山葡萄(Vitis amurensis Rupr)晚秋叶施^(15)N-尿素的吸收、运转、贮藏及再分配的研究

本文研究了山葡萄对晚秋叶施^(15)N-尿素的吸收、运转、贮藏及再分配利用的特点。结果表明,山葡萄幼树晚秋叶施尿素后可很快被叶吸收并运到树体各部分,Ndff从叶运到根中(距离40cm)只需48小时,且运到根中后即部分形成蛋白质。休眠期80.02％的Ndff贮藏在根中,特别是细根,52.39％Ndff以蛋白态形式贮藏。翌春葡萄开始生长后,Ndff较总N优先用于新梢和叶片等新生器官的生长。不同时期,Ndff％最高的器官不同,一般生长活跃器官的Ndff％高。次年秋季,叶中Ndff仍可部分回流到树体内,第3年春天仍有小量Ndff重新分配到新生器官。Ndff可被山葡萄重复利用3年。

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Analysis of Genetic Diversity of Vitis amurensis Rupr.(Vitaceae) Germplasm Resources

[ Objective] This study aimed to explore the genetic diversity of Vitis amurensis Rupr. （Vitaceae） germplasm resources. [ Method ] Out of total 245 pairs of primers, 18 were selected for SSR amplification of 360 V. amurensis experimental materials. [Result] The number of bands amplified by each primer ranged from 4 to 13 with a mean of 9.44. The length of bands ranged from 150 to 1 000 bp, concentrated at 200 -750 bp. The 18 pairs of primers amplified 170 bands totally, of which 167 bands were polymorphic with a polymorphism ratio of 98.2%. The Shannon＇s diversity index （I） is 1. 778 051. With the SSR-PCR am- plification of 360 V. amurensis varieties （ strains）, 5 specific bands were amplified by certain primers in several varieties（strains） accounting for 2.95% of the total bands. [ Conclusion] SSR molecular marker technique was an efficient method to detect the genetic diversity of V. amurensis and thereby is an effective tool for pedigree analysis and variety identification of A. amurensis varieties（strains）.

山葡萄(Vitis amurensis)资源核心种质的初步构建

采用省份分组-花型分组-聚类-分组法构建了山葡萄核心种质,在此基础上,考虑到山葡萄品种在生产上和雄株山葡萄在科学研究上的作用,把未进入核心种质的山葡萄品种和雄株山葡萄也并入核心种质。该核心种质包括48份资源(占资源总数的31%),其中来自吉林省的资源18份(占37.5%),黑龙江省的资源29份(占60.4%),辽宁省的资源1份(占2.1%);两性花资源22份(占45.8%),雌能花资源21份(占43.8%),雄株5份(占10.4%)。除出汁率外,其他性状的符合度达72%以上;除果粒质量外,其他性状的变幅吻合度达79%以上。以上结果表明,所构建的核心种质较好地代表了评价的山葡萄资源。

Synthesis of Amurensin H, a New Resveratrol Dimer from the Roots of Vitis Amurensis

Amurensin H(1) is a new resveratrol dimer isolated from the roots of Vitis amurensis Rupr. Its structure was determined by spectroscopic methods. II was synthesized from resveratrol with an oxidative coupling reaction as a key step.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures.However,the accumulation of metabolites during cold acclimation(CA)in V.amurensis remains largely unknown.In this study,plantlets of V.amurensis and V.vinifera cv.Muscat of Hamburg were treated at 4℃ for 24 and 72 h,and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry.Most of the identified metabolites,including carbohydrates,amino acids,and organic acids,accumulated in the two types of grape after CA.Galactinol,raffinose,fructose,mannose,glycine,and ascorbate were continuously induced by cold in V.amurensis,but not in Muscat of Hamburg.Twelve metabolites,including isoleucine,valine,proline,2-oxoglutarate,and putrescine,increased in V.amurensis during CA.More galactinol,ascorbate,2-oxoglutarate,and putrescine,accumulated in V.amurensis,but not in Muscat of Hamburg,during CA,which may be responsible for the excellent cold tolerance in V.amurensis.The expression levels of the genes encodingβ-amylase(BAMY),galactinol synthase(GolS),and raffinose synthase(RafS)were evaluated by quantitative reverse transcription-PCR.The expression BAMY(VIT_02s0012 g00170)and RafS(VIT_05s0077 g00840)were primarily responsible for the accumulation of maltose and raffinose,respectively.The accumulation of galactinol was attributed to different members of GolS in the two grapes.In conclusion,these results show the inherent differences in metabolites between V.amurensis and V.vinifera under CA.

