香蕉抗寒相关功能基因研究进展

香蕉是世界上产量最大的三大水果之一,也是世界上第四大粮食作物。低温是影响中国香蕉生产的重要因素之一,尤其是除海南和广东西南等地区外的香蕉产区常年遭遇冬春寒流侵袭。近年来随着抗寒基因工程的发展,有关香蕉抗寒性的分子水平研究得到了较大进展。本文从香蕉抗寒相关的功能基因入手,归纳了近几年国内外关于香蕉的抗寒相关功能基因(冷胁迫相关蛋白基因,膜脂相关蛋白基因,抗氧化酶基因等)研究并提出了展望,旨在为香蕉抗寒主效基因资源的发掘及香蕉抗寒品种的选育提供思路。     

低温胁迫下甘蓝型冬油菜蛋白质组学及光合特性分析

为了探明低温胁迫对甘蓝型冬油菜光合特性的影响,本研究采用抗寒性不同的两种甘蓝型冬油菜,在低温(-4℃)处理下对其蛋白质组学进行分析.结果 表明,低温处理下,甘蓝型冬油菜叶片萎蔫下垂,叶绿体膜质出现不同程度的破裂,叶绿体基质出现断裂,保卫细胞淀粉粒由多变少、由大变小.气孔处于关闭或半关闭状态,胞间二氧化碳浓度(Ci)增加,气孔导度(Gs)、净光合速率(Pn)和蒸腾速率(Tr)减小,相较于强抗寒品种,冷敏感品种受害症状更明显.2-DE和PDQuest8.0.1软件对强抗寒品种分析表明,低温胁迫下具有显著差异蛋白点414个,对表达变化量在2倍丰度以上的24个蛋白点进行质谱分析鉴定.成功鉴定出的20个蛋白参与能量代谢、氧化磷酸化、氨基酸代谢、光合生物的固碳作用和光合作用蛋白等过程.综上,低温胁迫下,冷敏感品种气孔关闭,叶绿体受损,进而使其净光合速率下降,淀粉积累量减少,导致其越冬困难;强抗寒品种气孔不完全关闭,叶绿体受损较小,且与光合作用相关的蛋白上调表达,净光合速率下降较小,淀粉粒积累较多,能安全越冬.本研究揭示了甘蓝型冬油菜低温胁迫下光合适应机制,为强抗寒甘蓝型冬油菜育种提高理论依据.     

白菜型冬油菜反复低温与恢复过程中叶片的差异蛋白分析

为了从蛋白质分子生物学方面研究白菜型冬油菜不同抗寒品种低温逆境及恢复条件下相关蛋白的响应机制,揭示大田生产中冬油菜抵御突发骤变低温伤害的抗寒机理,以超强抗寒冬油菜品种陇油7号和弱抗寒品种天油2号为材料,在培养箱培养至幼苗五叶期时,模拟北方寒旱区冬油菜返青期倒春寒环境,运用双向电泳结合质谱技术,分析其幼苗在低温(4℃)-恢复(20℃)-低温(4℃)-恢复(20℃)过程中存在的蛋白及其差异表达量。研究表明低温胁迫与恢复过程中,有2个蛋白点发生了显著差异的丰度变化,它们分别是半胱氨酸型氧化还蛋白过氧化物酶(定义为1号蛋白)和一种未知蛋白(2号蛋白)。且不同抗寒性品种存在2种不同蛋白的差异表达机制,1号蛋白在抗寒性较弱的天油2号低温胁迫恢复过程中表现出上调-下调-上调的变化趋势,抗寒性较强的陇油7号中此蛋白在第一次低温胁迫后消失,而出现了另外一种未知蛋白(蛋白2),且在低温胁迫恢复过程中也表现出上调-下调-上调的变化趋势,并且表达量高于天油2号中的蛋白1,因此,这种未知蛋白2可能是陇油7号抗寒性强于天油2号的原因之一。     

白菜型冬油菜叶片质外体蛋白提取及响应低温胁迫的蛋白表达分析

为了研究质外体蛋白在低温胁迫下白菜型冬油菜抗寒的作用机理,以陇油7号五叶期叶片为试材,采用2种不同提取液(提取液A:0.1 mol/L p H值=7.6 Tris-HCl、0.2 mol/L KCl、1 mmol/L PMSF,提取液B:0.05 mol/L p H值=7.6 Tris-HCl、0.01 mol/L EDTA-Na2、0.02 mol/L Vc、1 mmol/L PMSF)进行质外体蛋白提取效果比较。结果表明:提取液A的蛋白提取率达到了(0.073±0.004 6)mg/g,比提取液B提高了42.88%;经六磷酸葡萄糖脱氢酶(G6PDH)活性检测,提取液A提取的蛋白质污染率较提取液B低0.91%;双向电泳第一向等电聚焦IEF中,提取液A提取的蛋白质不仅除盐时间较提取液B短4.5 h,且获得的凝胶图谱清晰;通过丰度计算,得到陇油7号低温胁迫(4℃,48 h)后具有显著差异的蛋白点339个,其中表达量变化在2倍以上的蛋白点为46个,包括上调表达蛋白点18个,下调表达蛋白点21个,特异性表达蛋白点7个,推测这些差异蛋白点可能与低温胁迫有关。对特异表达的7个蛋白点进行质谱分析鉴定,得到与低温胁迫相关的蛋白,为白菜型冬油菜超强抗寒品种陇油7号抗寒相关分子机理的研究奠定了基础理论。     

4种杜鹃幼苗高温胁迫下蛋白表达差异

为进一步明确杜鹃幼苗的耐热机制,为耐热杜鹃育种提供分子依据,本研究比较了映山红、满山红、马银花和云锦杜鹃4种耐热性不同的杜鹃幼苗在高温胁迫下蛋白质组分变化的差异。研究发现,高温胁迫导致4种杜鹃叶片蛋白质表达出现了明显差异,主要表现为蛋白质表达的下调或消失、蛋白质表达的上调或特异蛋白的出现及波动表达。其中耐热性最强的映山红有60个蛋白位点出现差异表达,其中包括30个特异表达蛋白位点和8个表达上调的蛋白位点,其次是马银花,有55个蛋白位点出现差异表达,10个蛋白质表达上调,27个特异蛋白。高温胁迫下映山红和马银花表达上调和特异表达的蛋白位点也明显高于另外2种热敏感杜鹃。     

香蕉BADH基因序列及表达特性分析

甜菜碱醛脱氢酶(BADH)是植物体内甜菜碱合成过程中的关键酶,对植物抵御逆境具有重要作用。为研究香蕉BADH基因序列及表达特性,本研究利用香蕉基因数据库对香蕉BADH基因序列及蛋白序列进行生物学信息分析,采用qTR-PCR技术对香蕉幼苗中BADH基因进行表达分析。结果表明:MaBADH基因属单克隆基因,含有15个外显子,14个内含子;顺式调控元件预测显示在MaBADH启动子区域存在15种激素和逆境胁迫相关的顺式调控元件;蛋白结构域和功能分析有一个甜菜碱醛脱氢酶结构域具有氧化还原酶活性;亚细胞定位在叶绿体中,多重序列比对发现其具有N-端信号肽和C-端信号肽两种信号肽;系统进化树显示其与水稻、玉米、菠萝亲缘关系最近。基因表达分析发现MaBADH具有组织特异表达特性,在叶片中相对表达量最高,假茎次之,根最低;此外,茉莉酸甲酯能够诱导香蕉幼苗根和叶中MaBADH差异表达,其中对叶片的诱导能力更强。本研究为激素信号调控甜菜碱分子保护机制和利用基因工程提高香蕉抗逆性提供了参考依据。     

干热胁迫条件下文冠果差异蛋白表达分析

为分析干热胁迫下文冠果幼苗的分子响应机制,本研究以三月龄文冠果幼苗为材料,采用非标定量技术,结合质谱鉴定及生物信息学分析,探究文冠果幼苗叶片在干热胁迫下的蛋白差异表达。本试验共鉴定得到蛋白组数1 442个,根据明显差异肽段确定标准分析得到差异蛋白100个,其中包括上调表达蛋白55个,下调表达蛋白45个。在100个差异蛋白中,通过数据库搜索分析得到了已知功能的蛋白29个,假定蛋白71个,其中上调差异表达蛋白主要参与抗氧化代谢、逆境防御、细胞内信号转导、光合作用等过程,下调差异表达蛋白主要参与蛋白加工修饰与水解、氧化还原反应、TCA循环、物质代谢等过程。本研究结果有助于阐明文冠果干热胁迫分子机制。     

