BnaUBP15s positively regulates seed size and seed weight in Brassica napus

Brassica napus(B.napus)is a globally significant oilseed crop,making a substantial contribution to both human oil and livestock feed production.Enhancing seed weight is crucial for improving rapeseed yield;however,only a limited number of seed weight-related genes have been functionally validated in B.napus thus far.UBIQUITINSPECIFIC PROTEASE 15(UBP15)belongs to the ubiquitin protease pathway and plays a maternal role in prolonging seed development in Arabidopsis.The potential utilization of UBP15 for enhancing seed yield in B.napus has remained unexplored until now.In this study,we identified the orthologs of UBP15 in B.napus and investigated its functionality using the CRISPR-Cas9 system.We generated mutant plants with multiple editing types targeting Bnaubp15s and successfully isolated T-DNA-free homozygous mutant lines that exhibited edits across four homologs of BnaUBP15 in T2 generation plants.Our preliminary data demonstrated that mutation of BnaUBP15s significantly reduced seed size,seed weight,and plant height while noticeably increasing the number of primary branches.These findings not only provide crucial evidence for further elucidating the molecular mechanism underlying the regulation of seed weight and size by BnaUBP15s but also offer promising novel germplasm for enhancing plant architecture.

Genetic dissection of and genomic selection for seed weight, pod length,and pod width in soybean

A biparental soybean population of 364 recombinant inbred lines(RILs)derived from Zhongdou 41×ZYD02.878 was used to identify quantitative trait loci(QTL)associated with hundred-seed weight(100-SW),pod length(PL),and pod width(PW).100-SW,PL,and PW showed moderate correlations among one another,and 100-SW was correlated most strongly with PW(0.64–0.74).Respectively 74,70,75 and19 QTL accounting for 38.7%–78.8%of total phenotypic variance were identified by inclusive composite interval mapping,restricted two-stage multi-locus genome-wide association analysis,3 variancecomponent multi-locus random-SNP-effect mixed linear model analysis,and conditional genome-wide association analysis.Of these QTL,189 were novel,and 24 were detected by multiple methods.Six loci were associated with 100-SW,PL,and PW and may be pleiotropic loci.A total of 284 candidate genes were identified in colocalizing QTL regions,including the verified gene Seed thickness 1(ST1).Eleven genes with functions involved in pectin biosynthesis,phytohormone,ubiquitin-protein,and photosynthesis pathways were prioritized by examining single nucleotide polymorphism(SNP)variation,calculating genetic differentiation index,and inquiring gene expression.The prediction accuracies of genomic selection(GS)for 100-SW,PL,and PW based on single trait-associated markers reached 0.82,0.76,and 0.86 respectively,but selection index(SI)-assisted GS strategy did not increase GS efficiency and inclusion of trait-associated markers as fixed effects reduced prediction accuracy.These results shed light on the genetic basis of 100-SW,PL,and PW and provide GS models for these traits with potential application in breeding programs.

Detection of new candidate genes controlling seed weight by integrating gene coexpression analysis and QTL mapping in Brassica napus L.

Seed weight is a component of seed yield in rapeseed(Brassica napus L.).Although quantitative trait loci(QTL)for seed weight have been reported in rapeseed,only a few causal quantitative trait genes(QTGs)have been identified,resulting in a limitation in understanding of seed weight regulation.We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species.Among the 10 modules in this gene coexpression network,modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein,and photosynthesis.A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19(SCPL19)was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines.Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Meta-Analysis of 100-Seed Weight QTLs in Soybean

100-seed weight is a very complicated quantitative trait of yield. The study of gene mapping for yield trait in soybean is very important for application. However, the mapping result of 100-seed weight was dispersed, the public map should be chosen which was suitable for the published results integrated, and to improve yield. In this research, an integrated map of 100-seed weight QTLs in soybean had been established with soymap2 published in 2004 as a reference map. QTLs of 100-seed weight in soybean were collected in recent 20 yr. With the software BioMercator 2.1, QTLs from their own maps were projected to the reference map. From published papers, 65 QTLs of 100-seed weight were collected and 53 QTLs were integrated, including 17 reductive effect QTLs and 36 additive effect QTLs. 12 clusters of QTLs were found in the integrated map. A method of meta-analysis was used to narrow down the confidence interval, and 6 additive QTLs and 6 reductive QTLs and their corresponding markers were obtained respectively. The minimum confidence interval （C.I.） was shrunk to 1.52 cM. These results would lay the foundation for marker-assisted selection and mapping QTL precisely, as well as QTL gene cloning in soybean.

