甜樱桃(Prunus avium L.)品种S基因型鉴定

根据蔷薇科S-RNase基因(S基因)高度保守区C2和RC4区设计一对特异引物PruC2和PruC4R,对甜樱桃品种的基因组DNA进行S基因特异PCR扩增。克隆S基因的扩增片段,核酸序列在GenBank上搜索,确定了4种S基因的核酸序列和大小。结果表明,在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因。4种S基因扩增片段的大小分别是：S1为677bp,S3为762bp,S4为945bp,S6为456bp。参试的自交不亲和品种的S基因型分别是：红灯、红艳、早红宝石和先锋相同,为S1S3;抉择、红丰和那翁相同,为S3S4;大紫为S1S6;长把红为S1S4;养老为S2S6;自交亲和品种外引7号和斯太拉为S3S4。     

基于cDNA芯片的梨品种S基因型鉴定及新S-RNase基因进化分析

梨品种S基因型鉴定对梨栽培中授粉品种选择和遗传育种都具有重要意义。本研究利用梨S-RNase基因荧光标记的特异引物PCR扩增获得梨品种荧光标记的cDNA特异产物;进一步完善梨S-RNase基因cDNA芯片,以被检测梨品种cDNA特异序列与梨S-RNase基因cDNA芯片杂交检测不同梨品种S基因型,并发现新的S-RNase基因。结果表明:利用梨S-RNase基因cDNA芯片鉴定了泸定王皮梨、兴山24号、弥渡百合等35个未知S基因型梨品种,确定了各品种的S基因型。结合PCRRFLP及DNA克隆和测序等技术,发现了7个新的S-RNase基因资源,获得了新S-RNase基因序列。序列分析表明各新S-RNase基因均具有S-RNase基因特异区域序列的典型特征;进化分析显示7个新S-RNase基因主要属于蔷薇科苹果亚科S-RNase类群,且存在种间和属间比种内和属内进化关系更近的现象。7个新的S基因分别命名为:PpS_(53)(Pyrus pyrifolia S53)、PpS_(54)、PpS_(55)、PpS_(56)、PpS_(57)、PpS_(58)和PpS_(59),GenBank登录号分别为:KX581753、KX581754、KX581755、KX581756、KX581757、KX581751和KX581752。     

梨自交不亲和及其亲和突变品种花柱内S4（S4^SM）基因的表达与作用的比较

提取梨 (PyrusserotinaRehd .)自交不亲和品种“二十世纪”(基因型为S2 S4 )、自交亲和的突变品种“奥嗄二十世纪”(S2 SSM4 ,SM =Stylar_partmutant;花柱部分突变 )及其亲和后代花柱的可溶性蛋白。经等电聚焦电泳 (IEF_PAGE)分析表明 ,“奥嗄二十世纪”及其后代花柱仍存在SSM4 蛋白 ,但其含量逐代减少 ,同时发现“奥嗄二十世纪”的SSM4 基因仅在柱头表达 ,而“二十世纪”的S4 基因表达的部位除了柱头外 ,还包括花柱上部及花柱下部 ,且表达量呈现从柱头到花柱下部下降的趋势。S蛋白经等电聚焦电泳的凝胶板进行RNase活性染色处理 ,也得到相同的结果。从花柱 (包括柱头 )中纯化出的S蛋白经SDS_PAGE电泳后进行RNase活性染色的结果表明 ,S4 与SSM4 蛋白的分子量相近 (约 30kD) ,并且均具有RNase活性。进一步以酵母RNA为基质测定的比活性也基本相等 ,约为 2 75U·min-1·mg-1蛋白。在离体条件下 ,上述两种S蛋白 (S_RNase)也以相同的程度抑制S4 或SSM4 花粉发芽及花粉管伸长。研究证明 ,自交亲和突变品种“奥嗄二十世纪”的SSM4 基因也具有原始自交不亲和品种“二十世纪”S4 基因的功能。因此 ,其自交亲和的原因可归结为SSM4 基因的表达量较少及SSM4 基因仅在柱头中表达的缘故。     

确定梨自交不亲和基因型研究的技术进展

综述了运用杂交授粉试验和分子生物学方法等技术确定梨品种自交不亲和基因型研究的技术进展,分析了这些技术在确定梨品种自交不亲和基因型方面的优点和不足之处,并初步探讨了研究前景。因为HV区氨基酸的不同,不同S基因型也有所差异。因此,除了在分子生物学的水平上进行研究外,其他方法如mRNA、蛋白质和杂交授粉等水平上的研究在确定S基因型上也同样重要。     

白梨和砂梨S基因型确定及S34新基因的分离

梨是配子体型自交不亲和植物,确定不同品种的S基因型是科学杂交授粉及提高梨产量和品质的基础。本文根据砂梨S1-9等位基因一级结构特征,设计特异引物PF和PR,以白梨（Pyrus bretschneideri）种鹅梨（Pyrus bretschneideri‘Eli’）和砂梨（Pyrus pyrifolia）品种博多青（Pyrus pyrifolia‘Hakataao’）的叶片基因组DNA为模板,通过PCR·RFLP系统检测、克隆测序以及生物信息学分析,分离鉴定了它们的片段大小相似的2条S等位基因,从中获得1条新的S基因,命名为S34-RNase基因,并确定了这2个梨品种的S基因型,分别为鹅梨S13S34和博多青S22S34。     

白梨和砂梨S基因型确定及S34新基因的分离

梨是配子体型自交不亲和植物, 确定不同品种的S基因型是科学杂交授粉及提高梨产量和品质的基础。本文根据砂梨S1-9等位基因一级结构特征, 设计特异引物PF和PR, 以白梨(Pyrus bretschneideri)品种鹅梨(Pyrus bretschneideri ‘El i’) 和砂梨(Pyrus pyrifolia)品种博多青(Pyrus pyri folia ‘Hakataao’) 的叶片基因组DNA为模板, 通过 PCR-RFLP系统检测、克隆测 序以及生物信息学分析, 分离鉴定了它们的片段大小相似的2条S等位基因, 从中获得1条新的S基因, 命名为S34-RNase基因, 并确定了这2个梨品种的S基因型, 分别为鹅梨S13S34和博多青S22S34。     

24份甜樱桃材料自交不亲和S基因型鉴定

甜樱桃品种绝大部分自交不亲和,限制了甜樱桃的正确评价和合理利用,因此自交不亲和基因型的鉴定对于生产具有重要意义。以24个甜樱桃主栽品种为材料,用5对蔷薇科李属引物组合对24个甜樱桃品种进行了S等位基因的PCR扩增,克隆S基因的扩增片段,用核酸序列在Gen Bank上搜索,确定了5种S基因的核酸序列和大小。结果表明:Pru C2+Pru C4R引物组合扩增效果最好;在琼脂糖凝胶上位置相同的扩增带其核酸序列相同,是同一种S基因;5种S基因扩增片段的大小分别是S1为800 bp,S3为762 bp,S4为962bp,S5为300 bp,S6为456 bp,S9为650 bp;24个甜樱桃S基因型是红手球、早红宝石为S1S3,拉宾斯S1S4',红宝石S1S6,布鲁克斯S1S9,那翁S3S4,秦林、泰安大紫、先锋、早大果、丽珠、美早、5-106、左滕锦、桑提娜为S3S6,黑珍珠、红灯、萨米脱、秦樱为S3S9,胜利为S5S9,明珠、红蜜、雷尼、滨库为S6S9。     

小麦品种品质性状的基因型因子分析

用陕西省关中小麦品种区域试验所选用的20个小麦品种(品系)在12个试点1996／97、1997／98两年度的数据资料,通过基因型因子分析,探讨小麦品种籽粒品质性状间相互关系的内在规律。结果表明：可将12个品质指标用4个“公共因子”表示。因子1对蛋白质含量、沉淀值、形成时间、稳定时间、软化度与评价值等品质性状起支配作用,主要反映的是蛋白质的质与量;因子2对湿面筋含量、容重和出粉率起支配作用,反映了湿面筋含量与磨粉品质之间存在着较密切的、直接的联系;因子3对籽粒硬度与吸水率起支配作用,反映了小麦籽粒的质地结构特性;因子4中,只有千粒重有较大载荷。表明千粒重与其它品质性状的直接联系较小,受相对较为独立的遗传因素控制。     

