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.

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.

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.

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.

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）.

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.

Genome-wide profiling of histone H3 lysine 27 trimethylation and its modification in response to chilling stress in grapevine leaves

Histone H3 lysine 27 trimethylation(H3K27me3) is a histone modification associated with transcriptional repression. However, insights into the genome-wide pattern of H3K27me3 in grapevines are limited. Here, anti-H3K27 chromatin immunoprecipitation(ChIP), high-throughput sequencing, and transcriptome analysis were performed using leaves of Vitis amurensis. The leaves were treated at 4°C for 2 h and 24 h and used to investigate changes in H3K27me3 under chilling treatment. The results show that H3K27me3 is well-distributed both in gene regions(-50%) and in the intergenic region(-50%) in the grapevine genome(Vitis vinifera ‘Pinot Noir PN40024'). H3K27me3 was found to be localized in8 368 annotated gene regions in all detected samples(leaves at normal temperature and under chilling treatments) and mainly enriched in gene bodies with the adjacent promoter and downstream areas. The short-term chilling treatments(4°C for 2 h) induced 2 793 gains and 305losses in H3K27me3 modification. Subsequently, 97.3% of the alterations were restored to original levels after 24 h treatment. The ChIP-qPCR for five differential peaks showed similar results to the data for ChIP-seq, indicating that the chilling-induced H3K27me3 modification is reliable.Integrative analysis of transcriptome and ChIP-seq results showed that the expression of H3K27me3 target genes was significantly lower than those of non-target genes, indicating transcriptional repression of H3K27me3 in grapevine leaves. Furthermore, histone methylation alterations were detected in 82 genes and were related to either repression or activation of their expression during chilling stress. The findings provide the genome-wide H3K27me3 patterns in grapevines and shed light on uncovering its regulation in chilling stress responses.

山葡萄转录组中果皮着色机理初探

为探究山葡萄在转色不同时期的相关差异表达基因,本研究以不同转色时期的果皮为试验材料,用Illumina HiSeq平台进行测序,共获得39.83 Gb Clean Data。根据基因在不同样品中的表达量识别差异表达基因,并对其进行功能注释和富集分析,在获得的29315个Unigenes中,通过与Nr、GO、COG、SwissProt和KEGG数据库进行比对分析发现,山葡萄Unigenes与葡萄亲缘性最高,差异基因数量在COG数据库中随着转色程度的加深而呈上升趋势,其不同时期行使生物学过程中的细胞杀伤和分子功能中的营养贮存活性也呈增加趋势。本研究结果为进一步探究山葡萄不同时期的差异表达基因及分子机制提供了可参考数据。

山葡萄耐盐愈伤组织筛选及其抗氧化水平分析

以山葡萄叶片外植体诱导出的愈伤组织为试材,氯化钠(sodium chloride,NaCl)为选择因子,筛选耐盐愈伤组织。通过比较不同浓度NaCl处理后愈伤组织相对生长率及生长情况,初步明确了山葡萄耐盐愈伤组织筛选的临界NaCl浓度为1.5%;且1.5%NaCl培养基上处理的愈伤组织白藜芦醇含量也最高,处理3周后含量高达1580μg/g FW,是未经NaCl处理愈伤的3.13倍,但当NaCl浓度为2.0%时,白藜芦醇含量降至652μg/g FW。进一步在无盐和含1.5%NaCl培养基交替培养3次,每4周继代1次后,初步获得了山葡萄耐盐愈伤组织。经1.5%NaCl处理后,耐盐愈伤组织的相对生长率大幅提高,是对照愈伤组织的6.09倍,其H2O2含量则大幅下降,而抗氧化酶活性也明显升高。说明山葡萄耐盐愈伤组织可能通过提高抗氧化水平来降低盐分造成的伤害。