Dt1 inhibits SWEET-mediated sucrose transport to regulate photoperiod-dependent seed weight in soybean

Soybean is a photoperiod-sensitive short-day crop whose reproductive period and yield are markedly affected by day-length changes.Seed weight is one of the key traits determining the soybean yield;how-ever,the prominent genes that control the final seed weight of soybean and the mechanisms underlying the photoperiod's effect on this trait remain poorly understood.In this study,we identify SwW19 as a major locus controlling soybean seed weight by QTL mapping and determine Dt1,an orthologous gene of Arabidopsis TFL1 that is known to govern the soybean growth habit,as the causal gene of the SW19 locus.We showed that Dt1 is highly expressed in developing seeds and regulates photoperiod-dependent seed weight in soybean.Further analyses revealed that the Dt1 protein physically interacts with the sucrose transporter GmSWEET10a to negatively regulate the import of sucrose from seed coat to the embryo,thus modulating seed weight under long days.However,Dt1 does not function in seed development under short days due to its very low expression.Importantly,we discovered a novel natural allelic variant of Dt1(H4 haplotype)that decouples its pleiotropic effects on seed size and growth habit;i.e.,this variant remains functional in seed development but fails to regulate the stem growth habit of soybean.Collectively,our findings provide new insights into how soybean seed development responds to photoperiod at different latitudes,offering an ideal genetic component for improving soybean's yield by manipulating its seed weightandgrowth habit.

Differential SW16.1 allelic effects and genetic backgrounds contributed to increased seed weight after soybean domestication

Although seed weight has increased following domestication from wild soybean(Glycine soja) to cultivated soybean(Glycine max), the genetic basis underlying this change is unclear. Using mapping populations derived from chromosome segment substitution lines of wild soybean, we identified SW16.1 as the causative gene underlying a major quantitative trait locus controlling seed weight.SW16.1 encodes a nucleus-localized LIM domaincontaining protein. Importantly, the GsSW16.1 allele from wild soybean accession N24852 had a negative effect on seed weight, whereas the GmSW16.1 allele from cultivar NN1138-2 had a positive effect. Gene expression network analysis,reverse-transcription quantitative polymerase chain reaction, and promoter-luciferase reporter transient expression assays suggested that SW16.1 regulates the transcription of MT4, a positive regulator of seed weight. The natural variations in SW16.1 and other known seed weight genes were analyzed in soybean germplasm. The SW16.1 polymorphism was associated with seed weight in 247 soybean accessions, showing much higher frequency of positive-effect alleles in cultivated soybean than in wild soybean. Interestingly,gene allele matrix analysis of the known seed weight genes revealed that G. max has lost 38.5%of the G. soja alleles and that most of the lost alleles had negative effects on seed weight. Our results suggest that eliminating negative alleles from G. soja led to a higher frequency of positive alleles and changed genetic backgrounds in G. max,which contributed to larger seeds in cultivated soybean after domestication from wild soybean.Our findings provide new insights regarding soybean domestication and should assist current soybean breeding programs.

A loss-of-function mutant allele of a glycosyl hydrolase gene has been co-opted for seed weight control during soybean domestication

The resultant DNA from loss-of-function mutation can be recruited in biological evolution and development.Here,we present such a rare and potential case of“to gain by loss”as a neomorphic mutation during soybean domestication for increasing seed weight.Using a population derived from a chromosome segment substitution line of Glycine max(SN14)and Glycine soja(ZYD06),a quantitative trait locus(QTL)of 100-seed weight(q HSW)was mapped on chromosome 11,corresponding to a truncatedβ-1,3-glucosidase(βGlu)gene.The novel gene hsw results from a 14-bp deletion,causing a frameshift mutation and a premature stop codon in theβGlu.In contrast to HSW,the hsw completely lostβGlu activity and function but acquired a novel function to promote cell expansion,thus increasing seed weight.Overexpressing hsw instead of HSW produced large soybean seeds,and surprisingly,truncating hsw via gene editing further increased the seed size.We further found that the core 21-aa peptide of hsw and its variants acted as a promoter of seed size.Transcriptomic variation in these transgenic soybean lines substantiated the integration hsw into cell and seed size control.Moreover,the hsw allele underwent selection and expansion during soybean domestication and improvement.Our work cloned a likely domesticated QTL controlling soybean seed weight,revealed a novel genetic variation and mechanism in soybean domestication,and provided new insight into crop domestication and breeding,and plant evolution.

