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 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.

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.

环塔盆地不同山葡萄品种果实品质间的差异

为山葡萄在环塔盆地生态区域的选育、推广应用提供科学理论依据,建立8个新疆山葡萄果实综合评价体系。以2019-2021年田间表现良好的山葡萄‘北冰红’‘双红’‘左优红’‘雪兰红’‘双丰’‘北国红’‘左山1’和‘左山2’为试材,测量果实23项外观及理化品质指标,按照测量结果进行描述性统计和主成分分析,根据综合得分进行排名。结果表明,23项果实品质指标变异程度不同,其中白藜芦醇、果形指数、Zn含量和花色苷变异程度较大,超过50%;果穗质量、原花青素、固酸比和Fe含量变异程度一般,为30%~50%;其他果实品质指标变异程度较小(<30%)。因子分析提取出6个特征根>1的公因子,累计方差贡献率达97.839%,公因子PC1贡献率达22.154%,主要由pH、果实纵径、横径、单果质量、单果体积和果穗质量决定,主要反映果实外观品质;公因子PC_(2)贡献率达20.987%,由可溶性固形物、总糖、糖酸比、固酸比和Mn含量5个因子决定,主要反映果汁的糖度;公因子PC_(3)贡献率为19.820%,由Ca、Mg和Cu含量3个因子决定,主要反映矿质元素含量。公因子PC_(4)贡献率为19.117%,由花色苷、总酸和果形指数3个因子决定,主要反映果汁色泽、酸度和果实形状;公因子PC_(5)贡献率为8.293%,由原花青素和总黄酮2个因子决定,主要反映果实的营养状况;公因子PC_(6)贡献率为7.467%,由白藜芦醇和Zn含量2个因子决定,主要反映白藜芦醇和Zn含量的高低情况。山葡萄果实品质综合得分排序由高到低依次是:‘北冰红’‘左优红’‘雪兰红’‘左山2’‘北国红’‘双丰’‘双红’‘左山1’。

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。

山葡萄基因VaEXO11及其启动子的克隆与序列分析

【目的】探究欧洲葡萄基因VvEXO11在山葡萄中的同源基因VaEXO11的表达及功能,为揭示葡萄抗寒分子通路、培育新的抗寒葡萄品种提供理论依据。【方法】克隆并分析VaEXO11及其启动子PVaEXO11序列,通过瞬时转化烟草探究PVaEXO11与低温胁迫的关系。【结果】从VaEXO11克隆到的cDNA序列全长为957 bp,其中ORF为957 bp,编码318个氨基酸。该基因仅由1个外显子构成,其蛋白含有一个高度保守的Phi_1结构域和一个信号肽,丝氨酸含量丰富,经预测为疏水脂溶性蛋白。对克隆到的P_(VaEXO11)进行顺式作用元件预测,分析结果表明,PVa EXO11不仅含有CAAT-box、TATA-box等核心启动子元件,还具备参与干旱胁迫、昼夜节律调控和创伤响应等功能响应元件。瞬时转化结果表明,低温激活P_(VaEXO11)的活性,VaEXO11的相对表达量迅速上升,直接或间接促进细胞内抗氧化酶的产生。【结论】VaEXO11除了参与低温调控,还可能参与多种逆境相关的调控,从而响应多种生物与非生物胁迫。

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。

山葡萄不同代际间杂交群体抗寒性差异调查分析

于2021—2023年以山葡萄F1~F3代共14个杂交群体为材料,通过调查不同代间及代内群体越冬后的抗冻等级差异,探讨山葡萄抗冻性遗传特性。结果表明,抗冻性随山葡萄血统稀释而减弱,F1代抗冻性最强,有87%以上的后代可以免埋土越冬,且无明显母系遗传特点;F2代抗冻性减弱,F2代中正反交组合的抗冻性表现出母系遗传特点,以抗冻的F1代做母本可以获得更多抗冻后代;F2代与欧亚种杂交的F3代抗冻性较F2代更弱,但与美洲种或其他含有抗寒基因的亲本杂交后提升了F3代的抗冻能力。

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。

响应面优化超声辅助提取山葡萄皮渣花色苷

以山葡萄皮渣为试材,采用单因素和响应面试验,研究了超声提取时间、超声提取温度、液料比、乙醇体积分数对山葡萄皮渣花色苷提取量的影响,优化了山葡萄皮渣花色苷的最佳提取工艺,以期为山葡萄皮渣花色苷的提取提供参考依据。结果表明:最佳工艺条件为超声提取时间21.20 min、超声提取温度37.64℃、液料比15.62∶1 mL·g^(-1)、乙醇体积分数61.81%,该条件下测得山葡萄皮渣花色苷提取量为(0.358±0.005)mg·g^(-1),与理论预测值误差仅为0.56%。

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。