美洲拟鲽抗冻蛋白基因遗传转化草莓的研究

以草莓品种星都2号和全明星的无菌苗叶盘和叶柄基部为外植体,采用携带美洲拟鲽编码pre、pro和mature 3种抗冻蛋白（AFP）基因的双元载体系统的根癌农杆菌进行转化,获得7个Km抗性株系。PCR扩增及PCR-Southern杂交检测6个株系呈阳性,结果表明,编码pre、pro和mature的AFP基因分别整合到了星都2号和全明星染色体DNA中,其中获得pre、pro和mature转星都2号株系各1个,转化率分别为1.56%、1.49%、1.67%;mature基因转全明星株系3个,转化率为4.05%.     

Characterizing NAD-dependent alcohol dehydrogenase enzymes of Methylobacterium extorquens and strawberry (Fragaria x ananassa cv. Elsanta)

The methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The microbial biotransformation of 1,2-propanediol to 2-hydroxypropanal (lactaldehyde) was studied. The bacterial alcohol dehydrogenase (ADH) enzymatic activities were assessed, and the optimum pH for ADH activity was found to be pH 6.0. Enzyme assays were carried out for both the bacterial and the strawberry extracts to define the best substrate specificity. For Methylobacterium extorquens, the best substrates were found to be methanol (Km = 0.78 mM) and 1,2-propanediol (Km = 15.84 mM), whereas for strawberries, 1-propanol (Km = 3.54 mM) and ethanol (Km = 6.66 mM) were the best substrates. A wide variety of metals as well as EDTA were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE experiments showed molecular weights of 45.0 and 24.6 kDa for the alcohol dehydrogenases of Methylobacterium extorquens and Fragaria x ananassa, respectively.     

EMS离体诱变及抗草莓灰霉病愈伤组织的筛选

试验用甲基磺酸乙酯（EMS）处理草莓愈伤组织后通过草莓灰霉病病原毒素（Toxin）离体筛选获得抗病愈伤组织并诱导成苗。EMS诱变处理‘幸香’草莓离体叶片产生的愈伤组织,用经毒力检测的草莓灰霉菌毒素粗提液进行离体筛选,通过在愈伤组织继代培养中附加不同浓度毒素粗提液,以确定适宜的抗病选择压,获得抗灰霉病突变体材料。结果表明,将愈伤组织直接进行EMS处理的成活率高于将愈伤组织切割成小块继代培养再经EMS处理的愈伤组织;且0.2% EMS处理1h为最适剂量。在含80%毒素粗提液的培养基培养10 d为抗性筛选的适宜选择压。诱变处理后的867块愈伤组织,在经不连续二次筛选后有195块成活,成活率22.5%,其中有82块再生出了不定芽,再生率为42.1%。不定芽经生根后移栽至田间进一步进行抗性鉴定。     

Effects of ozone treatment on postharvest strawberry quality

The effect of ozone treatment on the postharvest quality of strawberry was evaluated. Strawberry fruits (Fragaria x ananassa Duch. cv. Camarosa) were stored at 2 degrees C in an atmosphere containing ozone (0.35 ppm). After 3 days at 2 degrees C, fruits were moved to 20 degrees C to mimic retail conditions (shelf life). The changes in several quality parameters such as fungal decay, color, sugar and acids distribution, and aroma were evaluated during the strawberries' shelf life. Ozone treatment was ineffective in preventing fungal decay in strawberries after 4 days at 20 degrees C. Significant differences in sugars and ascorbic acid content were found in ozone-treated strawberries. At the end of cold storage, the vitamin C content of ozonated strawberries was 3 times that of control fruits. A detrimental effect of ozone treatment on strawberry aroma was observed, with a 40% reduced emission of volatile esters in ozonated fruits.     

Biosynthesis of mono- and sesquiterpenes in strawberry fruits and foliage: 2H labeling studies