A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean l O0-Seed Weight

Cultivated soybeans may lose some useful genetic loci during domestication. Introgression of genes from wild soybeans could broaden the genetic background and improve soybean agronomic traits. In this study, through whole-genome sequencing of a recombinant inbred line population derived from a cross between a wild soybean ZYD7 and a cultivated soybean HN44, and mapping of quantitative trait loci for seed weight, we discovered that a phosphatase 2C-1 （PP2C-1） allele from wild soybean ZYD7 contributes to the increase in seed weight/size. PP2C-1 may achieve this function by enhancing cell size of integument and activating a subset of seed trait-related genes. We found that PP2C-1 is associ- ated with GmBZR1, a soybean ortholog of Arabidopsis BZR1, one of key transcription factors in brassi- nosteroid （BR） signaling, and facilitate accumulation of dephosphorylated GmBZR1. In contrast, the PP2C-2 allele with variations of a few amino acids at the N-terminus did not exhibit this function. More- over, we showed that GmBZR1 could promote seed weight/size in transgenic plants. Through analysis of cultivated soybean accessions, we found that 40% of the examined accessions do not have the PP2C-1 allele, suggesting that these accessions can be improved by introduction of this allele. Taken together, our study identifies an elite allele PP2C-1, which can enhance seed weight and/or size in soybean, and pinpoints that manipulation of this allele by molecular-assisted breeding may increase production in soybean and other legumes/crops.

不同化控剂处理对杂交大豆制种催熟效果的影响

为解决杂交大豆种子商业化生产过程中,母本持绿,无法机械化收获、种子皱缩,品质较差的问题,从而推进杂交大豆产业化的进程,系统研究了不同类型化控剂及处理方法对杂交大豆制种过程中母本催熟效果的影响。结果表明:3种不同类型化控剂均可加速茎叶脱水,促进植株成熟,催熟速率排序为:乙烯利<草甘膦<敌草快,化控剂浓度越大,催熟效率越高;R6期喷施草甘膦和敌草快导致籽粒无法正常发育,若晚于R7期喷施,各化控剂催熟效果均不理想,R6+7 d为最佳喷施时期,可解决不育系茎叶持绿、籽粒皱缩、百粒重过大等问题。草甘膦为内息传导型催熟药剂,喷施草甘膦会严重降低种子芽率,无法应用于种子生产,乙烯利、敌草快无此负面作用。综合考量催熟效果、环境安全及操作便利性等方面,敌草快在R6+7 d时期处理可有效促进植株成熟,加速茎叶脱水落叶,降低百粒重,保证种子品质,符合机械化收获要求。

大豆株型与产量相关性状QTL定位及候选基因预测

为探究与大豆株型和产量相关QTL位点及候选基因,对以东农42(♀)和东农50(♂)为亲本,与168个家系构建的F_(2:12)、F_(2:13)重组自交系(Recombination inbred lines,RILs)群体的株高、分枝数、四粒荚数、百粒重性状测定表型数据,运用IBM SPSS Statistics、R语言进行统计和相关性分析,并利用完备区间作图法(Inclusive composite interval mapping,ICIM)进行加性效应及上位效应分析,共计定位到43个QTL位点,贡献率超过10%的主效位点为14个,包括株高3个、分枝数8个、四粒荚数1个和百粒重2个;其中11个位点与前人已报道位点重合,分别位于4、6、8、16和19号染色体上;qBN-6-2(13.21%)、qBN-6-5(19.96%)和qBN-6-6(13.69%)为3个环境重复定位到的位点,qHSW-19-1与多个已报道位点均有重合。通过上位性分析,获得株高、分枝数、四粒荚数和百粒重位点分别为3、6、6和62对。根据所定位到的物理区间和定量预测,筛选到Glyma.04G238800、Glyma.03G181600、Glyma.08G271900、Glyma.18G278800和Glyma.19G187000等5个与株高、分枝数、四粒荚数和百粒重性状相关的候选基因,为分子辅助育种奠定基础。

Rapid Determination of Seed Number and Thousand Kernel Weight of Asparagus officinalis L.Using Image Processing Method

Asparagus officinalis L.is favored by its high health function,but its hybrid seeds are expensive.The amount of seed,seed plumpness and germination rate are related to the production costs of breeding enterprises and large growers.Therefore,it is necessary to investigate the seed number and thousand kernel weight of A.officinalis L.This study developed a quick and accurate method to measure the seed number and thousand kernel weight of A.officinalis L.using image processing technology.Seed sample of A.officinalis L.was scanned with 200 dpi resolution,and the seed number was then obtained using Image-ProPlus software.After weighing the seeds,thousand kernel weight was finally calculated.By recording‘macro’,the batch processing of the samples can also be realized.This method is simple and accurate,and can greatly save the time of investigation.