甜菜叶片应答干旱胁迫的差异蛋白质组学分析

甜菜是我国主要糖料作物之一,为从蛋白质组学水平了解甜菜对干旱胁迫的应答,进一步明确甜菜的杭旱分子机理,通过双向电泳技术对甜菜耐旱品种HI0466正常供水和干旱胁迫条件下叶片差异表达蛋白质组进行了对比研究,质谱鉴定共获得了17个差异表达蛋白点,包括丰度上调的10个,新出现的蛋白点6个,丰度下调的1个口功能分类分析表明干旱胁迫蛋白在光合作用、物质与能量代谢、活性氧清除、细胞结构、物质运输和蛋白质合成等方面发挥作用。     

表油菜素内酯影响水稻幼苗响应低温胁迫的蛋白质组学分析

表油菜素内酯(2,4-Epibrassinolide, EBR)是一种被广泛研究和应用的油菜素内酯类(brassinosteroids, BRs)植物生长调节剂, 它能有效增强植物对低温的耐受性, 但EBR在蛋白质组水平上对水稻幼苗响应低温胁迫的影响尚不清楚。本研究用0.1 mg L ^-1 EBR和蒸馏水分别浸泡萌发的日本晴种子, 然后提取4°C胁迫培养和26°C正常培养幼苗的总蛋白, 进行质谱非标(label-free)定量分析和平行反应监测(parallel reaction monitoring, PRM)验证。最终共鉴定出5778个蛋白质, 其中, 在有定量信息的4834个蛋白中, 401个上调和220个下调蛋白与EBR影响水稻幼苗响应低温胁迫有关。功能分析和代谢通路富集分析发现, 上调蛋白主要与RNA结合或水解酶活性等分子功能相关, 并富集在碳代谢、叶酸合成和氨基酸生物合成等途径中; 下调蛋白主要与催化活性和氧化还原酶活性相关, 主要涉及卟啉和叶绿素代谢等代谢途径。PRM验证结果和文献证据显示, 分布在碳代谢和苯丙素代谢通路中的NADP-苹果酸酶、过氧化物酶、3-磷酸甘油酸脱氢酶、烯醇化酶、甘油醛-3-磷酸脱氢酶和丙酮酸激酶参与了EBR对低温胁迫水稻幼苗的调控, 提示BRs可通过多种途径影响水稻幼苗对低温胁迫的响应。     

低温胁迫下白菜型冬油菜差异蛋白质组学及光合特性分析

分离鉴定白菜型冬油菜低温差异表达蛋白质, 从蛋白质组角度揭示白菜型冬油菜抗寒机理奠定基础。以强抗寒白菜型冬油菜陇油7号为材料, 采用双向电泳和质谱分析技术, 比较低温(4°C、–4°C)和常温(25°C/20°C)下叶片蛋白质组差异;对差异蛋白进行KO和KEGG功能分析。结果表明,低温下陇油7号生长点下陷、植株匍匐, 气孔处于关闭或半关闭状态。2-DE和PDQuest8.0.1软件分析表明, 常温和4°C低温下叶片蛋白质斑点数分别726、738;相对于常温处理, 4°C低温下陇油7号叶片10个蛋白点特异表达、5个蛋白点未表达;MALDI-TOF-TOF MS质谱分析鉴定出11个蛋白质, 参与碳水化合物代谢、糖代谢、氨基酸代谢、有机酸代谢、核酸代谢、信号转导与细胞通讯等细胞过程。冰晶形态显微观察结果表明低温处理后陇油7号叶片蛋白质提取液中含有高活性抗冻蛋白(antifreeze proteins, AFPs)。鉴定的11个蛋白质中, 有5个蛋白质点与光合作用有关, 低温下陇油7号叶片1,5-二磷酸核酮糖羧化酶(RuBPCase)活性和净光合速率Pn下降。叶片Pn下降与RuBPCase表达抑制和活性降低有关, 非气孔限制是Pn下降的主要因素;高活性抗冻蛋白在白菜型冬油菜抗寒中发挥重要作用。     