The biosynthesis of the monoterpene (S)-linalool and the sesquiterpene trans-(S)-nerolidol in fruits of Fragaria x ananassa Duch. cv. Eros and Florence and of the monoterpene (-)-alpha-pinene in Fragaria vesca was investigated by in vivo feeding experiments with [5,5-2H2]mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-d-xylulose (d2-DOX). The feeding experiments indicate that (S)-linalool and trans-(S)-nerolidol in Fragaria x ananassa Duch. and (-)-alpha-pinene in F. vesca are exclusively synthesized via the cytosolic mevalonic acid pathway without any contribution from the plastidial 1-deoxy-D-xylulose/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) route. Inhibition experiments revealed that even the presence of mevastatin, an export of plastid-derived isopentyl diphosphate/dimethylallyl diphosphate, cannot be induced. However, the enantioselective analysis shows that in Fragaria x ananassa Duch. cv. Eros and Florence both linalool enantiomers are present and that only (S)-linalool is labeled after administration of d2-MVL. Therefore, the origin of (R)-linalool in these fruits remains unknown. Contrarily, in Fragaria x ananassa Duch. foliage (R)-linalool is the dominant enantiomer. Feeding experiments revealed an incorporation of d2-MVL and d2-DOX at equal rates exclusively into (S)-linalool. Only in F. vesca foliage, where (R)-linalool is present at high enantiomeric purity (ee > 90%), is a de novo biosynthesis of the (R)-enantiomer via the DOXP/MEP pathway detectable. These results demonstrate a complex intraplant variation of (R)- and (S)-linalool biosynthesis via the cytosolic and plastidial route.     

Catalytic properties of alcohol acyltransferase in different strawberry species and cultivars

The substrate specificity of alcohol acyltransferase (AAT) enzymes from different strawberry varieties was studied. Proteins with AAT activity from fruits of Fragaria x ananassa Duch. cv. Oso Grande were purified to apparent homogeneity and used for kinetic studies with different straight-chain alcohols and acyl-CoAs. K(m) values obtained for Oso Grande enzyme with six different alcohols, using acetyl-CoA as cosubstrate, decreased with increasing length of the alcohol chain. In similar experiments the increase in the acyl-CoA carbon chain was also found to be correlated with a higher substrate specificity. Heptanol (K(m) = 0.73 mM) and hexanoyl-CoA (K(m) = 0.41 mM) were the best substrates for Oso Grande AAT. Comparative catalytic studies were carried out with AAT partially purified extracts from the wild type Fragaria vesca and five commercial strawberry varieties: Tudnew, Carisma, Camarosa, Sweet Charlie, and Eris. The specificities of these enzymes toward five selected alcohols and acyl-CoAs reflected interesting cultivar differences.     

两轮多重反转录巢式PCR方法扩增全长GalUR基因

研究了两轮多重反转录巢式PCR扩增全长基因的方法.先用多重巢式引物和cDNA进行第1轮PCR扩增;再用纯化的第1轮PCR产物和相同的引物进行第2轮扩增;最后通过改变Mg^2＋浓度、退火时间和退火温度进行PCR条件的优化,以增加PCR扩增的产量,用于克隆.结果显示该方法方便和快速地扩增出了草莓（Fragaria ananassa cv.SweatCharlie）高产量和高质量的全长GalUR基因,也非常快速地扩增出了用于基因构建的带有酶切位点的全长GalUR基因.该方法也可以用于其它全长基因的克隆.     

影响根癌农杆菌介导的丰香草莓遗传转化因素分析

将构建的携带NPTⅡ和AP-D基因的植物表达载体pBI121导入根癌农杆菌EH105中后,用其对草莓主栽品种丰香叶盘进行遗传转化,探讨了卡那霉素(Kan)的浓度、菌液浓度、侵染时间、预培养时间、共培养时间和筛选方式等因素对转化率的影响。结果表明:丰香草莓适宜的转化条件是Kan筛选浓度为20 mg/L,预培养时间为2~3 d,菌液浓度OD6000.3~0.5,侵染时间10~20 min;共培养2~3 d后将外植体接到含有5 mg/L Kan和500 mg/L Carb的诱导培养基上进行培养,以后每次继代逐步提高Kan选择压力至终浓度20 mg/L(每次增加5 mg/L);或共培养2~3 d后将外植体接到含有500 mg/L Carb的诱导培养基上,培养2周后继代到含20 mg/L Kan和400 mg/L Carb的培养基上进行选择培养。根据PCR检测结果,抗性愈伤组织转化率高达18.5%。抗性愈伤组织经进一步诱导,最后获得了17株抗性植株。本文探讨了影响丰香草莓转化的多个因素,以期建立高效的遗传转化体系,为草莓属植物种质的遗传转化奠定基础。     

