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Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Journal of Integrative Agriculture 2024年第5期23卷 1557-1567页

作者： Jiali Ying Yan Wang Liang Xu Tiaojiao Qin Kai Xia Peng Zhang Yinbo Ma Keyun Zhang Lun Wang Junhui Dong lianxue fan Yuelin Zhu Liwang Liu National Key Laboratory of Crop Genetics&Germplasm Enhancement and Utilization/Key Laboratory of Horticultural Crop Biology and Genetic Improvement(East China)of Ministry of Agriculture and Rural Affairs/College of Horticulture Nanjing Agricultural UniversityNanjing210095China College of Horticulture and Landscape Architecture Yangzhou UniversityYangzhou 225009China College of Life Sciences Nanjing Agricultural UniversityNanjing 210095China

Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

关键词： Raphanus sativus L. VIGS CRISPR/Cas9 Agrobacterium rhizogenes A.tumefaciens RsPDS

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A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L.

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Horticulture Research 2020年第1期7卷 793-805页

作者： lianxue fan YanWang Liang Xu Mingjia Tang Xiaoli Zhang Jiali Ying Cui Li Junhui Dong Liwang Liu National Key Laboratory of Crop Genetics and Germplasm Enhancement Key Laboratory of Horticultural Crop Biology and Genetic Improvement(East China)of MOARCollege of HorticultureNanjing Agricultural University210095 NanjingPR China

Radish(Raphanus sativus L.)taproot contains high concentrations of flavonoids,including anthocyanins(ATCs),in redskinned genotypes.However,little information on the genetic regulation of ATC biosynthesis in radish is available.A genome-wide association study of radish red skin color was conducted using whole-genome sequencing data derived from 179 radish genotypes.The R2R3-MYB transcription factor production of anthocyanin pigment 2(PAP2)gene was found in the region associated with a leading SNP located on chromosome 2.The amino acid sequence encoded by the RsPAP2 gene was different from those of the other published RsMYB genes responsible for the red skin color of radish.The overexpression of the RsPAP2 gene resulted in ATC accumulation in Arabidopsis and radish,which was accompanied by the upregulation of several ATC-related structural genes.RsPAP2 was found to bind the RsUFGT and RsTT8 promoters,as shown by a dual-luciferase reporter system and a yeast one-hybrid assay.The promoter activities of the RsANS,RsCHI,RsPAL,and RsUFGT genes could be strongly activated by coinfiltration with RsPAP2 and RsTT8.These findings showed the effectiveness of GWAS in identifying candidate genes in radish and demonstrated that RsPAP2 could(either directly or together with its cofactor RsTT8)regulate the transcript levels of ATC-related genes to promote ATC biosynthesis,facilitating the genetic enhancement of ATC contents and other related traits in radish.

关键词： Raphanus critical finding

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RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.)

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Horticulture Research 2023年第3期10卷 223-234页

作者： Cui Li Baozhen Mao Kai Wang Liang Xu lianxue fan Yan Wang Ying Li Yinbo Ma Lun Wang Liwang Liu National Key Laboratory of Crop Genetics&Germplasm Enhancement and utilization Key Laboratory of Horticultural Crop Biology and Genetic Improvement(East China)of MOARCollege of HorticultureNanjing Agricultural UniversityNanjing 210095China College of Horticulture and Landscape Architecture Yangzhou UniversityYangzhou 225009China

The growth and development of taproots are inhibited by cold stress in radish(Raphanus sativus L.).Ethylene-responsive element binding factors(ERF)are key participators in the cold stress response and growth regulation of plants.However,the function of ERF genes in cold tolerance and root development in radish remains elusive.Here,we showed that the secondary growth of radish taproots was inhibited by cold stress.Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation.The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved.Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish.Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots.RT-qPCR analysis showed that the expression of the cold-related genes(CORs)RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings.Yeast one-hybrid(Y1H)and dual-luciferase reporter assays(DLA)revealed that RsERF40 directly regulates RsCOR78,RsCOR413PM1,RsCESA6 and RsEXPB3 expression,illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish.In this study,the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth.These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

关键词： stress Raphanus roots

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Comparative proteomic analysis provides insight into a complex regulatory network of taproot formation in radish (Raphanus sativus L.)