A review on adaptation of banana (Musa spp.) to cold in subtropics

Global production of bananais adversely affected by abiotic stresses, namely, drought, salinity and low temperature which cause changes in morphology, anatomy, physiology and biochemical metabolism in plant. Different banana cultivars respond differently when grown in subtropical regions due to changes in temperature mainly at the time of flowering and fruiting. The subtropical regions persist cold in winter which become a major constraint for proper growth and development of banana. Only few cultivars are identified for cold tolerance. The cold tolerance is a complex phenomenon, involving numbers of interrelated metabolic pathways. With the onset of cold, growth decrease through alteration in the synthesis of metabolites which are regulated by expression of genes and their interactions. Recently, complete genome sequencing, mutation and transgenesis provided deep insight into the complex mechanism of cold tolerance in banana. In this review, efforts are made to compile the findings and to interpret the better application of technologies in understanding the cold tolerance which may assist in banana breeding.     

宁夏设施番茄主栽品种芽期和苗期抗寒性评价

以9个不同番茄品种在低温下的发芽特性及苗期的冷害指数,综合评估其耐冷性,以期为生产上筛选抗寒性强的品种提供依据。试验结果,表明“嘉粉8346”和“爱吉168”均表现为耐低温较强的品种。     

江南牡丹叶片耐热差异蛋白的鉴定与分析

为探究江南牡丹在高温胁迫下的应答模式,以进一步获得可能的耐热目的蛋白,本研究以江南牡丹耐热品种‘羽红’的小苗为实验材料,运用同位素标记相对定量与绝对定量技术(iTRAQ),结合质谱鉴定与相应生物学分析,探究40℃高温处理和25℃正常生长的江南牡丹叶片中蛋白差异表达情况。一共鉴定得到的蛋白总数1 916个,大于等于1.5倍的差异蛋白有100个,表达上调的有55个,表达下调的有45个。基因本体(GO)分子功能分析发现差异蛋白富集前三的分别是分子功能中的催化活性和结合功能以及生物学进程中的代谢过程。KEGG通路分析发现差异蛋白富集程度最高的是包括丙酮酸代谢、碳代谢等过程的代谢通路,其次是内质网中的蛋白质合成和光合作用通路。同时发现了肽基脯氨酰基异构酶、APX、HSPs、FBA、CaM、RubisCo、乙酰辅酶A羧化酶、GS等蛋白可能是江南牡丹应对高温胁迫中发挥重要作用的目的蛋白。本研究结果为进一步探究江南牡丹在高温胁迫下的应答模式提供了理论基础。     

ITRAQ-based quantitative proteomic analysis of japonica rice seedling during cold stress

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.     

棉花幼苗根系响应NaCl胁迫的差异蛋白组学分析

为了探讨盐胁迫对棉花幼苗根系蛋白表达谱的影响,丰富棉花在分子水平上应对盐胁迫的机理,本研究对盐胁迫下棉花幼苗根系进行差异蛋白组学分析。试验利用1%NaCl模拟盐胁迫,对陆地棉品种‘中棉69’进行72 h处理,利用磷酸盐缓冲液研磨后再用月桂酸辅助三氯醋酸沉淀的方法获得棉花幼苗根系蛋白样品,并进行双向电泳分离,获得了分辨率较高、背景清晰的差异蛋白表达图谱。图谱分析显示,共有36个棉花幼苗根系蛋白点响应盐胁迫,其中16个表达量显著上调,20个表达量显著下调。对其中的10个表达差异的蛋白进行了质谱分析和同源蛋白鉴定,结果显示它们分别为抗坏血酸过氧化物酶、烯醇化酶、谷胱甘肽-S-转移酶、醌氧化还原酶、膜联蛋白AnxGb3、叶绿体Rubiso大亚基蛋白结合α亚基、P23蛋白、果糖激酶、乙醇脱氢酶、腺嘌呤磷酸转移酶1亚型1。本研究结果显示盐胁迫下有部分棉花幼苗根系蛋白显示显著差异表达,表明这些蛋白可能参与棉花抗（耐）盐的信号、代谢途径中。     