草莓抗炭疽病遗传图谱及其QTL初分析

为获得与草莓炭疽病密切相关的分子标记,需构建高密度与抗病相关的遗传连锁图,本研究以易感草莓炭疽病品种宝交早生（Hokowase）与高抗草莓炭疽病品种甜查理（SweetCharlie）杂交的210个F2代群体材料为作图群体,构建了包含34个AFLP标记和109个SSR标记的分子遗传图谱,并对抗草莓炭疽病相关因素进行了QTL分析。该图谱共包括7个连锁群和133个遗传标记,平均每个连锁群有19个遗传标记。遗传图谱总覆盖长度为451．8cM,标记间平均距离为3．4cM。经复合区间作图法分析得到3个与草莓炭疽病抗性相关基因座（QTL）,其中2个与Colletotrichumacutatum抗性相关,分布在LG3和LG5连锁群上,可解释表型变异的31．6％;1个与Colletotrichumgloeosporioides抗性相关,分布在LG6连锁群上,可解释表型变异的68．4％。     

Effects of cyclamen mite (Phytonemus pallidus) and leaf beetle (Galerucella tenella) damage on volatile emission from strawberry (Fragaria x ananassa Duch.) plants and orientation of predatory mites (Neoseiulus cucumeris, N. californicus, and Euseius finlandicus)

Volatile emission profile of strawberry (Fragaria x ananassa Duch.) plants (cvs. Polka and Honeoye) damaged by cyclamen mite (Phytonemus pallidus Banks) or leaf beetle Galerucella tenella (L.) (cv. Polka) was analyzed to determine the potential of these strawberry plants to emit herbivore-induced volatiles. The total volatile emissions as well as emissions of many green leaf volatiles (e.g., (Z)-3-hexen-1-ol and (Z)-3-hexenyl acetate) and methyl salicylate were greater from cyclamen mite-damaged strawberry plants than from intact plants. Leaf beetle feeding increased emissions of monoterpenes (Z)-ocimene and (E)-beta-ocimene, sesquiterpenes (E)-beta-caryophyllene, (E,E)-alpha-farnesene, and germacrene-D, and a homoterpene (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) significantly. Nevertheless, the na?ve generalist predatory mites, Neoseiulus cucumeris, Neoseiulus californicus, and Euseius finlandicus did not prefer P. pallidus- or G. tenella-damaged plants over intact plants in a Y-tube olfactometer, suggesting that these predatory mite species are not attracted by the herbivore-induced volatiles being released from young strawberry plants.     

Elevated carbon dioxide increases contents of antioxidant compounds in field-grown strawberries

The effects of elevated CO2 concentrations on the antioxidant capacity and flavonoid content in strawberry fruit (Fragaria x ananassa Duch.) were studied under field conditions. Increased CO(2) (300 and 600 micromol mol(-1) above ambient) concentrations resulted in increases in ascorbic acid (AsA), glutathione (GSH), and ratios of AsA to dehydroascorbic acid (DHAsA) and GSH to oxidized glutathione (GSSG), and a decrease in DHAsA in strawberry fruit. High anthocyanin and phenolic content were also found in fruit of CO(2) treated plants. Growing strawberry plants under CO(2) enrichment conditions significantly enhanced fruit p-coumaroylglucose, dihydroflavonol, quercetin 3-glucoside, quercetin 3-glucuronide, and kaempferol 3-glucoside contents, as well as cyanidin 3-glucoside, pelargonidin 3-glucoside, and pelargonidin 3-glucoside-succinate content. Fruit of strawberry plants grown in the CO(2) enrichment conditions also had high oxygen radical absorbance activity against ROO(*), O(2)(*-), H(2)O(2), OH(*), and (1)O(2) radicals.     

Characterization of phenolic compounds in strawberry (Fragaria x ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity

Phenolic compounds in strawberry (Fragaria x ananassa) fruits were identified and characterized by using the complementary information from different high-performance liquid chromatography detectors: diode array, mass spectrometer in positive and negative mode, and coulometric array. Electrochemical profiles obtained from the coulometric array detector contributed to the structural elucidation suggested from the UV-vis and mass spectra. About 40 phenolic compounds including glycosides of quercetin, kaempferol, cyanidin, pelargonidin, and ellagic acid, together with flavanols, derivatives of p-coumaric acid, and ellagitannins, were described, providing a more complete identification of phenolic compounds in strawberry fruits. Quercetin-3-malonylhexoside and a deoxyhexoside of ellagic acid were reported for the first time. Antioxidative properties of individual components in strawberries were estimated by their electrochemical responses. Ascorbic acid was the single most important contributor to electrochemical response in strawberries (24%), whereas the ellagitannins and the anthocyanins were the groups of polyphenols with the highest contributions, 19 and 13% at 400 mV, respectively.     

Compost as a soil supplement increases the level of antioxidant compounds and oxygen radical absorbance capacity in strawberries

Compost as a soil supplement significantly enhanced levels of ascorbic acid (AsA) and glutathione (GSH) and ratios of AsA/dehydroascorbic acid (DHAsA) and GSH/oxidized glutathione (GSSG) in fruit of two strawberry (Fragaria x ananassa Duch.) cultivars, Allstar and Honeoye. The peroxyl radical (ROO(*)) as well as the superoxide radical (O(2)(*)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH(*)), and singlet oxygen ((1)O(2)) absorbance capacity in strawberries increased significantly with increasing fertilizer strength and compost use. The planting medium (compost) x fertilizer interaction for phenolics and flavonoids was significant. Fruit from plants grown in full-strength fertilizer with 50% soil plus 50% compost and 100% compost yielded fruit with the highest levels of phenolics, flavonol, and anthocyanin content. A positive relationship between antioxidant activities and contents of AsA and GSH and ratios of AsA/DHAsA and GSH/GSSG existed in fruit of both strawberry cultivars. Correlation coefficients for the content of antioxidant components versus antioxidant activity [against ROO(*), O(2)(*)(-), H(2)O(2), OH(*), or (1)O(2)] ranged from r( )()= 0.7706 for H(2)O(2) versus GSH/GSSH in cv. Allstar to r = 0.9832 for O(2)(*)(-) versus total flavonoids in cv. Allstar.     

草莓遗传转化研究进展

草莓是一种重要的小型浆果,近年来育出很多优良品种,种植面积逐渐扩大。然而仍有很多因素限制了草莓生产的发展。基因工程技术为植物育种、种质资源开发利用等领域开辟了新的途径,可以获得常规育种难以或无法得到的新类型。本文综述了近年来国内外在草莓抗病虫、抗逆、抗除草剂、品质特性、耐贮运性等基因工程的研究进展以及基因型、试管苗生理状态、共培养时间、侵染时间和菌液浓度、抗生素等因素对草莓遗传转化的影响。对草莓转基因研究中存在的主要问题和今后的研究方向及应用前景进行了探讨。     

Characterization of polyphenol oxidase and peroxidase and influence on browning of cold stored strawberry fruit

Polyphenol oxidase and peroxidase were extracted from two different varieties of strawberry fruit (Fragaria x ananassa D, cv. 'Elsanta' and Fragaria vesca L, cv. 'Madame Moutot') and characterized using reliable spectrophotometric methods. In all cases, the enzymes followed Michaelis-Menten kinetics, showing different values of peroxidase kinetics parameters between the two cultivars: Km = 50.68 +/- 2.42 mM ('Elsanta') and 18.18 +/- 8.79 mM ('Madame Moutot') mM and Vmax = 0.14 +/- 0.03 U/g ('Elsanta') and 0.05 +/- 0.01 U/g ('Madame Moutot'). The physiological pH of fruit at the red ripe stage negatively affected the expression of both oxidases, except polyphenol oxidase from 'Madame Moutot' that showed the highest residual activity (68% of the maximum). Peroxidase from both cultivars was much more thermolable as compared with PPO, losing over 60% of relative activity already after 60 min of incubation at 40 degrees C. The POD activation energy was much lower than the PPO activation energy (DeltaE = 97.5 and 57.8 kJ mol-1 for 'Elsanta' and 'Madame Moutot', respectively). Results obtained from d-glucose and d-fructose inhibition tests evidenced a decreasing course of PPO and POD activities from both cultivars as the sugar concentration in the assay medium increased. Changes in CIE L*, a*, b*, chroma, and hue angle values were taken as a browning index of the samples during storage at 4 degrees C. A decrease in L* was evident in both cultivars but more marked in 'Elsanta'. PPO and POD activities from cv. 'Elsanta' were very well-correlated with the parameter L* (r2=0.86 and 0.89, respectively) and hue angle (r2=0.85 and 0.93, respectively). According to these results, the browning of the fruit seemed to be in relation to both oxidase activities.     