A New Oligostilbene from the Roots of Vitis Amurensis

A new resveratrol trimer. amurensin G (1), was isolated from the roots of Vitis amurensis Rupr. Its structure and relative configuration were established on the basis of spectral evidence. especially on HMBC spectrum and NOE difference experiments.

Expression of structural genes related to anthocyanin biosynthesis of Vitis amurensis

This research was designed to assess the changes in anthocyanin content in grape skins of Vitis amurensis and to explore m RNA transcriptions of 11 structural genes（PAL,CHS3, CHI1, F3H2, F30 H, F3050 H, DFR, LDOX, UFGT,OMT and GST） related to anthocyanin biosynthesis during grape berry development, by the use of HPLC-MS/MS and real-time Q-PCR analysis. Accumulation of anthocyanins began at veraison, continued throughout the later berry development and reached a peak at maturity. Veraison is the time when the berries turn from green to purple. Expression of PAL, CHI1, and LDOX were up-regulated from 2 to4 weeks after flowering（WAF）, down-regulated from6 WAF to veraison, whereas DFR was up-regulated at8 WAF, and then up-regulated from veraison to maturity.CHS3, F3050 H, UFGT, GST, and OMT were down-regulated from 2 WAF to veraison, and then up-regulated from veraison to maturity. The transcriptional expressions of the11 structural genes also showed positive correlations with the anthocyanin content from veraison to maturity. Positive correlations were also observed between OMT transcriptional level and the content of methoxyl-anthocyanins, and between F3050 H transcriptional level and the content of delphinidin anthocyanins. F3H2 and F30 H expression was up-regulated at 2 WAF. F3H2 expression was down-regulated from 4 WAF to veraison and then up-regulated again from veraison to maturity. F30 H expression was down-regulated at 4 WAF and then up-regulated again from 6 WAF to maturity. F30 H transcriptional level was correlated positively with the cyanidin anthocyanin concentration from veraison to maturity. These results indicate that the onset of anthocyanin synthesis during berry development coincides with a coordinated increase in the expression of a number of genes in the anthocyanin biosynthetic pathway.

Expressed sequence tags (ESTs) analysis of the ripening Vitis amurensis cv. Shuang Hong berry skins

Vitis amurensis is a valuable resource for wine production. Ripening of the grape berry is the key phase which determines the com- position of wine. To better understand the gene expression that manifest in V. amurensis berry skins during the ripening, cDNA library of V. amurensis berry skins was constructed. A total of 935 high quality ex- pressed sequence tags （ESTs） were obtained from the library. These ESTs represent 636 unigenes, including 108 contigs and 528 singletons. The EST analysis was performed and genes were assigned to functional categories according to their primary BLAST match. Of these 25.35% were involved with metabolism, 6.27% with cell rescue and defense, 6.84% energy, 11.68% protein synthesis, 18.8% protein activity regula- tion, 11.11% cell structure, 7.98% transport, 6.27% transcription and the remaining 5.7% were signal transduction. The generated ESTs were characterized by the gene ontology analysis and were categorized ac- cording to its cellular component, molecular function and biological process. In the cDNA library, some genes are relevant to the biosynthesis of anthocyanins, while some genes are related to grape berry maturation.

