山葡萄转录因子CBF1基因的克隆及其遗传转化体系

根据山葡萄的CBF1基因序列设计一对特异引物,克隆出山葡萄(Vitis amurensis)栽培品种‘双优’的抗寒转录因子CBF1基因,并将其构建到pBI121表达载体中,获得山葡萄转录因子CBF1基因的植物表达载体,命名为pBI121-ViCBF1,利用农杆菌介导叶盘转化‘巨峰’葡萄以研究高效的‘巨峰’葡萄转化体系。结果表明,以农杆菌LBA4404为介导菌株,以‘巨峰’葡萄叶片为外植体、3 d为预培养时间和4~5 d共培养时间、400 mg·L-1羧苄青霉素,50 mg·L-1卡那霉素筛选浓度转化效果较好,并已筛选到6株转基因植株,PCR检测均为阳性,为提高‘巨峰’葡萄的抗寒性建立了初步的技术基础。     

葡萄NAC转录因子的生物信息学分析

NAC基因家族是高等植物所特有的一类转录因子家族,广泛参与植物生长发育、逆境胁迫应答等反应。通过对71种葡萄NAC蛋白与22种其他物种NAC蛋白的生物信息学分析,对与非生物逆境胁迫相关的12种NAC蛋白的染色体定位、理化性质、亲/疏水性、二级结构及亚细胞定位等进行了分析和预测。结果显示,葡萄NAC蛋白共分为5大类,与非生物逆境相关的NAC蛋白聚集在第Ⅲ类。分析显示不同NAC蛋白间氨基酸、疏水性及稳定性之间存在一定差异,二级结构主要为随机卷曲等。分析结果可为进一步研究通过葡萄NAC基因调控葡萄的生长与抗逆机制提供参考。     

酿造干红山葡萄酒新品种“雪兰红”区域试验的研究

在我国内蒙古和东北地区进行新品种"雪兰红"区域试验研究。在无霜期短的吉林省柳河县三源堡镇周家村、吉林省吉林市河湾镇梨树村、黑龙江省泰来县和平乡宏丰村、黑龙江省宝清县七星泡镇民主村和内蒙古扎兰屯市成吉思汗镇光明村栽培,试验期间,每年的9月16~9月19日早霜来临,果实未达到充分成熟、含糖15.6%~18.2%、总酸14.2g~16.1g/L、单宁0.449g~0.512g/L,未达到酿造干红山葡萄酒的工艺标准,类似上述气候地区不宜大面积发展"雪兰红"。在无霜期较长的吉林省集安市青石镇下套村、吉林省珲春市哈达门乡太平村、辽宁省本溪县东营坊乡石湖村、辽宁省朝阳县六家子镇马石井子村和内蒙古喀喇沁旗锦山镇马鞍山村栽培,表现抗寒、抗病、穗粒大、产量高,果实含糖20.2%~22.4%、总酸12.4%~13.9%、单宁0.333%~0.360%、出汁率62.1%~64.1%,是"雪兰红"适宜栽培区域。通过区域试验可以确定,"雪兰红"适宜年无霜期145d以上、降雨量400mm~800mm、活动积温≥3000℃、砂壤土或黑壤土或暗森林土的山区及半山区栽培。     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ''Moldova''

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape 'Moldova' (Vitis vinifera L. 'Moldova'). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0mg'L-1 2,4-D and 0.5 mg'L-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg-L-1 6-BA and 2 mg.L-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg.L-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dy-namic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic em-bryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Vine-1 retrotransposon-based sequence-specific amplified polymorphism for Vitis vinifera L. genotyping

The sequence‐specific amplification polymorphism (S‐SAP) method, derived from the amplified fragment length polymorphism (AFLP) technique, produces amplified fragments containing retrotransposon long terminal repeat ( LTR ) sequence at one end and a host restriction site at the other. The development and application of this procedure to the LTR of the Vine‐1 element from grapevine is reported. Two primers derived from one of the LTR sequences flanking the retrotransposon were used in combination with MseI degenerated primers on 15 grapevine accessions. S‐SAP results were compared with AFLP data. The heterozygosity and gene diversity values were higher for S‐SAP than for the AFLP procedure. Results show that S‐SAP amplification is effective in identifying polymorphisms and defining genetic distances among cultivars, and could be used for fingerprinting and for ‘Traminer’ clone identification. To the contrary Vine‐1 retrotransposon‐based S‐SAP was not able to distinguish ‘Pinot’ clones.     

Anatomy of Vitis Berries During Their Coloring

During 1998-1999, the course of the berry coloring and the development of the pigment cells from veraison to ripeness were studied by freeze sectioning 43 accessions of 12 Vitis species (including 10 Chinese wild species). External observation showed that the berries of most species began coloring on the fruit top surface or on the sun-lit surface, and the berry surface color was evenly distributed when the berry was ripe.Internal observation revealed that the pigment cells in a few layers between cuticle and sub-cuticle colored first, the cuticle colored from inner layers to outer layers while the sub-cuticle from outer to inner, and the cuticle cells began coloring a little earlier than the sub-cuticle ones in most species. The pigment cells developed unevenly during the berry ripening. In the beginning of berry coloring, the cell pigment density among the layers or among the cells in the same layer was different. Both the numbers of the pigmented cells and the cell pigment density increased during the berry coloring, while the former lasted a short time; however, the latter kept increasing from veraison to ripeness, and they reached the deepest color when the berry was ripe.     

山葡萄资源性状评价

统计了156份山葡萄资源的产地、花型,分析了果实经济性状。结果表明,来自黑龙江省的种质107份(占68.6%)、吉林省的种质44份(占28.2%)、辽宁省的种质5份(占3.2%);雌能花种质80份(占51.3%)、两性花种质71份(45.5%)、雄株5份(占3.2%);山葡萄种质果皮颜色均为紫黑色;果穗质量最大为140.8g,最小为4.1g,平均为63.4g;果粒质量最大为1.70g,最小为0.41g,平均为0.78g;出汁率最高为65.0%,最低为46.7%,平均为49.6%;浆果可溶性固形物质量分数最高为18.0%,最低为7.5%,平均为13.3%;浆果可溶性糖质量浓度最高为153.2g.L-1,最低为46.9g.L-1,平均为90.9g.L-1;浆果滴定酸质量浓度最高为36.9g.L-1,最低为14.5g.L-1,平均为24.4g.L-1。评价性状表明,吉林省山葡萄资源果实经济性状要稍优于黑龙江省的资源,雌能花山葡萄资源果实经济性状要稍优于两性花资源,但差异不明显。     

欧亚种葡萄对霜霉病与白粉病的抗性及关系

通过田间自然感病调查，利用配对设计方法，研究了7个欧亚种葡萄品种对霜霉病与白粉病的抗性关系。结果表明，在7个供试品种中，以藤稔、红宝石无核、矢富罗莎最抗霜霉病和白粉病，其它品种对这两种病害均无明显的抗性。欧亚种葡萄对霜霉病与白粉病的抗性之间存在极显著的直线正相关。     

山葡萄果实成熟过程中呼吸强度和主要营养成分的变化规律

对山葡萄品种“双优”和“左山一”果实成熟过程中呼吸强度、浆果百粒重和主要营养成分的变化进行了分析。结果表明：山葡萄果实成熟过程中，呼吸强度先呈上升趋势，然后逐渐下降或者一直呈下降趋势；百粒重在成熟前期呈上升趋势，成熟后期呈下降趋势；总糖、维生素C、色素和游离氨基酸呈上升趋势；而酒石酸和单宁呈下降趋势。     

利用GenBank中大量葡萄EST序列分离有效基因的电子表达分析平台

 【目的】充分利用GenBank上的大量葡萄EST序列,建立本地化基因电子表达分析平台,实现基因表达模式的大规模快速分析。【方法】利用生物信息学方法对GenBank上的362 193条葡萄EST序列进行本地化及后续处理,建立电子分析平台,并通过RT-PCR技术对其电子分析准确性进行验证。【结果】建立了包含叶、花等11个组织类别和GA3等5个胁迫类型的基因电子表达分析平台。通过VvAG、VvCHS、VvF3H、VvLDOX等4个葡萄基因的RT-PCR和电子表达分析结果比较发现,平台预测效率较高,在预测EST来源数较多的果实、花序、花组织的表达效果较好,而对叶等EST来源较少的组织的预测效果需结合试验判断。随着dbEST库中源于葡萄各组织EST序列数量的大量增加,平台的预测效果将更加符合基因表达的实际情况。【结论】葡萄基因电子表达分析平台预测效率较高,为葡萄基因大规模快速表达分析以及重要相关信息的挖掘提供了很好的分析工具。