播种密度和深度对机播菠菜发芽和产量的影响

为了确定菠菜机械播种的适宜密度和深度,本试验使用百粒重、发芽率有明显差别的4个菠菜品种‘539A’、‘539B’、‘530’、‘532’,播种前在人工气候箱内测定各个品种种子的发芽率,在青岛市农业科学研究院蔬菜试验基地进行露地播种试验,研究使用简易机械播种时,机械播种密度和深度对菠菜发芽、产量的影响。结果表明:菠菜百粒重小于1.0 g且芽率较高时使用孔径5 mm的播种轮可以得到理想的播种密度,而芽率偏低时需要使用孔径6 mm的播种轮;菠菜百粒重大于1.8 g时需要使用孔径7 mm的播种轮才能获得理想的播种密度。在1.5~3 cm的播种深度内,随着播种深度的增加,菠菜发芽时间延迟、发芽数量减少、产量降低,播种深度1.5 cm时发芽数最多、产量最高。综合比较,使用大粒种子时,1.5 cm是理想的播种深度,播种时还需要根据种子大小选用适宜的播种轮孔径才能得到适宜的密度。理论出苗数和实际出苗数的差别随种子大小、播种深度而变化,还需要进行深入研究。通过研究分析,以期为菠菜播种机械的广泛推广提供指导依据。

Diversity in seed oil content and fatty acid composition in Acer species with potential as sources of nervonic acid

Nervonic acid(NA,cis-15-tetracosenoic acid)is a very long-chain monounsaturated fatty acid that has been shown to be a core component of nerve fibers and nerve cells.It can be used to treat and prevent many neurological diseases.At present,commercially available NA is mainly derived from Acer truncatum seeds,which contain about 5%e6%NA in their seed oil.The aim of this study were to identify and analyze NA-containing Acer species that could be used as NA resource plants.For this purpose,46 Acer species seeds were collected in China and in some or all of the seed oils from these species 15 fatty acids were detected,including linoleic acid,oleic acid(C18:1D9,C18:1D11),erucic acid,palmitic acid,NA,linolenic acid(C18:3D6,9,12,C18:3D9,12,15),eicosenoic acid(C20:1D11,C20:1D13),stearic acid,behenic acid,tetracosanoic acid,arachidic acid,and docosadienoic acid.Nervonic acid was detected in all samples,but the content was highly variable among species.NA content over 9%was detected in eleven species,of which Acer elegantulum had the highest levels(13.90%).The seed oil content,seed weight,and fatty acid profiles varied among species,but the comprehensive evaluation value(W)showed that A.coriaceifolium could be a new potential NA resources plant.The results also showed that NA was significantly negatively correlated with palmitic acid,oleic acid,and eicosenoic acid,but positively correlated with eicosadienoic acid,behenic acid,erucic acid,and tetracosanoic acid,which indicate the probable pathway for NA biosynthesis in Acer plants.This study has identified Acer species that may serve as NA resources and will help guide subsequent species breeding programs.

Effects of grazing intensity on seed production of Caragana stenophylla along a climatic aridity gradient in the Inner Mongolia Steppe,China

As the increases of climatic aridity and grazing intensity, shrubs play an increasingly important role in grassland ecosystem in arid and semi-arid regions, and its abundance also generally increases. However, the effects of climatic aridity and grazing intensity on sexual reproduction of shrubs in grassland remain largely unclear. In order to understand the effects of grazing intensity and climatic drought stress, and their interaction on seed production of shrub species, we examined the seed number, seed weight and seed yield of Caragana stenophylla under three grazing intensities （fenced, mild grazing and severe grazing） across a climatic aridity gradient （semi-arid, arid, very arid and intensively arid zones） in the Inner Mongolia Steppe, northern China during 2012-2013. The seed number, seed weight and seed yield gradually increased from the semi-arid to the very arid zones, but decreased from the very arid to the intensively arid zones in fenced plots. The seed number and seed yield decreased from the semi-arid to the intensively arid zones in mild and severe grazing treatment plots, therefore, grazing enhanced the suppression effect of climatic aridity on seed production of C. stenophylla. The seed number and seed yield gradually decreased as grazing intensity increased. The seed weight was highest in severe grazing plots, followed by the mild grazing plots and then the fenced plots. Precipitation varied interannually during the study period. We observed that the seed number seed weight and seed yield were lower in the low precipitation year （2013） than in the high precipitation year （2012）. As climatic drought stress increased, the negative effects of grazing on seed production of C. stenophylla also gradually increased. Our results indicated that climatic drought stress may contribute to the encroachment of C. stenophylla shrub in arid zones by promoting its seed production. However, grazing had negative effects on sexual reproduction of C. stenophylla, and the combined effects of drought stress and grazing seriously suppressed sexual reproduction of C. stenophylla in the intensively arid zone.

