油果实中查尔酮合成酶基因PsCHS的克隆表达分析及其启动子的分离

从成熟果实均一化全长cDNA文库中分离了编码CHS基因的全长cDNA序列,命名为PsCHS,根据其序列设计引物,采用Genome Walking方法从基因组DNA中分离获得PsCHS基因上游的调控序列,命名为PsCHSp,PsCHS基因全长1 442bp,其中ORF 1 176bp,编码392个氨基酸;采用APA-Walking技术,获得该基因的5ˊ端调控区,经在线软件预测,启动子序列含有典型的结构特征元件TATA-box和CAAT-box,还包含光响应元件、厌氧诱导元件、胚乳表达相关元件、MYB结合位点以及激素响应元件;RT-PCR结果显示,PsCHS基因在果实发育的前期表达量较高,花后40d表达量最高,随后开始下降,果实成熟期表达量较低。分离获得的PsCHS基因属于查尔酮合成酶基因家族成员之一,查尔酮合成酶(Chalcone synthase,CHS,EC 2.3.1.74)是类黄酮合成途径中的一个重要酶,该基因可能对类黄酮的合成起到调控作用。     

重瓣榆叶梅PrtSHP的基因克隆与序列结构分析

采用同源克隆法结合3′-RACE技术从重瓣榆叶梅(Prunus triloba var.Plena)中分离得到了1个雌蕊和果实发育调控基因SHP的同源基因PrtSHP的全长cDNA(GenBank登录号:JF911701).其cDNA全长970 bp,包括1个编码244个氨基酸的735 bp的完整开放阅读框.蛋白质序列比对和分子系统发生分析表明,PrtSHP是拟南芥的SHP的同源基因,其编码的蛋白质的C末端结构域具有2个高度保守的模体(AG Ⅰ模体和AG Ⅱ模体),属C类转录因子中PLE进化系.     

梅花品种资源同工酶多态性分析

该文采用聚丙烯酰胺凝胶电泳法,选用过氧化物酶、酯酶、苹果酸脱氢酶、超氧化物歧化酶、α 淀粉酶和过氧化氢酶等６种同工酶,对１１４个梅花品种进行了分析,以期分析各栽培品种在同工酶水平上所表现出的多态性．结果表明：梅花品种资源在同工酶这一水平上表现出了很好的多态性,既有着明显的特征谱带,又有着众多的变异．所得结果与形态标记等的分类有较好的相似性,但也表现出了一定的差异性．     

寿星桃种质资源的RAPD分析

该研究利用RAPD技术,从分子生物学角度对寿星桃种质进行分析.采用从200个10碱基随机引物筛选的22个引物对10个寿星桃类型的基因组DNA进行扩增,通过对180个扩增位点的谱带的聚类,分析供试寿星桃的系统发育;运用特殊谱带,建立了寿星桃的分子检索表;并对扩增的特殊位点进行统计,提出了重点保存的寿星桃种质.     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

Development and applicability of GBS approach for genomic studies in Japanese plum (Prunus salicina Lindl.)

Genotyping by sequencing (GBS) provides a large quantity of useful data suitable for the identification of single nucleotide polymorphisms (SNPs), facilitating accurate genomic studies in plant species. In this study, GBS-based SNPs were used to characterise 11 Japanese plum cultivars and to explore their natural allelic diversity in relation to the most important phenology events (flowering date, ripening date and fruit development period) and fruit quality traits (weight, shape, skin and flesh colour, over colour, skin and flesh chlorophyll index, flesh firmness and soluble solids concentration). GBS-based SNPs were shown to be a powerful tool for genetic diversity and other genomic studies where SNP markers were related to several traits, particularly for flowering date, ripening date, fruit development period, skin chlorophyll degradation, flesh chlorophyll degradation and flesh colour. These results represent a preliminary approach using GBS as a possible breeding tool in current and new Japanese plum breeding programmes.     

桃Aux/IAA家族基因鉴定及在果实成熟过程中的表达分析

通过生物信息学鉴定桃基因组中生长素/吲哚乙酸(Aux/IAA)基因家族,并对其在溶质型和硬质型桃果实成熟阶段的表达水平进行q RT-PCR检测。结果表明:桃Aux/IAA基因家族包含22个成员,这些基因集中分布在5条染色体上,以第1条染色体上含有最多,为8个。大部分Aux/IAA基因包含高度保守的Ⅰ、Ⅱ、Ⅲ和Ⅳ功能域,聚类分析表明其分为10类。基因结构分析显示这些基因包含2~5个外显子。荧光定量PCR分析表明10个Aux/IAA基因在溶质型桃果实的表达量高于对应时期硬质型桃果实,其中5个基因随着溶质型桃果实的成熟上调表达,特别是ppa010303m、ppa010871m和ppa020369m。试验结果表明桃Aux/IAA家族成员在结构上高度保守,其中多个成员(尤其是ppa010303m、ppa010871m和ppa020369m等)可能参与调控桃果实的成熟。     

桃PpSnRK1β1和PpSnRK1β2在番茄中超表达对光合碳代谢的影响

从毛桃[Prunus persica(Linn.)Batsch.]叶片中克隆了PpSnRK1蛋白激酶β亚基编码基因PpSnRK1β1和PpSnRK1β2,其c DNA全长分别为720 bp和867 bp。q RT-PCR组织表达分析表明,PpSnRK1β1在‘鲁星’桃叶片中的表达量最高,PpSnRK1β2在其果实中表达量最高,两者在其花中的表达量均较低。通过农杆菌介导转化番茄,获得超表达PpSnRK1β1的番茄株系T21、T23和超表达PpSnRK1β2的株系T91、T92。研究发现,与野生型(WT)相比,超表达株系T23和T91叶片的Sn RK1蛋白激酶活性分别增加9.84%和53.32%。T91叶片净光合速率比WT提高17.02%;叶片可溶性糖含量比WT增加34.00%。T91和T92叶片淀粉含量分别约是WT的2倍。T23、T91和T92果实维生素C含量分别比WT下降19.21%、38.49%和39.76%。因此说明PpSnRK1β1和PpSnRK1β2参与了光合作用过程,调控碳代谢及次生代谢过程。     

新疆野扁桃自交不亲和花粉SFB基因的克隆及生物信息学分析

旨在克隆获得新疆野扁桃自交不亲和花粉SFB新基因,以新疆野扁桃花药为试材,应用分子同源克隆及RT-PCR等技术,并进一步采用在线软件分析SFB基因的蛋白质理化性质,实验成功克隆到了PtSFB5与PtSFB6 2个新SFB基因的cDNA全长,2个基因的cDNA全长分别为1124 bp和1072 bp,分别编码了374和348个氨基酸,2个基因都具有F-Box基因结构,属于F-Box基因家族,预测的相对分子量分别为30787.26和44037.74,等电点为5.93和5.43,均属于水溶性不稳定蛋白。     

S_allele identification and genetic diversity analysis of apricot cultivars

In this study, the sexual incompatibility and S-allele diversity of 24 Turkish apricot cultivars, Paviot and Sak?t-1 as parents and 127 F₁ progenies were identified by polymerase chain reaction (PCR) and sequencing techniques. Additionally, genetic diversity and relatedness among the 24 cultivars were determined using 18 simple sequence repeat (SSR) markers from the genus Prunus. PCR for S_alleles identified nine different S-RNase alleles in the 24 apricot cultivars, namely S_c, S₁, S₂, S₈, S₉, S₂₀, S₂₄, S₅₂, and S₅₃. All primers amplified only one S_allele in the cultivars Adilcevaz-1, Adilcevaz-3, Ethembey, Pasamismisi, Canakkale, and Soganci. Most of the Turkish cultivars were self-incompatible. The S_c allele was present in only three cultivars (Canakkale, Ethembey, Imrahor) that are, therefore, self-compatible. The S_alleles of cultivars Paviot and Sak?t-1 displayed homology with the S_c, S₂ and S₂₀, and S₅₂ alleles. In the 127 F₁ genotypes, the two S_alleles of Paviot were inherited by roughly half of the offspring, while about 76% of the offspring inherited the S₅₂ allele from Sak?t-1, and less than 24% inherited S₂₀. The amplification using all SSR 18 primers was successful and produced 128 polymorphic alleles with an average of 7.11 alleles per locus. Among the apricot cultivars studied, expected heterozygosity (He) ranged from 0.33 to 0.72, observed heterozygosity (Ho) ranged from 0.42 to 1.00, PIC values were between 0.28 and 0.89, and similarity rates were between 0.30 and 0.68. The cultivars Levent and Ozal were genetically closest (0.68) while cultivars Sak?t-3 and Soganc? were the most distinct (0.30).