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Horticulture Research 2018年第1期5卷 249-262页

作者： Yang Xie Liang Xu Yan Wang lianxue fan Yinglong Chen Mingjia Tang Xiaobo Luo Liwang Liu National Key Laboratory of Crop Genetics and Germplasm Enhancement Key Laboratory of Horticultural Crop Biology and Genetic Improvement(East China)of MOACollege of HorticultureNanjing Agricultural UniversityNanjing210095PR China The UWA Institute of Agriculture UWA School of Agriculture and EnvironmentThe University of Western AustraliaPerthWA6001Australia

The fleshy taproot of radish is an important storage organ determining its yield and quality.Taproot thickening is a complex developmental process in radish.However,the molecular mechanisms governing this process remain unclear at the proteome level.In this study,a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach.In total,1862 differentially expressed proteins(DEPs)were identified from 6342 high-confidence proteins,among which 256 up-regulated proteins displayed overlapped accumulation in S1(pre-cortex splitting stage)vs.S2(cortex splitting stage)and S1 vs.S3(expanding stage)pairs,whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs.S3 and S2 vs.S3 pairs.Gene Ontology(GO)and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as“starch and sucrose metabolism”,“plant hormone signal transduction”,and“biosynthesis of secondary metabolites”.A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels.Furthermore,association analysis showed that 187,181,and 96 DEPs were matched with their corresponding differentially expressed genes(DEGs)in S1 vs.S2,S1 vs.S3,and S2 vs.S3 comparison,respectively.Notably,several functional proteins including cell division cycle 5-like protein(CDC5),expansin B1(EXPB1),and xyloglucan endotransglucosylase/hydrolase protein 24(XTH24)were responsible for cell division and expansion during radish taproot thickening process.These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening,and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops.

关键词： metabolism analysis Raphanus

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黄瓜氮素胁迫相关基因CsAHP1的克隆及功能分析

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河南农业科学 2018年第6期47卷 92-97页

作者：杜亚琳陈海燕徐丽琳王琛范莲雪农业部东北地区园艺作物生物学与种质创制重点实验室/东北农业大学园艺园林学院黑龙江哈尔滨150030 河南省农业科学院农业经济与信息研究所河南郑州450002

为研究黄瓜耐低氮素胁迫的生理与分子机制,克隆了1个黄瓜氮素胁迫相关基因CsAHP1,其cDNA全长为462 bp。序列分析表明,CsAHP1基因在不同物种中高度保守。进化树分析结果表明,CsAHP1蛋白与甜瓜的AHP蛋白亲缘关系较近。qRT-PCR结果表明,CsA... 详细信息

为研究黄瓜耐低氮素胁迫的生理与分子机制,克隆了1个黄瓜氮素胁迫相关基因CsAHP1,其cDNA全长为462 bp。序列分析表明,CsAHP1基因在不同物种中高度保守。进化树分析结果表明,CsAHP1蛋白与甜瓜的AHP蛋白亲缘关系较近。qRT-PCR结果表明,CsAHP1基因在低浓度氮素条件下表达量较高。功能分析结果表明,低浓度氮素胁迫条件下拟南芥Col-0植株根长明显变短,其质量也显著降低,而35S∶∶CsAHP1转基因拟南芥株系的根长缩短程度和质量下降率都显著小于Col-0植株,表明CsAHP1过表达拟南芥转基因植株表现出比Col-0植株更强的耐氮素胁迫能力。综上所述,黄瓜CsAHP1基因可以提高35S∶∶CsAHP1拟南芥过表达转基因株系的氮素胁迫能力。

关键词：拟南芥 CsAHP 1 黄瓜低氮素胁迫

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不同种质萝卜肉质根硫苷组分及含量分析

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南京农业大学学报 2019年第3期42卷 413-420页

作者：公茂勇王燕徐良王娟娟谢洋范莲雪陈微柳李旺南京农业大学作物遗传与种质创新国家重点实验室/农业农村部华东地区园艺作物生物学与种质创制重点实验室/园艺学院江苏南京210095 全国农业技术推广服务中心北京100125

[目的]本文旨在检测萝卜肉质根中硫苷组分与含量,分析硫苷总量与单个硫苷组分含量的相关性,为萝卜种质品质性状评价与优良新品种培育提供理论基础。[方法]通过液质联用(LC-MS)技术和高效液相色谱法(HPLC)对44份萝卜种质肉质根中硫苷组... 详细信息

[目的]本文旨在检测萝卜肉质根中硫苷组分与含量,分析硫苷总量与单个硫苷组分含量的相关性,为萝卜种质品质性状评价与优良新品种培育提供理论基础。[方法]通过液质联用(LC-MS)技术和高效液相色谱法(HPLC)对44份萝卜种质肉质根中硫苷组分与含量进行检测,并通过主成分分析研究了总硫苷与单个硫苷含量之间的相关性。[结果]在萝卜肉质根中共检测到12种硫苷,包括8种脂肪族硫苷和4种吲哚族硫苷,其中4-甲硫基-3-丁烯基硫苷(GRH)作为重要的硫苷组分在所有材料中含量最高。44个萝卜种质干样总硫苷平均含量10.73μmol·g^(-1),变化区间1.61～23.23μmol·g^(-1);硫苷含量占干质量的平均比例为0.4%,变化区间为0.14%～0.97%。筛选到4份4-甲基亚磺酰丁基硫苷(RAA)含量较高的种质,4份GRH含量较低的种质。分析单一硫苷相关系数矩阵显示,总硫苷含量与4-甲基亚磺酰基-3-丁烯基硫苷(GRE)、4-戊烯基硫苷(GBN)、4-甲硫基丁基硫苷(GER)的含量呈极显著正相关,且同族硫苷间相关性较强。[结论]萝卜不同种质肉质根中硫苷组分与含量存在较大差异,其中GRH是影响脂肪族硫苷及总硫苷含量差异的关键硫苷,筛选到的高含量RAA与低含量GRH种质可为萝卜品质性状遗传改良提供重要材料。

关键词：萝卜肉质根硫苷组分含量 LC-MS

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痛风宁胶囊对大鼠急性痛风性关节炎的抗炎作用及其机制

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吉林大学学报（医学版） 2018年第2期44卷 270-274页

作者：樊海瑞赵岩傅警龙张海波王德超郜玉钢张连学吉林农业大学中药材学院人参工程技术研究中心吉林长春130118 吉林省长春市长中风湿骨病医院风湿科吉林长春130000

目的:探讨痛风宁胶囊(TFN)对大鼠急性痛风性关节炎模型的影响,阐明TFN对急性痛风性关节炎大鼠的治疗作用及机制。方法:采用穿刺法对大鼠踝关节注射尿酸钠,建立大鼠急性痛风性关节炎模型。将48只大鼠分为空白对照组,模型组,低(100mg·kg... 详细信息

目的:探讨痛风宁胶囊(TFN)对大鼠急性痛风性关节炎模型的影响,阐明TFN对急性痛风性关节炎大鼠的治疗作用及机制。方法:采用穿刺法对大鼠踝关节注射尿酸钠,建立大鼠急性痛风性关节炎模型。将48只大鼠分为空白对照组,模型组,低(100mg·kg-1)、中(200mg·kg-1)和高(550mg·kg-1)剂量TFN组,秋水仙碱(0.63mg·kg-1)阳性药对照组,每组8只,均采用灌胃给药。造模后检测不同时间段大鼠踝关节肿胀度,检测各组大鼠血液中白细胞、淋巴细胞和中性粒细胞数量,检测各组大鼠血清及踝关节组织匀浆中一氧化氮(NO)水平及血清中尿酸(UA)水平,测定各组大鼠踝关节周围软组织及关节液中白细胞介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平。结果:与模型组比较,在0~8h内阳性药对照组和高剂量TFN组大鼠踝关节肿胀度明显降低(P0.05)。与模型组比较,阳性药对照组和不同剂量TFN组大鼠血液中白细胞、淋巴细胞和中性粒细胞数量降低,其中高剂量TFN组最为明显(P0.05)。与模型组比较,阳性药对照组和高剂量TFN组大鼠血清中UA水平明显降低(P0.05)。结论:TFN治疗痛风性关节炎疗效显著,其作用机制可能与抑制炎症反应有关。