Isolation and expression analysis of CBF4 from <i>Vitis amurensis</i>associated with stress

The C-repeat Binding Factor/Dehydration Responsive Element Binding factor (CBF/DREB) responsive pathway is involved in plant response to abiotic stress. In this study, we report the isolation of VaCBF4 (a complete cDNA) and its promoter from Vitis amurensis through rapid amplification of cDNA ends and genome walking techniques, respectively. The CBF4 transcript accumulation of V. amurensis increased under cold, salinity, and abscisic acid (ABA) and salicylic acid (SA) treatments, whereas that of Vitis vinifera showed a different change. The transcript levels of VaCBF4 inthe roots, stems, leaves, and petioles under cold, salinity, and ABA and SA treatments were up-regulated in a timedependent manner. The presence of the cis-elements MBC, ABRE, and as-2-box in the VaCBF4 promoter further confirmed that this promoter is a component of the CBF transduction pathway, which is involved in plant response to multistress.

Isolation and expression characterization of CBF2 in <i>vitis amurensis</i>with stress

The transcription factor VaCBF2, which interacts with C-repeat/DRE and its promoter, was isolated from Vitis amurensis. The VaCBF2 amino acid sequence contained a conserved AP2 domain of 56 amino acids and a potential nuclear localization sequence. The sequence of VaCBF2 showed a high level of homology with other CBF2 family members. Phylogenetic analysis showed that the amino acid sequences may be CBF2 proteins with evolutionary relationship. Quantitative reverse-transcription polymerase chain reaction analysis indicated that the expression of VaCBF2 gene in tissues (roots, stems, leaves, and petioles) was induced by low temperature, high salinity, and application of abscisic acid and salicylic acid in a time-dependent manner but to different extents in the cold-hardy V. amurensis and the less cold-hardy Vitis vinifera. The presence of cis-elements such as MYC and ABRE in VaCBF2 promoter further confirmed that this promoter was a component of the CBF transduction pathway involved in plant response to multiple stresses.

COR-Like Gene Is Involved in Induced-Expression Response to Multiple Abiotic Stresses in Grape Vine (<i>Vitis amurensis</i>) Tissues

Cold stress, which causes dehydration damage to plants, is one of the most common abiotic stresses that limit plant distributions and affect crop growth and development. To improve their cold tolerance, plants often upregulate the expression of some cold-related genes. In this study, a cold-regulated (COR) gene was isolated from Vitis amurensis and designated as VaCOR. RT-PCR analysis demonstrated that VaCOR was expressed at high levels in the roots, stems, leaves, and petioles under low temperature, but it was not detected under normal temperatures. Further analysis revealed that salinity and the application of exogenous abscisic acid and salicylic acid significantly induced VaCOR transcription, with apparent differences in its expression in different organs. The data also showed that COR gene expression was higher in cold-resistant wild V. amurensis than in cold-sensitive Vitis vinifera “Manicure Finger” under low temperature. These results suggest that the VaCOR gene in V. amurensis grapes is involved in multiple stresses and plays a central role in stress-induced and stress-tolerance.

不同山葡萄品种CO_(2)响应模型拟合及评价

为探索不同山葡萄品种叶片CO_(2)响应特征差异,以5 a生山葡萄‘北冰红’‘北国红’‘双红’和‘雪兰红’为试材,采用Li-6400便携式光合仪,测定果实膨大期山葡萄叶片光合-二氧化碳响应曲线(photosynthetic CO_(2)response curve,P_(n)-C_(i))以及胞间CO_(2)浓度(intercellular CO_(2)concentration,C_i)、气孔导度(stomatal conductance,G_s)、水分利用率(water use efficiency,WUE)和蒸腾速率(transpiration rate,T_r)等气体交换参数,基于直角双曲线模型、Michaelis-Menten模型和直角双曲线修正模型3种模型拟合山葡萄叶片P_(n)-C_(i)响应曲线。结果表明,直角双曲线修正模型拟合的山葡萄P_(n)-C_(i)响应曲线,其拟合参数与实测值最为接近,可直接计算CO_(2)饱和点(CO_(2)saturation point,CSP)。随大气CO_(2)浓度(atmospheric CO_(2)concentration,C_a)的增加,4个山葡萄品种C_i呈线性递增趋势;G_s和T_r总体呈先升后降趋势;WUE先降后升,呈“U”型变化趋势。主成分分析提取出2个主成分,累计贡献率达84.613%。综合评价‘雪兰红’得分最高,光能转化利用率最高,在低C_a环境下的适应性最佳;‘双红’在不同C_a水平下均可保持较高光合效率,排名第2。综上所述,直角双曲线修正模型拟合山葡萄叶片P_(n)-C_(i)响应曲线效果最优。