芥菜型油菜AHK家族成员的全基因组鉴定及调控粒质量性状的初步探索

为探究芥菜型油菜AHK家族蛋白的结构特征及其与粒质量性状相关的调控功能,采用生物信息学方法对芥菜型油菜AHK家族成员进行了全基因组鉴定、理化性质、系统发育树、蛋白结构、启动子顺式元件和不同组织表达谱等分析。结果表明,在芥菜型油菜基因组中共鉴定到19条BjAHK蛋白序列,系统发育树分析将BjAHK家族成员分为了4个分支,即G1(BjAHK2~BjAHK4)、G2(BjAHK5)、G3(BjAHK1)和G4(BjCKI1)。功能结构域研究发现,BjAHK家族成员具有6个核心基序(Motif1~Motif6),与HisKA、HATPase_c和REC保守结构域相对应,其中BjAHK1~BjAHK4和BjCKI1蛋白的N端含有较为保守的跨膜结构域,可能与其跨膜作用功能有关。不同组织表达谱研究发现,细胞分裂素受体蛋白基因(BjAHK2~BjAHK4)为泛表达基因,且在根组织中表达量最高。结合已有的芥菜型油菜粒质量性状转录组数据,成功挖掘出4个可能参与粒质量性状调控的基因BjA07.AHK3、BjB03.AHK3、BjA10.AHK5和BjB05.AHK5。其中,BjA07.AHK3和BjB03.AHK3可能正向调控种子发育,而BjA10.AHK5和BjB05.AHK5则负向调控。结合BjAHK基因家族分析和转录组测序结果,推测BjAHK3和BjAHK5基因在调控芥菜型油菜种子发育方面发挥重要功能。

巢湖流域不同栽培方式对水稻产量和氮肥利用率及经济效益的影响

为探究巢湖流域水稻高产和肥料高效利用的栽培技术,以实现技术与肥料高效匹配。在巢湖流域进行田间试验,以直播稻乔两优17号和移栽稻全香优19为材料,设置不施肥、普通复合肥和新型肥料3个处理,比较分析两种不同栽培方式在相同施肥处理下水稻产量、氮肥利用率、水稻成本和品质。直播稻和移栽稻施用增效控失肥产量均为最高,可达到了10 000 kg·hm^(-2)以上。直播稻方式中,施用普通肥料(CF)和新型肥料(IF)均能显著提高水稻的氮肥利用率,其中,施用普通肥料提高了氮素累积和农学利用率,而新型肥料对生理利用率有积极影响。移栽稻方式中,施用增效控失肥能够降低肥料成本,并提高稻谷产值和利润,且利润可达25 527元·hm^(-2)。移栽稻施用增效控失肥在氮肥利用率和经济效益方面表现优异,可为巢湖流域栽培技术与肥料高效匹配提供参考依据。

濒危植物百山祖冷杉种子活力与种子物理指标相关性分析

【目的】探究百山祖冷杉种子的形态特征和物理指标与种子活力之间的关系,为研究该濒危物种种群更新、保护及解释其濒危机制和其种子生物学提供理论依据。【方法】以极度濒危植物百山祖冷杉为材料,测定百山祖冷杉种子长度、宽度、厚度、单粒重、RGB值等特征常数,用TTC染色法测定其种子活力,并建立数学模型。【结果】百山祖冷杉有活力和无活力种子的R、B值和单粒重存在显著差异,单因素线性回归分析表明,百山祖冷杉种子活力与种子单粒重显著相关,R2为0.839;双因素线性回归分析表明,种子活力与种子单粒重和宽度显著相关,R2为0.895;多因素线性回归分析,R2为0.978;但其有活力种子和无活力种子长度、宽度、厚度和G值不存在显著差异。【结论】用百山祖冷杉种子的R、B值和单粒重等指标可筛选具有较高种子活力的种子,用多因素线性回归分析可更精确分析其种子活力。

Variability in cone and seed characteristics and seed testing in various provenances of Himalayan spruce (Picea smithiana)

