码区为465 bp,编码154个氨基酸。序列分析表明:该基因编码蛋白是非分泌性疏水蛋白,具有2个铜锌超氧化物歧化酶位点,属于Sod Cu基因家族,与其他植物超氧化物歧化酶的同源性为68.8%~86.8%。实时定量PCR检测结果显示,虽然GpSOD在干旱及复水条件下均有表达,但GpSOD受干旱诱导表达明显,且复水处理抑制其表达,表明GpSOD蛋白与毛尖紫萼藓响应干旱胁迫的过程密切相关,该结果为进一步寻找高效抗氧化胁迫基因提供前提条件。     

毛尖紫萼藓泛素延伸蛋白基因克隆及序列分析

目的:该文首次从毛尖紫萼藓中克隆到泛素延伸蛋白基因的全长,为深入研究其功能打下基础。方法:提取植物的总RNA,经反转录得到cDNA,根据实验室得到的毛尖紫萼藓泛素延伸蛋白基因的EST序列设计引物,基于3’RACE(Rapid Ampli-fication of cDNA End)技术克隆得到全长序列,使用分子生物学软件进行序列分析。结果:获得了cDNA全序列,GenBank登录号为JQ659260,序列全长为673bp,开放阅读框为471 bp,编码156个氨基酸残基。结论:通过Blast P对其编码的氨基酸序列进行比对结果显示:毛尖紫萼藓泛素延伸蛋白与小立碗藓、北美云杉、葡萄、蓖麻、毛果杨、紫茎泽兰的泛素延伸蛋白的同源性均在96%以上。     

微生境对耐旱石生毛尖紫萼藓水分保持与散失特性的影响

毛尖紫萼藓(Grimmia pilifera)是典型石生耐旱藓类,水分是其生存繁衍的关键影响因素。为探究微生境对毛尖紫萼藓水分生理的影响,在安徽省大龙山国家森林公园低山丘陵区选择3种典型微生境(竹林遮蔽S-1,向阳裸岩E,薜荔灌丛遮蔽S-2),对比研究毛尖紫萼藓个体大小、饱和含水量及脱水过程中的含水量指标在不同微生境间的差异性,综合判断微生境对苔藓水分胁迫耐受性的影响及其权衡特征。结果表明:3种微生境毛尖紫萼藓个体大小及饱和含水量差异显著,其中竹林遮蔽生境毛尖紫萼藓具有较小的个体但拥有较高的内、外含水量。向阳裸岩和薜荔灌丛遮蔽生境植株大小及外吸水量接近,但前者内含水量更高。两荫蔽生境苔藓外吸水量是内吸水量的7倍,显著高于向阳裸岩生境的5.8倍。脱水过程中相同时间点向阳裸岩生境苔藓各含水量指标均高于两遮蔽生境,且达到相同含水量的时间差也随脱水进程持续而逐渐增大,这为向阳裸岩生境苔藓的有效光合作用(即相对含水量不低于35%时)及脱水后期的生理和结构调整赢得更多时间。综合而言,向阳裸岩生境毛尖紫萼藓比荫蔽生境具有更强的脱水耐受性,但后者可以通过增加外部吸水量来弥补失水过快的缺陷,这可能是不同微生境毛尖紫萼藓对水分吸收和保持的权衡策略。     

束生刚毛藻脱水和复水过程中光合作用的变化

利用叶绿素荧光技术研究了束生刚毛藻（Cladophora fascicularis）在脱水和复水过程中光合作用的变化。自然干燥状态下,束生刚毛藻迅速脱水,干燥2．5h后,脱水达90％。脱水1．5h内。光合作用的最大量子产量Fv／Fm和最大相对电子传递速率Pm分别下降了约10％和11％;此后Fv／Fm和Pm的下降速度明显加快,脱水2．5h后,已下降到初始值的一半。脱水后半饱和光强Ik逐渐下降;初始斜率α在脱水初始阶段有轻微上升,此后逐渐下降。复水后Fv／Fm、Pm、Ik和α均迅速恢复。这表明脱水可引起束生刚毛藻光合作用的降低,但光合器官并未受到不可逆损伤,因此复水后光合活性迅速恢复。这种复水后光合能力的快速恢复,有利于束生刚毛藻适应潮间带特殊的生活环境。     

干旱和复水对大豆(Glycine max)叶片光合及叶绿素荧光的影响

选用河南省大面积种植的大豆品种豫豆29作为实验材料,通过研究逐步干旱和旱后复水条件下大豆叶片光合、叶绿素荧光等指标随土壤水分的动态变化规律,以期为大豆的水分高效利用提供理论依据。研究发现,在土壤相对含水量高于46.5%时,虽然随着土壤相对含水量的下降,豫豆29仍可以保持它的叶片水分状态;豫豆29的叶片净光合速率在土壤水分中等条件下最大,在土壤相对含水量为64.3%时,它比对照组高出11.2%(P<0.01);在实验的第3d,处理组的土壤相对含水量降为46.5%,叶片水势与对照组相比降低了7.2%(P>0.05),净光合速率为对照组的89.6%(P<0.05),但气孔导度却迅速下降为对照组的44.7%(P<0.01),这说明与叶片的光合和水分状况相比,豫豆29的气孔对土壤水分的匮缺更加敏感。复水后,豫豆29叶片的水势、净光合速率、气孔导度和叶绿素荧光等值都可以得到迅速的恢复,并在实验的最后接近对照组的水平,这表明豫豆29的叶片光合在水分胁迫解除后有迅速恢复的能力。     

Organ-dependent responses of Periploca sepium to repeated dehydration and rehydration

Plant responses to water deficit occur in a complex framework of organ interactions, but few studies focus on the effect of drought stress on all organs in a whole-plant. The effects of repeated dehydration and rehydration (DH) on physiological and biochemical responses in various organs of Periploca sepium Bunge (P. sepium) were investigated. The leaf relative water content decreased significantly during drought, but recovered and showed an increase when compared to well-watered control plants. The malondialdehyde (MDA) content increased in mature and old leaves, but decreased in young leaves, new stems and fine roots during drought, indicating that the young and vigorous tissues of a whole-plant are protected preferentially from the oxidative stress. Among all organs, the fine roots showed the highest levels of proline, total free amino acids (TFAA) and Na⁺, while the leaves showed the highest levels of total soluble sugars (TSS), soluble proteins (SP), Ca²⁺ and Mg²⁺. The response to DH differed in different organs, both in magnitude and in the type of solutes involved. Drought stress increased the contents of proline, TFAA, TSS, SP and K⁺ in all organs of P. sepium plants, while the accumulation amounts were obviously different among the organs. The storage starch in stems and roots plays an important role in providing carbohydrates for growth. Changes in Na⁺, Ca²⁺ and Mg²⁺ under DH presented a high degree of organ specificity. Our data indicates that response strategies are different between different organs; therefore, evidence the needs to integrate all the information in order to better understand plant tolerance mechanisms.     

Morphological and ultrastructural aspects of dehydration and rehydration in leaves of Sporobolus stapfianus

The resurrection species Sporobolus stapfianus Gandoger has been studied by LM, TEM and SEM in order to define the leaf morphology and fine structure and to analyse the cellular changes occurring during the processes of dehydration and rehydration of the plant. Some characteristics of the fully hydrated leaf and some ultrastructural and physiological events which take place during leaf wilting are discussed in relation to their possible role in plant desiccation-tolerance.The leaves of S. stapfianus show several characteristics common among xerophytic species. In the resurrection leaf they could play a role in slowing down the drying rate, thus leaving time to activate the mechanisms protecting the cell structures against drought damage. Actually, the S. stapfianus leaves do not undergo important cellular alterations during dehydration. The chloroplasts, in particular, retain part of their photosynthetic pigments and thylakoid membranes. Upon rewatering leaf recovery is rather fast and the tissue structure and cell organization of the fully hydrated state are already regained after two days.     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.     

干旱和复水对入侵植物三裂叶蟛蜞菊叶片叶绿素荧光特性的影响

选取入侵植物三裂叶蟛蜞菊(Wedelia trilobata)及其本地近缘种蟛蜞菊(W. chinensis)为实验材料,比较干旱和复水后二者叶片的叶绿素荧光特性和抗氧化酶活性等生理指标的变化规律,探讨入侵种三裂叶蟛蜞菊对干旱的响应和生态适应性.结果发现,在自然干旱处理过程中,入侵种三裂叶蟛蜞菊与本地种蟛蜞菊相比土壤含水量下降较快,对它们叶片气孔形态的比较发现,干旱胁迫11d后三裂叶蟛蜞菊叶片气孔开度明显大于蟛蜞菊,这可能是导致其失水较快的原因之一.干旱胁迫11d后三裂叶蟛蜞菊的PSⅡ最大光能转化效率(Fv/Fm)降低了43.8%,而蟛蜞菊只降低了3.7%;同时,与蟛蜞菊相比,三裂叶蟛蜞菊的PSⅡ实际光化学量子产量(Yield)、表观光合电子传递速率(ETR)和光化学猝灭系数(qP)也表现出较大幅度的降低,说明三裂叶蟛蜞菊对干旱胁迫较敏感;但复水后,三裂叶蟛蜞菊能够较快地恢复到正常水平,且与本地种不存在显著差异.这主要是由于入侵种在遭受干旱胁迫时提高了对其过量激发能的热耗散能力以及超氧化物歧化酶(SOD)和抗坏血酸还原酶(APX)的活性,保护光合机构少受不可逆的损伤,使其在干旱胁迫解除后光合功能得以迅速恢复.研究结果初步表明三裂叶蟛蜞菊容易受到水分条件的限制,它向干旱地区扩散的可能性较小.     

Surface reflectance properties of Antarctic moss and their relationship to plant species, pigment composition and photosynthetic function

In this study the variations in surface reflectance properties and pigment concentrations of Antarctic moss over species, sites, microtopography and with water content were investigated. It was found that species had significantly different surface reflectance properties, particularly in the region of the red edge (approximately 700 nm), but this did not correlate strongly with pigment concentrations. Surface reflectance of moss also varied in the visible region and in the characteristics of the red edge over different sites. Reflectance parameters, such as the photochemical reflectance index (PRI) and cold hard band were useful discriminators of site, microtopographic position and water content. The PRI was correlated both with the concentrations of active xanthophyll‐cycle pigments and the photosynthetic light use efficiency, F_v/F_m, measured using chlorophyll fluorescence. Water content of moss strongly influenced the amplitude and position of the red‐edge as well as the PRI, and may be responsible for observed differences in reflectance properties for different species and sites. All moss showed sustained high levels of photoprotective xanthophyll pigments, especially at exposed sites, indicating moss is experiencing continual high levels of photochemical stress.     

铁胁迫下大豆光合和磷/铁性状对磷素的响应

为了明确低铁胁迫下磷素用量对大豆光合和磷/铁性状的影响及基因型差异,为磷、铁肥的合理施用提供理论依据,以前期筛选的6个磷高效基因型和6个磷低效基因型大豆为供试材料,设4个P∶Fe配比处理,分别为0∶30、30∶30、150 ∶30和300∶30(μmol·L-1),对大豆叶绿素荧光特性和磷、铁利用率进行了测定,利用单株...     

S.stapfianus and E. curvula cv. Consol in vivo photosynthesis, PSII activity and ABA content during dehydration

Changes in photosynthetic activity, CO₂ assimilation rate, _PSII by fluorescence andABA content, were monitored in the grasses Eragrostis curvula cv. Consol and Sporobolusstapfianus Gandoger in response to dehydration. Thefirst being a warm season grass well adapted todrought and the second a desiccation-tolerant orresurrection plant. The trial was performed on intactleaves during a whole plant drying course. After acycle of dehydration (down to 5% RWC) andrehydration to full turgor the resurrection plantshowed recovery of photosynthetic capability. E.curvula is drought-resistant but notdrought-tolerant being not capable of recovering whendried to 20% RWC. The sensitivity of photosynthesisto the drying treatment was different in E.curvula and S. stapfianus. During dryingtreatment, up to a leaf water loss of 40%, E.curvula photosynthesis seemed to be inhibited bycarbon metabolism, because PSII activity was not yetaffected. In S. stapfianus at the same point ofdehydration photosynthesis still worked though adown-regulation of PSII activity (Fv/Fm) occurred ata higher RWC. Non-photochemical chlorophyllfluorescence quenching (qN) was analysed. Duringdrying qN increased in both plants, but more in theresurrection plant though its assimilation rate wasless affected. The importance of ABA in regulating CO₂ assimilation rate is discussed.     