山葡萄砧木提高葡萄耐寒性的转录调控研究

以山葡萄为砧木可提高葡萄的耐寒性,但其分子调控机制还不明确。本研究以山葡萄为砧木的‘黑比诺’异嫁接苗(Vv_Va)和‘黑比诺’自嫁接苗(Vv_Vv)为材料,利用转录组数据分析两者在低温胁迫下的基因表达差异。结果表明,低温胁迫下,与自接相比,异接中显著上调的基因数330个,显著下调的基因数190个;对差异表达基因的GO富集分析表明,自接和异接的差异表达基因与非生物胁迫相关,尤其是应答温度胁迫相关基因的表达产生了显著差异;在所有差异表达的基因中,有27个热激蛋白基因、3个渗透调节物质合成关键酶基因和6个活性氧清除关键基因的表达显著上调,且有WRKY、NAC、MYB、HSF、AP2/ERF、Zinc Finger和MYC 7个家族共23个转录因子发生了差异表达,这可能是Vv_Va在遭遇低温胁迫时,抗寒砧木嫁接对接穗具有更好的保护作用。本研究通过对低温胁迫下自接苗和异接苗相关基因的转录表达差异分析,为阐释山葡萄嫁接提高葡萄耐寒性的分子机制奠定了基础。

山葡萄VaERF095基因表达及其启动子功能分析

【目的】克隆山葡萄乙烯响应因子基因VaERF095及其启动子序列,分析在低温和外源激素诱导下该基因的表达模式和启动子活性,为深入探究VaERF095基因参与低温胁迫响应机理提供理论依据。【方法】采用同源克隆方法从山葡萄品种左山-1中获得VaERF095基因及其启动子,并通过实时荧光定量PCR检测VaERF095基因在低温(4℃)胁迫和外源激素诱导下及不同组织中的表达情况。同时构建VaERF095基因启动子融合GUS蛋白表达载体,瞬时转化葡萄叶片,通过β-葡萄糖苷酸酶(GUS)活性定量分析VaERF095基因启动子受低温胁迫和外源激素诱导的表达情况。【结果】从山葡萄克隆获得VaERF095基因,编码区(CDS)全长393 bp,编码130个氨基酸残基,该蛋白含有1个保守的AP2/ERF结构域,与欧洲葡萄和河岸葡萄的亲缘关系较近,氨基酸序列相似性分别为100.00%和93.08%。Va ERF095基因可响应低温和外源激素乙烯(ET)、水杨酸(SA)、茉莉酸甲酯(MeJA)和脱落酸(ABA)的诱导,呈现出不同的表达模式。VaERF095基因在老叶、茎、花序、卷须和果实均有表达,其中在卷须中的表达量最高,在幼叶中的表达量极低或几乎不表达。克隆获得长度为1360 bp的VaERF095基因启动子,含有2个低温响应元件(LTR)、1个ET响应元件(ERE)、1个SA响应元件(TCA-element)、2个MeJA响应元件(CGTCA-motif和TGACG-motif)、1个ABA响应元件(ABRE)、1个GA响应元件(P-box)和1个防御和胁迫响应元件(TC-rich repeats)。在低温胁迫及外源激素(ET、SA和ABA)诱导下,VaERF095基因启动子活性较对照极显著升高(P<0.01),在MeJA诱导下,VaERF095基因启动子活性显著增强(P<0.05)。【结论】VaERF095基因及其启动子正向响应低温(4℃)及外源激素(ET、SA、ABA和MeJA)的诱导,具有明显的组织表达特异性。

山葡萄致病致灾气象指标及其栽培管理技术

从园田气象基础数据积累和田间调查入手,对山葡萄致病致灾的气象指标进行了研究,对影响山葡萄产量和品质的气象因子进行了普查和筛选,对病害、灾害的防御措施及栽培和管理措施进行了归纳总结,形成了较为完整的山葡萄致病致灾气象指标及其栽培管理技术,以营造适宜山葡萄栽培的农田小气候,规避气象灾害,保证和提高山葡萄的产量和品质。

不同采收期北冰红葡萄中总酚含量变化规律研究

为综合评价北冰红葡萄品种特性,以3个采样点(阳坡、阴坡、半阴半阳坡)5个不同采收期(T1、T2、T3、T4、T5)的北冰红葡萄为研究对象,测定单粒果重、可溶性固形物等指标,以福林肖卡法重点考察了果实不同部位(果皮和果籽)总酚含量的变化,旨在研究不同采收期北冰红葡萄果实酚类物质的积累规律.结果表明,3个采样点在T5时期的北冰红葡萄果实中总酚含量分别为88.48 mg/g、88.06 mg/g、78.34 mg/g,均显著(p<0.05)高于其他时期的总酚含量,且单粒果重和可溶性固形物含量随采收期的延长而增长,确定了北冰红葡萄的最适采收期为T5.该研究结果将为实际生产中进一步调控酿酒葡萄总酚物质积累和果实品质提供一定的理论依据.

响应面法优化低共熔溶剂提取紫椴花总黄酮工艺及抗氧化活性研究

目的通过合成不同类型的低共熔溶剂,考察其对紫椴花中黄酮类化合物提取率的影响并优化其工艺,并对其总黄酮的抗氧化活性进行研究。方法在单因素实验基础上,选取对紫椴花总黄酮提取率影响较大的因素,采用响应面试验设计方法,建立以超声功率、超声时间、溶剂含水量为自变量,紫椴花总黄酮提取率为因变量的二次回归模型。根据紫椴花总黄酮对ABTS自由基和DPPH自由基的清除效果判断其体外抗氧化能力。结果紫椴花总黄酮的提取最优条件为氯化胆碱-乳酸(摩尔比1:2),超声功率200 W,提取时间30 min,含水量为50%。在此条件下,具有最高的提取率(10.61%)。紫椴花总黄酮对ABTS自由基、DPPH自由基清除率分别为93.27%和90.85%,IC50值分别为0.021和0.0045 mg/ml,具有较好的抗氧化能力。结论利用响应面法优化紫椴花总黄酮的提取工艺,建立了模拟回归方程,可用于提取工艺的参数优化。抗氧化实验结果表明,紫椴花总黄酮有较好的体外抗氧化活性,可用于开发新的抗氧化药物。

Ectomycorrhizal communities associated with Tilia amurensis trees in natural versus urban forests of Heilongjiang in northeast China

The deciduous linden tree （Tilia amurensis Rupr.） is protected at National Level II in China as a species of ecological and economic importance. The objective of this study was to assess and compare the ectomycorrhizal communities associated with T. amurensis in natural versus urban forests of central Heilongjiang Province. The percentage of T. amurensis colonisation by ectomycorrhiza was more than 60 % in urban forests, compared to 34-49 % in natural forests. Use of a combi- nation of morphological and molecular methods docu- mented 18 ECM （ectomycorrhizal） types among three sites; 12-13 ECM species were identified in the natural sites versus 9 species in the urban site. Four ECM species （Boletus sp., Tuber sp., Inocybe sp.2, Leccinum sp.1） were the dominant mycorrhizal symbionts, and Cenococcum geophilum and Russula sp. were found only in the natural forests.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.