Low temperature induced changes in activity and protein levels of the enzymes associated to conversion of starch to sucrose in banana fruit

Storage at low temperature is the most frequently used method to extend the shelf life of banana fruit, and is fundamental for extended storage and transport over long distances. However, storage and transport conditions must be carefully controlled because of the high susceptibility of many commercial cultivars to chilling injury. The physiological behavior of bananas at low temperatures has been studied to identify possible mechanisms of resistance to chilling injury. The aim of this work was to evaluate differences in the starch-to-sucrose metabolism of a less tolerant and susceptible (Musa acuminata, AAA cv. Nanicão) and a more tolerant (M. acuminata × Musa balbusiana, AAB, cv. Prata) banana cultivar to chilling injury. Fruits of these cultivars were stored in chambers at 13 °C for 15 d, at which point they were transferred to 19 °C, where they were left until complete ripening. The low temperature induced significant changes in the metabolism of starch and sucrose in comparison to fruit ripened only at 19 °C. The sucrose accumulation was slightly higher in cv. Prata, and different patterns of starch degradation, sucrose synthesis, activity and protein levels of the α- and β-amylases, starch phosphorylase, sucrose synthase and sucrose phosphate synthase were detected between the cultivars. Our results suggest that starch-to-sucrose metabolism is likely part of the mechanism for cold acclimation in banana fruit, and the cultivar-dependent differences contribute to their ability to tolerate cold temperatures.     

低温对香蕉幼苗的伤害及外源H_2O_2、CaCl_2对香蕉抗寒性的影响

以香蕉品种‘巴西蕉’为材料,用H2O2、CaCl2、H2O（CK）喷洒香蕉幼苗植株,在人工气候箱中模拟低温胁迫,观察香蕉幼苗对低温的反应,研究外源H2O2、CaCl2对香蕉抗寒性的影响。结果表明,巴西蕉对低温敏感,且随着温度的降低,幼苗冷害症状加剧严重,膜透性加大,叶绿素降解加剧;和对照（CK）相比,低温胁迫下,外源H2O2、CaCl2能够提高7℃低温胁迫期间香蕉叶片脯氨酸含量和POD、CAT活性,降低MDA的积累。外源H2O2、CaCl2能有效降低香蕉幼苗叶片膜透性,减缓叶绿素的降解,减轻冷害程度,提高香蕉幼苗的抗寒性。     

棕榈科植物抗寒生理研究进展及展望

本文综述了棕榈科植物在低温下的胁迫生理研究、防寒生理研究和低温对光合生理的影响等方面的研究进展与发展趋势。并简要概述了棕榈科植物抗寒生理机制等方面的研究进展。又进一步阐明了棕榈科植物的抗寒性研究意义和今后的发展方向。认为棕榈科植物相关研究会是今后研究和发展的热点,具有广阔的发展前景,对于发展我国热区经济具有重要作用。     

Biochemical and proteomic analysis of ‘Kyoho’ grape (Vitis labruscana) berries during cold storage

Low temperature storage is widely used to maintain the quality of postharvest fruit and extend their shelf-life. In this study, changes in specific metabolites and protein expression profiles of grape berries under cold storage were investigated by liquid chromatography and proteomic studies, respectively. During cold storage, total soluble solids and reducing sugars accumulation was accompanied by a decline in organic acids and phenols contents. A comparative analysis of the proteomes of grape berries during cold storage was performed using a two-dimensional electrophoresis (2-DE) proteomic approach. Seventy-nine differentially regulated proteins during cold storage were successfully identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and classified into eight main categories based on their biological function. Down-regulation of proteins associated with glycolysis and the Krebs cycle, and up-regulation of cell wall polysaccharide degradation-related enzymes provided molecular evidence that soluble sugar and carbohydrate metabolism play a crucial role in postharvest regulation. In addition, cold storage increased the expression abundance of several stress related proteins such as heat shock proteins, proteasome and antioxidant enzymes, suggesting a physiological adaptation to low temperature was induced in grape berries. This study contributes to a better understanding of the cellular events in grape berries under cold storage and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses.