草莓乙烯受体FaEtr1和FaErs1基因的克隆及其反义表达载体的构建

在已报道的FaEtr1和FaErs1基因序列的基础上,设计特异引物,分别克隆草莓(Fragaria ananassa)乙烯受体FaEtr1基因580bp部分特异序列和FaErs1基因445bp部分特异序列,将上述2个片段分别反向插入植物表达载体pBI121的CaMV35S启动子和Nos终止子之间,构建了反义表达载体pBI121Etr1和pBI121Ers1。将带有完整启动子和终止子的FaEtr1和FaErs1基因引入pCAMBIA1301中,最终获得FaEtr1和FaErs1的双价植物表达载体pFRS。将这3个重组表达质粒pBI121Etr1、pBI121Ers1和pFRS导入根癌农杆菌(Agrobactrium tumefaciens)EHA105中,PCR及限制性内切酶酶切确定质粒已被导入。     

反义乙烯受体LeFTR2基因对番茄的转化和功能分析

采用根癌农杆菌(Agrobacterium tumefaciens)介导法将LeETR2的部分反义序列导入番茄(Lycopersicon esculentum)子叶外植体，经抗性筛选和组织培养获得再生植株。通过对转基因植株中插入序列的PCR检测和Southern杂交，证明转化番茄成功。通过对LeETR2的功能的初步研究结果表明，转基因植株顶端优势丧失、极端矮化和番茄果实的内源乙烯合成量增加。这提示此基因在植物乙烯信号感受与转导系统中起着负调控的作用。     

反义乙烯受体LeETR2基因对番茄的转化和功能分析

摘要：采用根癌农杆菌（Agrobacterium tumefaciens）介导法将LeETR2的部分反义序列导入番茄(Lycopersicon esculentum)子叶外植体，经抗性筛选和组织培养获得再生植株。通过对转基因植株中插入序列的PCR检测和Southern杂交，证明转化番茄成功。通过对LeETR2的功能的初步研究结果表明, 转基因植株顶端优势丧失、极端矮化和番茄果实的内源乙烯合成量增加。这提示此基因在植物乙烯信号感受与转导系统中起着负调控的作用。     

＇早红＇草莓高效遗传转化受体系统的建立

本文以草莓主栽品种‘早红’组培苗离体叶片和叶柄为外植体,进行叶龄、暗培养、植物生长调节剂配比及抗生素敏感性研究,建立草莓高效遗传转化的受体系统。在含3.0mg/L6-BA与0.1mg/L2,4-D的MS培养基上,30d叶龄的叶片再生频率高达98.31%,平均每叶片再生芽数5.09个,叶柄切段的再生频率为89.25%,平均每叶柄切段再生芽数4.92个,叶片的再生频率略高于叶柄;不定芽在含0.2mg/L6-BA与0.2mg/LGA3的MS继代培养基上培养成苗。将生长状态良好的不定芽转至含0.2mg/LIBA的1/2MS培养基上生根,生根率达100%,平均生根数量16.27条,平均根长1.85cm。抗生素敏感性试验表明,草莓外植体适宜的卡那霉素选择压力为25mg/L,头孢霉素的筛选浓度为300mg/L。本研究建立的再生体系可作为草莓遗传转化的受体系统。     

乙烯受体基因LeFTR1和LeFTR4的克隆及在番茄果实中的表达

为研究野生型番茄(Lycopersicon esculentum Mill．cv．Lichun)和转反义ACS番茄果实中乙烯受体基因上eETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

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。     

乙烯受体基因LeETR1和LeETR4的克隆及在番茄果实中的表达

摘要：为研究野生型番茄（Lycopersicon esculentum Mill. cv . Lichun）和转反义ACS 番茄果实中乙烯受体基因LeETR1和LeETR4的表达与乙烯的关系，实验用RT-PCR方法扩增了乙烯受体基因LeETR1和LeETR4片段，用两片段作探针进行Northern 杂交。结果表明：两受体基因的表达在番茄果实成熟进程中变化不明显，LeETR4在同一时期的果实外果皮中表达水平低于其在辐射壁和中柱的表达。转反义ACS番茄果实中LeETR1和LeETR4的表达水平显著低于野生型番茄果实，外源乙烯处理转反义ACS番茄果实，促进两个受体基因的表达。可见果实中LeETR1和LeETR4的表达受乙烯调控的影响。     

香蕉乙烯受体基因RNA干扰表达载体的构建

设汁两对引物，PCR扩增香蕉乙烯受体基因cDNA序列自AUG启始密码子起长538bp区段的正向(记为5‘I)、反向(记为AI’)DNA区段。构建含该正、反向互补重复序列DNA片段的中间载体，经测序证实连接正确后。克隆到植物表达载体pCAMBIA-1301中的GUs基因位置并经双酶切证实。在所得表达载体pCAMBIA-1301-S5’I-AI’中，该插入正反互补重复片段处于35S启动子之后，由此转录的mRNA因两端序列反向互补而形成发夹式RNA，因此可应用于RNA干扰研究。同时，文中对构建含正、反向重复序列插入片段植物表达载体过程中出现的插入片段链内退火引起连接困难等问题进行了分析讨论。     

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%.     

草莓主栽品种Tudla遗传转化体系的建立

本研究建立起农杆菌介导的草莓主栽品种Ｔｕｄｌａ的转化体系,获得了转化植株,试管苗叶片与携双元载体ｐＭＯＧ４１０的农杆菌菌株ＥＨＡ１０５共培养３天,共培养后叶片在附加止那霉素４０ｍｇ．Ｌ＾－１的再生培养基（ＭＳ＋ＴＤＺ２．０ｍｇ．Ｌ＾－１＋ＩＢＡ０．１ｍｇ．Ｌ＾－１）上选择培养４周后,外植株再生出明显可见的转化芽,转化芽再生频率达４．５％,转化芽在附加卡那霉素２５ｍｇ．Ｌ＾－１草莓继代培养基上分化成     

预处理和激素对草莓花药离体培养的影响

花药培养是进行植物脱毒和获取单倍体的有效方法。为提高草莓花药培养的再生效率,采用低温和黑暗的方式对草莓花药进行预处理,以不同种类和浓度的激素诱导诱导愈伤组织形成、不定芽形成与增殖。结果表明,培养前低温处理可促进愈伤组织形成,以4℃低温处理72h的效果最好。但花药黑暗预处理对愈伤组织形成没有作用。较高浓度的2,4-D(MS+1.0mg·L-16-BA+2.0mg·L-12,4-D)最适合诱导且促进花药愈伤组织形成,有助于下一步分化培养。而NAA对不定芽分化起着重要作用,诱导成功率达到10%以上,其中以MS+1.0mg·L-16-BA+0.2mg·L-1NAA诱导效果最好。在添加NAA的基础上再加入KT也能提高不定芽分化率。不定芽增殖过程不宜添加过高浓度的细胞分裂素和生长素,以MS+0.7mg·L-16-BA+0.1mg·L-1NAA诱导增殖效率最高,激素浓度过高时易出现玻璃化,现象,形成畸形苗。本研究建立了高效的草莓花药培养再生体系,为获得无病毒草莓苗和单倍体提供了条件。     

Influence of Drought Stress on Leaf Traits of Different Strawberry (Fragaria ananassa L.) Varieties in Natural Environment

A study was carried out to explore leaf traits analysis of three strawberry varieties under different drought conditions in Malaysian upland environment in 2013. Plants of three strawberry varieties were grown in three different soil moisture levels including 25 percent (severe stress), 50 percent (mild stress), and 75 percent (normal irrigation) and remained for 60 days as a duration of stress to get appropriate observations of plants to drought stress. Significant differences were observed among varieties, treatments, and duration of drought stress in different traits (P < 0.05). Leaf area, leaf number, chlorophyll content, chlorophyll stability index, leaf moisture, leaf expansion rate, and leaf yield were diminished under stress especially when treated with 25 percent of soil moisture level and 60 days of duration. Moreover, there were remarkable differences among plants in terms of leaf thickness in 25 percent, 50 percent, and control. Severe stress reduced leaf thickness significantly compared to other treatments.     

Two-phase flavonoid formation in developing strawberry (Fragaria x ananassa) fruit

Flavonoids are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions. In addition, they are beneficial for human health. Previous studies have shown for selected enzymes from the flavonoid pathway that flavonoid biosynthesis shows two peaks during fruit development. We provide optimized protocols for the determination of the activities of the key flavonoid enzymes: phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, flavonoid 3-O-glucosyltransferase, and flavonoid 7-O-glucosyltransferase. Using these protocols we were able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes with the exception of flavonol synthase. The first activity peak corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation. The results indicate that flavonoid 3-O-glucosyltransferase activity is not essential for redirection from flavanol to anthocyanin formation in strawberry.     

Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa)

High performance liquid chromatography coupled with diode array and coulometric array detectors were used to characterize and quantify phenolic compounds in achenes and flesh of ripe strawberries (cv Totem and Puget Reliance). Total phenolics and total monomeric anthocyanins were measured and antioxidant activities were evaluated by the FRAP and the ORAC assays. Strawberries contained 1% achenes on a fresh weight basis; however, they contributed to about 11% of total phenolics and 14% of antioxidant activities in strawberries. Ellagic acid, ellagic acid glycosides, and ellagitannins were the main contributors to the antioxidant activities of achenes. The major anthocyanin in flesh was pelargonidin-3-glucoside, whereas achenes consisted of nearly equal amounts of cyanidin-3-glucoside and pelargonidin-3-glucoside. Phenolic content and antioxidant activity of strawberry achenes were reduced by industrial processing. However, the levels were still high and strawberry waste byproduct could thus be a possible source of nutraceuticals or natural antioxidants.     

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.     

Effects of yam starch films on storability and quality of fresh strawberries (Fragaria ananassa)

Yam starch films, formulated with yam starch (4.00 g/100 g of solution) and glycerol (1.30 and 2.00 g/100 g of solution) in filmogenic solution, were employed as packaging to extend storage life of strawberries stored at 4 degrees C and 85% RH. The effects of yam starch films on fruits were compared to the effect of PVC (poly(vinyl chloride)) packaging. Starch and PVC films significantly reduced decay of the fruits compared to control. Compared to starch films, PVC presented the better behavior on weight and firmness retention of fruits, especially in the last 7 days of storage. Considering microbiological counts, the shelf life of control fruits was 14 days, and of all packaged samples, stored at same conditions, was 21 days. Two different formulations of yam starch film were tested and had different mechanical properties as a function of glycerol content (1.30 and 2.00 g/100 g of solution) but showed no difference when employed as strawberries packaging.     

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria x ananassa)

An octaploid (Fragaria x ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria x ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways.