番茄ACC合酶反义基因对河套蜜瓜的转化

河套蜜瓜（CucumismeloLcvHetau）的子叶经预培养。芽诱导和生根培养,获得再生小植株,诱导率达58％。取带有番茄ACC合酶反义基因的双元载体pMQ6／JM109与农杆菌（Agrobacteriumtumefaciens）LBA4404经三亲融合后,与在MS0上萌发5d、并在MS＋1mg／LNAA培养基上预培养3d的子叶共培养48h,然后转入含50mg／L卡那霉素的MS＋6mg／LZT的芽诱导培养基中,1l个月后诱导生芽,待芽长1．5－2cm时转入生根培养基中,1－2周后可诱导产生大量的根,形成完整的转基因小植株。经PCR和分子杂交检测证明,目的基因已整合入河套蜜瓜的基因组中。     

番茄1-氨基环丙烷羧酸(ACC)合成酶基因的反义RNA-核酶嵌合DNA序列的构建

根据番茄ACC合成酶基因（LE—ACC2）DNA序列,以番茄(LycopersiconesculentumMill)果实的总DNA为模板,利用PCR技术扩增得到预期大小的该基因编码区内部分DNA序列,插入到质粒载体pGEM—3zf(＋)的BamHⅠ和HindⅢ位点之间后转化E．coliDH—5α,可选出重组子pRE,经酶切,PCR及DNA序列分析证明克隆成功;将pRE上的目的DNA序列以反义方式构建到我室已合成并克隆的含核酶DNA序列的重组质粒pRⅠ的BamHⅠ和HindⅢ之间,构成含有反义RNA-核酶嵌合DNA序列的重组质粒pREⅠ,经酶切及序列分析,结果与预期一致.     

ACC脱氨酶基因转化白兰瓜的初步研究

用带有ACC脱氨酶基因和卡那霉素抗性基因（NptⅡ,作为报告基因）的工程根癌农杆菌转化白兰瓜子叶。通过组织培养,得到具有卡那霉素（Km)抗性的再生小苗,经NptⅡ报告基因的PCR扩增,ACC脱氨酶基因的Southern点杂交以及ACC脱氨酶活性的生理生化检测可知,ACC脱氨酶基因已成功转入白兰瓜子叶再生小苗,而且不同植株的真叶中表现出不同水平的ACC脱氨酶活性。     

‘弥河银瓜’高效植株再生体系的建立

建立了以薄皮甜瓜品种‘弥河银瓜’子叶为外植体的高效再生体系。取生长5d的甜瓜子叶为外植体,置于MS+2.0mg·L-16-BA+0.5mg·L-1IBA+3.5%蔗糖+0.7%琼脂(pH5.8)的诱导培养基上培养,可获得100%芽分化频率。在不定芽伸长过程中,生长调节剂最佳浓度为0.5mg·L-16-BA+0.1mg·L-1IAA。潮霉素是较合适的筛选抗生素。     

番茄果实ACC合成酶的性质

测定了经4步纯化、比活性达20000U／mg蛋白质以上的番茄果实伤诱导ACC合成酶的一些酶学性质。酶反应最适PH值为9．5;酶在pH8．0下最稳定,pH7．5－10短时间处理不会使酶发生不可逆变性;酶在pH8．0和9．5的Km值分别为23和4Dμmol／L;根据酶反应不同时间的产物累积量,得出反应速度随时间的变化符合函数关系式Vt＝V0e－kt,并根据此式求出酶的半寿期为107min。光照对酶活性有抑制作用。酶的DTNB化学修饰结果表明,在酶活性中心的PLP结合部位很可能有半胱氨酸残基存在。     

河套蜜瓜ACC合成酶cDNA片段的克隆和序列分析

1-氨基环丙烷-1-羧酸（ACC）合成酶是高等植物中乙烯生物合成的关键酶。以成熟河套蜜瓜（CucumismeloL．cvHetau）果实的RNA为模板,经反转录和PCR扩增得到预期大小的DNA片段,插入到pUC19的SmaⅠ位点后转化E.coliJM109,筛选出重组子pHMAS1。序列分析表明获得了长627bp的ACC合成酶cDNA片段。与已报道的ACC合成酶基因相应序列比较有很高的同源性．     

2个甜瓜品种高效再生体系的建立

以甜瓜厚皮品种绿宝石和薄皮品种甘甜一号为材料,研究了5d龄无菌苗的子叶外植体在附加6-BA和IAA的不同浓度组合培养基上的分化情况.结果表明:2个品种在不定芽和愈伤组织的分化上存在着较大的差异,绿宝石在含有6-BA2.0mg·L-1的分化培养基上的不定芽诱导率最高,达93.75%;而甘甜一号品种在含有6-BA1.0mg·L-1和6-BA2.0mg·L-1的分化培养基上的不定芽分化频率均为100%.另外,随着IAA浓度的增大,疏松愈伤组织的分化均有加重的趋势;而提高6-BA的浓度,虽然分化进程有所加快,但玻璃化-褐化程度加重.本项研究为应用转基因技术改良甜瓜品种的重要育种性状奠定了组织培养方面的基础.     

番茄果实ACC合成酶的纯化

以伤诱导的番茄囊果皮为材料,改进了ＡＣＣ合成酶（ＥＣ４．４．１．１４）纯化方法。经改进的５步纯化后,ＡＣＣ合成酶被纯化７３００倍,比活性达到１１６８７０Ｕ／ｍｇ蛋白,回收率为７％．以较少的步骤得到了较高的纯化倍数。纯化的ＡＣＣ合成酶经ＳＤＳ－ＰＡＧＥ和银染检验为一条带,分子量为５０ｋＤ,等电点为ｐＨ６．５。     

番茄子叶外植体转化系统的优化

目的 :对番茄子叶外植体的转化进行优化 ,为进行转基因番茄的研究奠定基础。方法 :通过正交设计实验 ,以子叶外植体的存活天数为观察指标 ,分别对农杆菌稀释浓度、浸染时间和转化方式等条件进行优化。结果 :在子叶预培养 2d、活化农杆菌稀释倍数约 30倍、侵染时间为 1 0min、共培养时间为 2d的情况下 ,子叶存活时间最长 ,抗性芽产生频率达 2 5 %。结论 :获得了高效的番茄子叶外植体转化系统 ,为进行基因的转化奠定了基础     

Plastome data analysis of Cucumis melo subsp.agrestis

It is of interest to refine the taxonomic status of C. melo ssp. agrestis using its plastome data. The chloroplast size and GC% was found to be 1,56,016 bp and 36.92% respectively in Cucumis melo subsp. agrestis. The plastome of C. melo subsp. agrestis comprises of two inverted repeat (IR) regions of 25,797 bp each. It consisted of 133 genes with 88 protein-coding genes, 8 rRNA genes and 37 tRNA genes. Analysis of the C. melo ssp. agrestis plastome data will help breeders to improve the yield the crop.     

厚皮甜瓜(Cucumis melo var. reliculatus)的快速繁殖

以厚皮甜瓜 (Cucumismelovar.reliculatus)西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明 :在MS BA 0 .5～ 1 .0mg/L IAA 0 .1mg/L的培养基上利于诱导形成丛生芽 ,芽的月增殖系数达到 1 1以上 ;在 1 /2MS IAA 0 .5mg/L培养基上并经暗处理 3d最易生根 ,生根率 90 % ;在蛭石 :草炭土 =1 :1 (体积比 )基质中移栽驯化效果好。试管植株定植大田后种性不变 ,生长和结果习性优于种子苗。     

甜瓜乙烯信号转导途径关键因子基因CTR1的克隆及表达特性分析

在拟南芥中,CTR1在乙烯信号转导途径中发挥重要的负调控作用.目前的研究表明,在拟南芥中只存在一个CTR1基因.利用RT-PCR技术从甜瓜果实中克隆得到CTR1基因cDNA(Cm-CTR1).Cm-CTR1cDNA全长2 613 bp,编码870个氨基酸.氨基酸序列比对和同源性分析表明,所克隆的甜瓜CTR1基因与拟南芥CTR1基因相似度为53.69％.Cm-CTR1的C-末端具有明显的类似于哺乳动物和果蝇中的Raf( retro-viral protein,V-raf)蛋白激酶家族的丝/苏氨酸蛋白激酶(Serine/threonine protein kinase)的特征.该结构具有所有已知蛋白激酶所共有的11个亚结构域,其中包括ATP结合位点和丝/苏氨酸蛋白激酶位点结构域,说明CmCTR1与其他植物CTR1相似.实时荧光定量PCR分析结果表明,从授粉后第15天到第20天,甜瓜果实Cm-CTR1基因表达量显著增加,第20天的表达量是第15天的2.5倍,之后表达水平趋于稳定,第40天到第45天表达量有所下降.     

甜瓜离体再生继代培养中玻璃化现象的研究

为提高甜瓜离体培养的再生率和转基因效率,以优质甜瓜品种‘伽师瓜’(‘卡拉库赛’)离体再生不定芽为外植体,通过连续多代继代培养,对引起玻璃化苗现象的几个主要因素进行了研究。结果表明,在甜瓜离体再生继代培养中,外植体继代次数是影响玻璃化发生的主要因素,同时培养基中的6-BA浓度偏高、琼脂浓度偏低以及蔗糖浓度偏低或偏高等可导致玻璃化苗的增加。培养基中较低的6-BA浓度(0~0.2 mg/L),琼脂浓度为6 g/L,蔗糖浓度为25 g/L以及添加活性炭等措施可有效地降低甜瓜玻璃化苗的发生。     

牡丹ACC合成酶ACS1基因的克隆与原核表达

从牡丹‘洛阳红’(Paeonia suffruticosa‘ Luo Yang Hong’)花瓣中提取总RNA,根据GenBank上牡丹ACC合成酶基因PsACS的序列设计引物,通过RT-PCR获得牡丹PsACS1基因序列,包含一个1 479 bp的开放阅读框,编码492个氨基酸.将PsACS1基因cDNA片段与pET28a(+)构建原核表达载体pET-ACS1,转化大肠杆菌E.coli BL21 (DE3).0.4 mmol/LIPTG诱导3h后,在预期的蛋白分子量55 kD处出现1条表达加强的蛋白条带.为进一步目的蛋白的纯化和鉴定提供试验基础.     

甜瓜组织培养过程中的染色体数目变异

以甜瓜组织培养过程中产生的不定芽为材料,观察了不同阶段不定芽中染色体数目发生变异的频率,同时比较了在无外加诱变剂的情况下,甜瓜根尖与组培不定芽之间最大分裂相时间的变化。     

不同性别表型黄瓜基因组中雌性系特异的ACC合酶基因

利用一对引物（引物１和引物２）分别从雌性系黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）品种“ＣＯＲＯＮＡ”、“ＤＡＬＥＶＥ”和强雌性黄瓜品种“中农五号”、“欧洲八号”的基因组ＤＮＡ中扩增到一长约１０２５ｂｐ的ＡＣＣ合酶基因片段。序列分析表明：该基因片段与Ｔｒｅｂｉｔｓｈ等１９９７年发表的ＡＣＣ合酶基因片段的同源性大于９９％,认为这两个基因片段应该是同一个基因,不同品种来源的该基因的相同性说明了其高     

Ethylene production, shelf-life and evidence of RFLP polymorphisms linked to ethylene genes in melon (Cucumis melo L.)

Sixty three cultigens from eight market types of the melon (Cucumis melo L. subsp. melo) groups Cantaloupensis and Inodorus were evaluated for ethylene production rate, shelf-life (postharvest decay), and RFLP polymorphisms. The ethylene production rates of melon fruits at maturity and (after) postharvest decay were measured on individual genotypes. The ethylene production rates of individual genotypes ranged from undetectable to 103 nl/g per h. The mean ethylene production rates of the eight market types, ranked from highest to lowest, were Eastern U.S. type, Charentais, Western U.S. type, Long Shelf-Life cantaloupes (LSL), Galia, Ananas, Honeydew, and Casaba. Ethylene production and postharvest decay rating were positively significantly correlated (r ²=0.87, P=0.05). Orange-fleshed melon fruits produced significantly (P=0.05) more ethylene than did green- or white-fleshed types. Melon fruits with a netted rind had significantly (P=0.05 for orange-flesh fruits and 0.01 for green- or white-flesh fruits) higher ethylene production than did smooth-type fruits. Using probes made from cDNAs encoding ACC oxidase (MEL1) or ACC synthase (MEACS1) genes, RFLPs were detected melon cultigens of the eight marker types showing varying ethylene production rates and different flesh colors. Low ethylene production and green- and white-flesh color were associated (r ²=0.91; P=0.05) with the presence of a putative RFLP-MEL1 allele A ₀ (15-kb), whereas high ethylene production and orange-flesh color were associated with allele B ₀ (8.5-kb) in the homozygous condition, after probing MEL1 with EcoRV-digested genomic DNA. Also, after probing MEACS1 with NdeI-digested genomic DNA, RFLP polymorphism revealed five fragments denoted as A, B, C, D and E, with molecular sizes of 5.2-, 4.2-, 3.8-, 3.0- and 1.0-kb, respectively. A two-fragment pattern, AB, and a three-fragment pattern, ACE, the two predominant RFLP patterns, were also associated with low and high ethylene production, respectively. The ACE fragment pattern was also associated with orange-flesh melons. Scoring of both probes allowed for the unique classification of most melon market types consistent with ethylene production and the postharvest decay phenotypes. Therefore, these RFLPs might have utility in marker-assisted selection for the development of melons with enhanced postharvest keeping ability. Received: 26 March 1998 / Accepted: 12 January 2000     

AgNO3对网纹甜瓜试管苗糖代谢及抗氧化酶活性的影响

在网纹甜瓜继代培养过程中加入不同浓度的AgNO3,测定试管苗糖代谢及抗氧化酶活性等。结果表明,玻璃苗果糖、葡萄糖和可溶性糖含量显著降低,而蔗糖含量显著高于正常苗;抗氧化系统发生紊乱,O2产生速率降低,H2O2含量下降,MDA含量增加,木质素含量降低。加入30μmol·L^-1AgNO,使玻璃苗蔗糖合成酶、蔗糖转化酶活性和木质素含量与正常苗差异不显著。说明适当浓度AgNO3有利于维持试管苗糖代谢正常进行,降低试管苗脂质过氧化程度,从而降低或抑制玻璃化现象的发生。     

Studies on a Gynoecious-specific ACC Synthase Gene in Different Sexual Phenotypes of Cucumber Genome

Using a pair of primers (Primer Ⅰ and Primer Ⅱ), the authors have amplified a fragment of ACC synthase gene about 1025 bp from four varieties of gynoecious species of cucumber （Cucumis sativus L.） viz.:“CORONA”,“DALEVE”,“Zhongnong No.5”,and “Ouzhou No.8”. Sequence analysis revealed that this fragment of ACC synthase gene was more than 99% homologous with the gene reported by Trebitsh et al (1997). The authors regard them as the same gene, but it exhibited less homology with this ACC synthase gene when expressed by other induction. Southern blot analysis showed that this fragment of ACC synthase gene is associated with the sexual phenotype of cucumber,and it is the specific gene of gynoecium. However, the number of its copies has no direct correlation with the degree of female expression; this seems to indicate that there might be other genes associated with the degree of feminization.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.