苹果细胞分裂素氧化酶基因MdCKX7.2的克隆及抗性鉴定

采用同源克隆和PCR技术从‘嘎拉’苹果(Malus×domestica Borkh.)中克隆细胞分裂素氧化酶基因MdCKX7.2(基因序列号:MDP0000279125)。该基因含有1 542 bp的完整开放阅读框,编码513个氨基酸。利用Plant CARE数据库对MdCKX7.2启动子顺式作用元件进行预测分析,发现MdCKX7.2启动子序列中存在光、干旱、脱落酸、水杨酸、茉莉酸甲酯等响应元件。基因表达分析发现,‘嘎拉’幼苗中MdCKX7.2的表达明显受干旱和ABA的诱导。通过农杆菌介导的遗传转化获得MdCKX7.2转基因拟南芥植株。抗性试验表明,异位表达MdCKX7.2基因明显提高了拟南芥对干旱胁迫的抗性。拟南芥种子萌发试验表明,在拟南芥中异位表达MdCKX7.2提高了植株对ABA的敏感性,种子萌发率和幼苗鲜质量明显下降,与种子萌发相关基因的表达量明显上调。以上试验结果表明,MdCKX7.2在植物非生物胁迫响应中发挥重要作用。     

杂草环境下转EPSPS基因大豆NZL06-698的生态适应性研究

为研究转EPSPS基因大豆NZL06-698在杂草环境中的生态适应性,试验设置杂草处理模拟野外环境,研究转基因大豆NZL06-698(GT698)的生存竞争能力以及外源EPSPS蛋白表达情况。结果表明:在相同处理下转基因大豆GT698的株高(R8期)、百粒重显著高于亲本大豆蒙豆12(MD12),但单株产量、单株籽粒数、结实率显著低于亲本大豆,说明外源基因的存在并未增强转基因大豆的生存竞争力,且转基因大豆在野外的生存竞争能力与亲本大豆相比较弱。试验注意到,杂草环境对转EPSPS基因大豆的生长有明显的限制作用,杂草处理下转基因大豆的单株产量、单株籽粒数、百粒重、结实率等指标均显著低于对照处理。ELISA检测结果表明,转基因大豆叶片及籽粒中外源EPSPS蛋白在杂草处理及对照中均正常表达,且两种处理下的EPSPS蛋白表达量无显著差异。以上结果表明杂草环境中转基因大豆的EPSPS蛋白正常表达,正常提供抗草甘膦性状,但是外源基因的存在并没有增加转基因大豆的生存竞争能力,且转基因大豆的生长受到杂草环境的显著抑制,由此得出结论:与亲本大豆相比,转EPSPS基因大豆NZL06-698在野外环境中生态适应能力较弱。     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

苹果赤霉素信号转导因子MdGAMYB的克隆和表达分析

以‘长富2号’苹果为试验材料,从其短枝顶芽中克隆得到1个赤霉素信号转导因子MdGAMYB,对其进行生物信息学和表达分析。结果表明,MdGAMYB的开放阅读框(ORF)长度为1 656bp,编码551个氨基酸,蛋白质分子量为59.741 kD。生物信息学分析表明MdGAMYB编码的蛋白存在多个糖基化位点和磷酸化位点;序列分析表明,Md GAYMB和其他物种的GAMYB蛋白有很高的相似性,均含有保守的R2R3 DNA结合域和GAMYB家族所特有的Box1,Box2和Box3保守区域;系统进化分析表明,Md GAYMB与梨、梅花、草莓、枣和葡萄等的GAMYB蛋白具有较高的同源性。实时荧光定量PCR分析表明,Md GAYMB具有组织表达特异性,在叶片、花和芽中的表达量较高。外源GA3处理抑制了花芽孕育和翌年成花,抑制MdGAMYB的表达。在易成花品种‘烟富6号’中的表达量高于难成花品种‘长富2号’。     

苹果‘长富2号’开花调控转录因子基因MdIDD7的克隆及表达分析

以‘长富2号’苹果为试验材料,采用RT-PCR的方法,从其芽中克隆得到INDETERMINATE DOMAIN(IDD)转录因子基因MdIDD7,其开放阅读框长度为1 626 bp,编码541个氨基酸。序列比对和结构域分析表明,该转录因子含有1个核定位信号和4个高度保守的锌指蛋白结构域。系统进化树分析表明,MdIDD7与白梨(Pyrus×bretschneideri,XP_009364602.1)、桃(Prunus persica,XP_007225628.1)和梅(Prunus mume,XP_008220893.1)聚在一起。实时荧光定量PCR表明,MdIDD7在‘长富2号’不同组织(茎、叶、花、果和芽)中均有表达,其中芽的表达量最高。花后40~60 d,MdIDD7在易成花品种‘烟富6号’芽中表达量显著高于难成花品种‘长富2号’。"小年"树顶芽组织中MdIDD7的表达量显著高于"大年"树。在花芽诱导前期,外源GA处理诱导MdIDD7下调表达,而蔗糖处理诱导其上调表达,说明MdIDD7响应激素和糖信号,促进苹果成花。     

Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple

Apple Glomerella leaf spot (GLS) is a severe fungal disease that damages apple leaves during the summer in China. Breeding new apple varieties that are resistant to the disease is considered the best way of controlling GLS. Fine mapping and tightly linked marker are critically essential for the preselection of resistant seedlings. In this study, a population of 207 F₁ individuals derived from a cross between ‘Golden Delicious’ and ‘Fuji’ was used to construct a fine simple sequence repeat (SSR)‐based genetic linkage map. The position of R_gls, a locus responsible for resistance to GLS, was identified on apple linkage group (LG) 15 using SSR markers CH05g05 and CH01d08, which was adapted from a published set of 300 SSR markers that were developed using the bulked segregant analysis (BSA) method. These two SSR markers flanked the gene, and its recombination rate was 8.7% and 23.2%, respectively. A total of 276 newly developed SSR markers around the target region and designed from the genome apple assembly contig of LG15 were screened. Only nine of these were determined to be linked to the R_gls locus. Thus, a total of 11 SSR markers were in linkage with R_gls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the R_gls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene R_gls was constructed, and the locations of the 11 effective primers in the ‘Golden Delicious’ apple genome sequence were anchored. This result facilitates better understanding of the molecular mechanisms underlying the trait of resistance to GLS and could be used in improving the breeding efficiency of GLS‐resistant apple varieties.     

苹果种植区的土壤养分含量情况研究—以白水县为例

为了稳定和提高苹果产量,通过对2013 年渭南白水县500 个点农业土壤区进行分析,并对土壤pH、全氮、有机质、碱解氮、速效钾、有效磷、有效铁和有效锰、锌、铜进行有效的对比研究。利用Microsoft Excel 2010 软件进行数据整理统计。结果发现：白水县苹果生长的土壤酸碱度为中性偏碱,并且土壤中的全氮和有机质的含量丰富,速效钾较丰富;有效锌、有效磷含量适中;有效铜含量高,有效铁、碱解氮、有效锰含量低。     

基于高光谱成像的苹果品种快速鉴别

以"乔纳金"苹果,"红富士"苹果和"秦冠"苹果共90个试验样本为试材分别采集865~1 711 nm的近红外波段高光谱图像,选取苹果图像感兴趣区域(ROI),以分辨率2.8 nm提取其平均反射光谱数据,分别利用K近邻法(KNN)和径向基核函数支持向量机(RBF-SVM)进行品种判别,5折交叉检验。结果表明,3种苹果的近红外高光谱图像均在波长941~1 602 nm之间变得清晰,该区域200个波段下的平均反射光谱数据经KNN法中的10种距离算法评判,当K取值3和5时,切比雪夫距离、欧几里得距离和明可夫斯基距离3种距离算法的识别正确率均达到100%;SVM-RBF核函数模型中,γ取值为2-8~1的范围内识别正确率均在92%以上,当γ取值2-5,C取值为16和32时,识别正确率最高,为96.67%。故利用近红外高光谱图像技术结合KNN计算对苹果品种进行快速鉴别是优异和可靠的方案。     

Characterization and pathogenicity of six Cytospora strains causing stem canker of wild apple in the Tianshan Forest,China

Cytospora species are capable of causing destructive cankers of stems belonging to a wide range of woody plant species. In severe cases, cankers may lead to dieback of twigs and branches. Little is known about the Cytospora species causing canker disease of wild apple (Malus sieversii) trees in the Wild Fruit Forest Reserve in Tianshan Forest, Xinjiang Uygur Autonomous Region, China. In this study, six Cytospora isolates belonging to two species were isolated from cankerous lesions of wild apple twigs. Based on multi‐locus phylogenetic analysis using three DNA markers (ITS, tef1‐α and tub2) and morphological characterization, these isolates were identified as Cytospora mali and Cytospora parasitica. Temperature trials (15, 20, 25 and 30°C) showed that the optimal growth temperature for six isolates was 25°C. At a variety of temperatures, C. mali isolates tended to grow faster than isolates of C. parasitica, with the C. mali isolate, EGI1 performing better than others with regard to growth rate. Morphological observations showed that these species exhibited a single locule without conceptacles, and the conidia length was 3–5 μm. In vitro inoculation trials of twigs and leaves of M. sieversii seedlings revealed that the C. mali isolates were highly virulent phytopathogenic fungi, whereas the C. parasitica isolates were less virulent. The isolate EGI1 was the most virulent isolate among the six isolates. This paper presents the first report of pathogenic Cytospora spp. of the M. sieversii Tianshan Wild Fruit Forest Reserve of Yili, Xinjiang in China. It will aid in the understanding of how apple tree cankers are induced and provide disease management guidelines for M. sieversii forest conservation.     

山楂海棠RAPD亲缘关系研究

应用RAPD分子标记技术,对山楂海棠的分类地位进行了探讨。根据RAPD分子标记聚类分析,将14份苹果材料分为5类,8份山楂材料分为4类,山楂海棠的亲缘关系位于山楂和苹果之间,但与苹果较近,认为山楂海棠可能是苹果和山楂的属间杂交种。