Abstract： We investigated the effect of provenance variation on cone and seed morphology and germination behaviunr under different pretreatments in Picea smithiana. Three categories of cones were recognized： large （13.18 cm×3.30 cm-15.42 cm × 4.35 cm）, medium （10.85 cm × 3.93 cm-12.18 cm× 3.98 cm） and small （7.69 cm× 3.06-10.98 cm× 3.39 cm）. Significant variation was observed for various cone and seed characteristics. Seeds moisture content varied from 44.48% to 56.91%, seed size from 0.64 cmx 0.31 cm （largest seed） to 0.49 cm× 0.10 cm （smallest seed）, and the seed weight from 2.45 g per 100 seeds to 1.36 g per 100 seeds on fresh weight basis of P. smithiana. The highest seed germination （72.0±7.53%） at 10℃ was observed under chilling treatment in Tapovan provenance, while the minimum （15.0±5.71%） seed germination was recorded at 25℃ under control set in Tapovan provenance. Further among all the sources Pandukeshwar consistently had the highest average （38%） percent germination across all treatments. Compared to other temperature regimes and pretreatments, seeds subjected to 10℃ and chilling treatment had the highest germination and took the least time for germination irrespective of provenance effects. Altitude （provenance） had little or no relationship with germination following laboratory treatments. Seeds from Tapovan provenance subjected at 10℃ following chilling treatment may be suggested for further multiplication of this species.

Extra-Large Seed Germplasms of Brassica Napus Created through Microspore Culture

Thousand Seed Weight （TSW） is one of the major yield components of rapeseed （Brassica napus L.）. Here reports an extra-large seed germplasm GM01 which was obtained through isolated microspore culture. Three-way cross was made： H8--a Yunnan spring early-maturing rapeseed variety, ＂Legeney＂--a Canadian canola variety and ＂020010＂--a semi-winter late-maturing rapeseed variety. One hundred and forty eight doubled hyploid lines were obtained from the F~ plants of three-way cross through isolated mierospore culture. Among them, the TSW of GM01 amounted to 8.68 g and the TSW of 53 lines were above 5.0 g. The TSWs of GM01 were relatively stable among the multi-location field trials from 2007 to 2014 with variation being only 10%-15% among the locations and years. Compared with H8, GM01 had larger flowers, stigmas, siliques and seed diameters, but less branches, siliques per plant and seeds per silique.

基于数据库挖掘、网络药理学和试验验证探究葡萄籽多酚缓解溃疡性结肠炎的作用机制

本试验旨在探究葡萄籽多酚缓解葡聚糖硫酸钠诱导的溃疡性结肠炎的机制。试验选用GEO数据库中的两个数据集,使用sva包对其进行整合后,通过加权基因共表达网络的方法鉴定与溃疡性结肠炎相关的基因模块,并利用基因本体论数据库和京都基因与基因组百科全书数据库对基因功能进行注释;使用液相色谱-质谱联用技术检测葡萄籽多酚主要成分,选取含量前10的成分,利用TCMSP、DrugBank、SwissTargetPrediction等数据库和靶点预测工具获取其作用的靶点,并与先前鉴定的溃疡性结肠炎相关模块的基因取交集,使用蛋白质-蛋白质相互作用网络分析,得到葡萄籽多酚缓解溃疡性结肠炎的关键靶点;使用分子对接验证葡萄籽多酚主要成分与关键靶点的连接活性;利用Raw264.7细胞系,通过荧光定量PCR鉴定葡萄籽多酚对关键靶点基因的影响。结果表明,有3个基因模块与溃疡性结肠炎表型高度相关,且模块内的基因主要与炎症反应、细胞周期和代谢、细胞的趋化等功能相关;葡萄籽多酚的主要成分分别为儿茶素、表儿茶素、原花青素B2、刺柄芒花素等,这些成分作用的靶点基因与溃疡性结肠炎相关模块的基因有15个相同,而在这15个基因中,前列腺素内过氧化物合酶2、基质金属酶9、白介素-6、肿瘤坏死因子-α因为在网络中有最高的连接度而被认为是关键靶点;葡萄籽多酚含量最高的10个成分与这4个靶点均具有良好的连接活性,荧光定量PCR也证明了葡萄籽多酚能影响这4个靶点基因的表达。综上,葡萄籽多酚最有可能通过影响前列腺素内过氧化物合酶成2、基质金属酶9、白介素-6和肿瘤坏死因子-α来缓解溃疡性结肠炎。