梨自交不亲和基因cDNA芯片杂交条件优化及应用北大核心

[目的]优化梨自交不亲和基因(S-RNase或S基因)c DNA芯片杂交条件,利用芯片检测梨品种S基因型。[方法]提取梨品种雌蕊RNA,Cy3标记引物RT-PCR获得S基因荧光标记特异c DNA序列。设置不同杂交条件,用已知S基因型品种荧光标记的PCR产物在不同条件下分别与芯片杂交,杂交信号分析芯片杂交效果。用芯片优化杂交体系鉴定梨品种未知S基因型,DNA测序验证芯片鉴定结果。[结果]芯片杂交最佳条件:杂交温度42℃,杂交时间8~9 h,PCR纯化产物终浓度为200 ng·μl-1。优化杂交条件下芯片鉴定晚咸丰、秀水、丽江马占梨1、湘菊、木通梨、甘甜、弥渡小红梨、丽江大中古、金晶和弥渡火把等梨品种S基因型分别为:Pp S15Pp S52、Pp S4Pp S5、Pb S22Pp S37、Pp S1Pp S2、Pp S1Pp S3、Pp S13Pp S15、Pp S12Pb S42、Pb S21Pb S22、Pp S3Pp S60和Pp S5Pp S5。DNA测序验证各品种所含S基因与芯片鉴定结果一致。[结论]梨自交不亲和基因c DNA芯片优化杂交条件后可准确鉴定梨品种所含已鉴定的S基因资源。     

梨不同品种花粉生活力测定及授粉特性研究

以19个梨品种为试材,用离体培养法测定在不同贮藏温度下花粉生活力,并调查6个杂交组合和15个梨品种自交的结实特性。结果表明,在培养基中加入硼酸和赤霉素可提高花粉生活力;通过镜检发现大慈梨、八月红、黄金梨3个品种花粉败育;在不同的贮藏条件下,温度越低花粉发芽率下降越缓慢,因此在低温条件下适合花粉长久贮藏;在贮藏过程中,均存在"短期被迫休眠现象",且随温度的降低被迫休眠恢复时间向后延伸。6个杂交组合的花朵坐果率和花序坐果率平均值为82.54%和94.82%,与自然授粉没有明显差异,因此金二十世纪可以作为鸭梨、雪花梨的授粉树,雪花梨、鸭梨、红安久、红茄梨可以作为黄金梨的授粉树;15个梨品种自交结实率较低,而大慈梨、八月红、黄金梨、秦丰、秀玉、新星、雪花梨、丰水8个品种不结实,在生产上均需配置授粉树才能达到产量需求。     

PCR-based method for identifying the S-genotypes of Japanese pear cultivars

 Japanese pear (Pyrus pyrifolia Nakai), a member of the Rosaceae, shows gametophytic self-incompatibility that is controlled by the S-locus. The S-genotype of Japanese pear cultivars is an important factor for crossing and breeding. We report a rapid reliable method to identify these S-genotypes. It consists of PCR amplification of the S-RNase gene from genomic DNA and subsequent digestion of the PCR fragments with S-allele-specific restriction endonucleases. Using this method, we determined the unknown S-genotypes of nine Japanese pear cultivars and selected self-compatible varieties from the offspring of the self-compatible cultivar, ‘Osa-Nijisseiki’. Received: 8 June 1998 / Accepted: 19 October 1998     

Determination of S-genotypes of pear (Pyrus pyrifolia) cultivars by S-RNase sequencing and PCR-RFLP analyses

The pear (Pyrus pyrifolia) has gametophytic self-incompatibility (GSI). To elucidate the S-genotypes of Korean-bred pear cultivars, whose parents are heterozygotes, the PCR amplification using S-RNase primers that are specific for each S-genotype was carried out in 15 Korean-bred pear cultivars and 5 Japanese-bred pear cultivars. The difference of the fragment length was shown in the following order: S6 (355 bp) < S7 (360 bp) < S1 (375 bp) < S4 (376 bp) < S3 and S5 (384 bp) < S8 (442 bp) < S9 (1,323 bp) < S2 (1,355 bp). We analyzed the sequence of the S-RNase gene, which had introns of various sizes in the hypervariable (HV) region between the adjacent exons with a fairly high homology. The sizes of the introns were as follows: S1 = 167 bp, S2 = 1,153 bp, S3 = 179 bp, S4 = 168 bp, S5 = 179 bp, S6 = 147 bp, S7 = 152 bp, S8 = 234 bp, S9 = 1,115 bp. There were five conservative and five hypervariable regions in the introns of S1, S3, S4, S5, S6 and S-RNases. A pairwise comparison of these introns of S-RNases revealed homologies as follows: 93.7% between S1- and S4-RNases, 93.3% between S3- and S5-RNases and 78.9% between S6- and S7-RNases. PCR-RFLP and S-RNases sequencing determined the S-genotypes of the pear cultivars. The S-genotypes were S4S9 for Shinkou, S3S9 for Niitaka, S3S5 for Housui, S1S5 for Kimizukawase, S1S8 for Ichiharawase, S3S5 for Mansoo, S3S4 for Shinil, S3S4 for Whangkeumbae, S3S5 for Sunhwang, S3S5 for Whasan, S3S5 for Mihwang, S5S? for Chengsilri, S3S5 for Gamro, S3S4 for Yeongsanbae, S3S4 for Wonhwang, S3S5 for Gamcheonbae, S3S5 for Danbae, S3S4 for Manpoong, S3S4 for Soowhangbae and S4S6 for Chuwhangbae. The information on the S-genotypes of pear cultivars will be used for the pollinizer selection and breeding program.     

Nectar secretion dynamics of Hungarian local pear cultivars

 The dynamics of daily nectar secretion was studied in 28 local pear cultivars in a Hungarian cultivar collection and three main types were distinguished. 1. Cultivars secreting nectar continuously are the most favourable for pollinators, rewarding them both with nectar and pollen. 2. Cultivars with discontinuous nectar secretion have sufficient insect attraction only if anther dehiscence is continuous during the day. In the first two types nectar secretion peaks usually appeared at 9:00 or 10:00, 14:00 or 15:00 and 18:00 or 19:00, with 4–5-hour intervals. 3. In some cultivars no nectar secretion was observed in any or some of the studied years, these being the least attractive for pollinators. The cultivars studied did not necessarily show the same type of nectar secretion dynamics in each season. From the viewpoint of pollination it is also of high importance, in which developmental phase nectar secretion begins. Received August 27, 2002; accepted January 7, 2003 Published online: June 2, 2003     

Identification of S-genotypes in Chinese cherry cultivars (Prunus pseudocerasus Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae, and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility. In the present study, we successfully isolated nine S-RNase alleles from cultivars of Chinese cherry by PCR amplification from genomic DNA and stylar cDNA combining with cleaved amplified polymorphic sequence marker. Analysis of amino acid sequences revealed five novel S-alleles, S ₂ , S ₄ , S ₆ , S ₈ , and S ₉ , with respective accession numbers in the NCBI database of EF541168, EF541173, EF541172, FJ628598, and FJ628599. Results showed that “Dongtang” and “Yinzhu” contained six S-alleles (S ₁ , S ₃ , S ₅ , S ₇ , S ₈ , and S ₉ ); “Taishanganying” contained four S-alleles (S ₁ , S ₂ , S ₄ , and S ₆ ); “Daiba”, “Dayingzui”, and “Xiaomizi” contained four S-alleles (S ₁ , S ₂ , S ₅ , and S ₈ ); “Laiyangduanzhi”, “Shuangquanchangba”, and “Daqingye” contained three S-alleles (S ₁ , S ₂ , and S ₈ ). It is interesting that different cultivars collected from the same place hold the same S-genotypes. Moreover, pollination tests and pollen tube growth assays showed that nine cultivars were self-compatible. Chinese cherry presented in this article are naturally polyploidy, which is a very useful material for the study of self-compatibility, and much of this information will be valuable for further work on self-(in)compatibility of fruit tree in Rosaceae.     

PbrmiR397a regulates lignification during stone cell development in pear fruit

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.     

慢性乙型肝炎患者血清HBV基因分型

为了解长春市慢性乙型肝炎患者血清中的乙型肝炎病毒(HBV)基因型情况及其与临床特点的相关性,应用型特异性引物进行巢式PCR方法对长春市69例慢性乙型肝炎患者血清HBV进行基因分型检测。在69例血清标本中,B型10例(占14.5%);C型41例(占59.4%);B C混合型8例(占11.6%);未分型的患者共10例(占14.5%)。C基因型患者的HBV-DNA定量、HBeAg阳性率明显高于B基因型患者(HBV-DNA:P<0.01;HBeAg:χ2=3.98,P<0.05),C基因型患者肝功检查指标谷丙转氨酶(ALT)和总胆红素(TB IL)均较B基因型患者高(P<0.01)。长春地区存在HBV B基因型、C基因型、B C混合基因型及未分型,C基因型为优势基因,引起的肝脏活动性炎症较B基因型明显。     

云南泡梨中乳酸菌的分离鉴定及其应用

【背景】泡梨是云南省常见的一种腌渍水果,在云南加工食用已经有一百多年的历史,因其味道酸甜可口、风味独特而深受人们喜爱,而目前对泡梨中微生物种群的系统分析和发酵原理的研究尚未见报道。【目的】研究乳酸菌在云南泡梨中的分布及应用,阐明乳酸菌种类对泡梨发酵中风味物质的影响。【方法】从云南省4个不同地区采集12份泡梨样品,经菌落菌体形态、生理生化特性和16SrRNA基因序列分析进行菌种分离与鉴定。利用分离的乳酸菌为菌种进行泡梨的制备,采用GC-MS技术对人工接种的复合乳酸菌发酵与自然发酵泡梨进行风味物质的分析与感官评价。【结果】分离鉴定出79株植物乳杆菌(Lactobacillus plantarum)、 3株类植物乳杆菌(Lactobacillus paraplantarum)、1株戊糖乳杆菌(Lactobacillus pentosus)、1株干酪乳杆菌(Lactobacillus casei)、2株副干酪乳杆菌(Lactobacillus paracasei)和1株短乳杆菌(Lactobacillus brevis),植物乳杆菌为泡梨发酵中的优势菌。将分离所得乳酸菌用于泡梨制备的结果表明,...     

水晶梨病虫害防治预测模型

[目的]为提高水晶梨病虫害防治工作效率,进一步提升病虫害的预测效果和精度。[方法]深入研究了灰色模型(GM),利用GM对水晶梨环境因子数据进行建模得到病虫害预测公式,通过差分方程推导出时间响应式和参数估计,建立了优化初始值的灰色模型(OIVGM),将OIVGM与BP神经网络预测模型(BP)进行组合,建立了优化初始值的灰色BP神经网络预测组合模型(OIVGM-BP)。[结果]通过单位根检验法测量模型的稳定性,OIVGM-BP一阶差分处理后,T统计量(-5.487654)小于5%临界值(-2.878073),数据序列表明平稳,OIVGM-BP可以稳定进行预测。通过白噪声检验方法测量OIVGM-BP的适应性,OIVGM-BP的残差P值从第二阶开始,均大于0.05,说明OIVGM-BP的适应性较好,各阶均通过了白噪声检验。LRM、GM、TSM、BP、OIVGM-BP对梨锈病、白粉病、腐烂病、梨黄粉蚜、梨二叉蚜、梨木虱6种病虫害的预测准确率的平均值分别为70.81%、70.09%、69.74%、65.64%、83.01%,OIVGM-BP的预测准确率优于其他4种预测模型。[结论]OIVGM-BP能够对水晶梨病虫害进行有效预测,能够更好地指导农业生产。     

福建桃、梨果蛀虫及梨小食心虫初步研究

本文报道福建省桃、梨果蛀虫概况及梨小食心虫的初步研究和防治意见。