Carbohydrate differences in strawberry crowns and fruit (Fragaria x ananassa) during plant development

Carbohydrates accumulation and mobilization are highly relevant in plants because they have been related to yield and quality. Therefore, the aims of this work were to determine soluble carbohydrates and starch in strawberry (Fragaria x ananassa cv. Camarosa) crown sections (basal, middle, and upper) at three different plant growth stages (vegetative, blooming, and fruiting), and in fruit varieties (cv. Camarosa, Seascape, and Oso Grande) grown on the same field and in a different geoclimate. The main soluble carbohydrates found were glucose, sucrose, and fructose. Concentration differences were found among crown sections and time. The lowest levels of glucose, fructose, and sucrose were present at the beginning of fruit formation (6.2, 1.8 mg/g, and trace, respectively). Starch increased in basal and middle sections at the same time (8.6 to 109.6 and 6.6 to 93.5 mg/g, respectively). There appears to be a relationship between crown and fruit soluble carbohydrates. The most abundant fruit monosaccharides in all varieties were glucose (160-190 mg/g), fructose (90-180 mg/g), and sucrose (30-120 mg/g), followed by myo-inositol (10-23 mg/g). Strawberry crowns are an important source of carbohydrates and they might play a role during plant development specifically related to fruit sweetness. Fruit quality is highly influenced by a combination of several factors such as genotype, geoclimate, and probably carbon partitioning.     

短波紫外线处理对采后草莓果实腐烂和抗氧化能力的影响

以"凤冠"草莓为材料,研究了0、1.5、3.0和4.5kJ.m-2UV-C处理对草莓果实在10℃,12d贮藏期间腐烂和果实抗氧化能力的影响。结果表明,3kJ.m-2UV-C处理能显著抑制草莓果实采后腐烂的发生和MDA的积累,延缓果实失重、硬度和可溶性固形物含量的降低,促进果实CIRG值的提高,增强果实采后着色能力,从而保持果实的感官和食用品质。同时,3kJ.m-2UV-C处理能促进贮藏期间果实总花色苷、总酚含量和DPPH自由基清除能力的增加,减少草莓果实中抗氧化组分含量的下降,维持果实较高的营养品质。说明适宜剂量的UV-C处理(3kJ.m-2)在草莓果实采后后贮运保鲜和抗氧化能力调控中具有潜在的应用前景。     

高温处理对结果期草莓叶片衰老特征的影响

以‘日本丰香’(Fragaria ananassa Duch.cv Toyonaka)草莓品种为试材,于2010-12-2011-02在人工气候箱进行环境控制试验,以25℃为对照,设30、32、34、36、38℃共5个高温处理,分别在处理1、3、5d后,测定与叶片衰老特征密切相关的生理指标,包括叶片中超氧化岐化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性以及丙二醛(MDA)含量、可溶性蛋白质和叶绿素含量.结果表明,在30 ～ 38℃范围内,随着温度升高及处理时间的延长,草莓叶片中MDA含量呈持续增加趋势;经30 ～ 34℃高温处理后,可溶性蛋白质含量、SOD、POD、CAT酶活性值均高于对照,其中SOD、POD酶活性在34℃处理3d后达到最高,可溶性蛋白质含量、CAT酶活性在34℃处理5d后达到最高.36℃及38℃高温处理后,可溶性蛋白质含量、SOD、POD、CAT酶活性明显低于对照(P＜0.05);高温处理中叶片的叶绿素a、b和类胡萝卜素含量均低于对照,而且随着高温时间的延长,草莓叶片叶绿素a、b及类胡萝卜素含量都呈持续下降趋势.表明高温胁迫降低了草莓叶片保护酶活性,导致体内活性氧大量积累,抗氧化能力降低,加速了草莓叶片的衰老,研究结果为设施草莓高温气象灾害的防御提供了依据.     

Clarifying the identity of the main ellagitannin in the fruit of the strawberry, Fragaria vesca and Fragaria ananassa Duch

Although the composition of strawberry fruit has been extensively studied, especially for the most abundant phenolic compounds, agrimoniin has never been univocally identified as one of the most abundant phenolic compounds in the fruit. In this study agrimoniin was isolated in the fruit of Fragaria vesca and its structure characterized. Furthermore, its presence was definitively established to be the main ellagitannin in both F. vesca and Fragaria ananassa D. fruit. The presence of sanguiin H-6 and lambertianin C as minor compounds was confirmed in both F. vesca and F. ananassa D. samples. For the first time here is reported the full NMR assignments for agrimoniin. These data should represent a point of reference for NMR analysis of this and other structurally related ellagitannins. Finally, the establishment of an HPLC protocol for separation provided information making it possible to avoid confusion with sanguiin H-6, the main ellagitannin in Rubus species, which is also present in strawberries but at a much lower concentration.