持续脱水下海藻叶绿素瞬变荧光模式的三阶段转换及临界水量的发现

在自然干燥条件下,跟踪研究了海洋红藻带形蜈蚣藻（Grateloupia turuturu Yamada),绿藻石莼（Ulva pertusa Kjellm)和褐藻海带（Laminaria japonica Aresch)的叶绿素荧光动力学曲线的变化,并用脉冲调制荧光仪分析了藻体脱水过程中的各个荧光参数。结果表明,在持续脱水过程中,3类海藻的荧光动力学曲线都显示出3个阶段的变化,即：1）在脱水开始时正常的峰形曲线;2）荧光上升至最高时持平、形成了高原型曲线;3）荧光跌落变成平台型曲线,发现荧光跌落前藻体的含水百分数是一个重要的生理指标,称为临界水量（CWC）。一旦藻体失水至低于此临界水量时,即便复水也不能使原来的峰形曲线得以恢复,说明脱水可能引起了叶绿体类囊体膜的不可逆损伤。临界水量可以用来衡量不同海藻的耐旱力,其数值高低随不同海藻而不同,并与其耐旱力成负相关。如在上述3大类海藻中,海带的CWC值最高（约90％）,但其耐旱性最差。此外,在红藻（只有红藻）经受干燥后作复水处理时,观察到有荧光急剧上升（爆发）的现象。对海膜脱水前后的几个荧光参数（F0、Fv、Fv/Fm)作了测定和比较,发现F0和Fv在脱水的第一阶段都上升,而v/Fm值维持基本不变。因此可以得出结论,活体叶绿素荧光对干旱的最初响应是立即增强,当继续失水时,F0保持上升而Fv却下降并越来越低,故此时总荧光的变化（包括荧光的跌落和爆发）主要是由F0的变化所引起的,因而进一步研究F0与脱水时类囊体膜状态变化的关系具有重要意义。     

Changes in the content of individual lipid classes of a lichen <Emphasis Type="Italic">Peltigera aphthosa</Emphasis> during dehydration and subsequent rehydration

A lichen Peltigera aphthosa (L.) Willd. was subjected to a short-term (7 days) or a long-term (180 and 540 days) dehydration followed by rehydration. Then the composition and content of lipids, as well as the rate of their metabolism (the rate of sodium 2-¹⁴C-acetate incorporation) were investigated. The long-term dehydration resulted in a dramatic decrease in the content (per dry wt) of major extrachloroplastic phospholipids, mainly phosphatidylcholines and phosphatidylethanolamines. The rehydration of lichen thalli after a short-term and long-term dehydration also resulted in an enhanced breakdown of these lipid molecules; however, it was accompanied by their rather intense in vivo synthesis, which was decreased after long-term dehydration. In contrast to phospholipids, the betaine lipids, diacylglyceroltrimethylhomoserines (DGTSs), were involved in metabolic processes to a far lesser extent. In the course of rehydration, their content was virtually unchanged and decreased only after 540 days of dehydration. The rate of incorporation of sodium 2-¹⁴C-acetate into the DGTS molecules was moderate and did not change even after long-term dehydration. Glycolipids were characterized by a fair tolerance to hydrolytic processes and by an increase in the rate of their synthesis after 540 days of the lichen dehydration. Responses of neutral lipids to dehydration turned out to be different. The long-term dehydration (for 540 days) was accompanied by a decrease in the contents of free sterols and sterol esters, whereas the contents of di- and triacylglycerols remained unchanged. Rehydration resulted in a decrease in diacylglycerol and sterol ester contents. All neutral lipids were characterized by a dramatic decrease in the rate of de novo synthesis after long-term dehydration. It was suggested that the tolerance of lichen to long-term dehydration was appreciably determined by the tolerance of its phycobiont, in this case, a green alga Coccomyxa sp.; the bulk of its lipids was characterized by a minimum rate of breakdown and, at the same time, by a stable synthesis.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 43–50.Original Russian Text Copyright © 2005 by Kotlova, Sinyutina.