关键词：痛风宁胶囊痛风性关节炎尿酸钠炎症因

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痛风宁胶囊对小鼠高尿酸血症和肾脏功能的改善作用

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吉林大学学报（医学版） 2017年第5期43卷 893-896,I0003页

作者：樊海瑞赵岩傅警龙张海波王德超郜玉钢张连学吉林农业大学中药材学院人参工程技术研究中心吉林长春130118 长中风湿骨病医院风湿科吉林长春130000

目的:探讨痛风宁胶囊(TFN)对高尿酸血症模型小鼠血清中尿酸(UC)、肌酐(Cr)和尿素氮(BUN)水平以及小鼠肝脏匀浆液中黄嘌呤氧化酶(XOD)和腺苷脱氨酶(ADA)活性的影响,观察TFN对高尿酸小鼠肾脏组织病理学变化的改善作用,阐明其作用机制。方... 详细信息

目的:探讨痛风宁胶囊(TFN)对高尿酸血症模型小鼠血清中尿酸(UC)、肌酐(Cr)和尿素氮(BUN)水平以及小鼠肝脏匀浆液中黄嘌呤氧化酶(XOD)和腺苷脱氨酶(ADA)活性的影响,观察TFN对高尿酸小鼠肾脏组织病理学变化的改善作用,阐明其作用机制。方法:采用酵母膏合并氧嗪酸钾盐诱导建立小鼠高尿酸血症模型。将70只小鼠分为空白对照组,模型组,低(200mg·kg^(-1))、中(400mg·kg^(-1))和高剂量(800mg·kg^(-1))TFN组,别嘌醇(50 mg·kg^(-1))和痛风舒(TFS,600 mg·kg^(-1))阳性药对照组,每组10只,灌胃给药。实验结束后检测小鼠血清中UC、Cr和BUN水平以及小鼠肝脏匀浆液中XOD和ADA活性,HE染色观察小鼠肾脏组织病理表现。结果:与空白对照组比较,模型组小鼠血清中UC、Cr和BUN水平明显升高(P<0.01),肝组织中XOD和ADA活性亦升高(P<0.01);与模型组比较,阳性药对照组,低、中和高剂量TFN组小鼠血清中UC、Cr和BUN水平明显降低(P<0.01),肝组织中XOD和ADA活性降低(P<0.05或P<0.01),其中高剂量TFN组效果最为明显。光镜下观察,与模型组比较,阳性药对照组和高剂量TFN组小鼠肾脏肾小球萎缩、肾间质纤维化和肾小管扩张等病理变化得到一定程度上改善。结论:TFN对小鼠高尿酸血症具有一定的改善作用,其机制与抑制UC生成和促进UC排泄有关。

关键词：痛风宁胶囊高尿酸血症酵母膏氧嗪酸钾盐

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痛风宁胶囊对痛风性肾病模型小鼠主要脏器病理损伤的改善作用

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吉林大学学报（医学版） 2018年第4期44卷 741-745,892页

作者：樊海瑞赵岩傅警龙张海波王德超郜玉钢张连学吉林农业大学中药材学院人参工程技术研究中心吉林长春130118 吉林省长春市长中风湿骨病医院风湿科吉林长春130021

目的:探讨痛风宁胶囊(TFN)对痛风性肾病模型小鼠血清中尿酸(UA)、肌酐(Cr)、单核趋化蛋白(MCP-1)、肿瘤坏死因子(TNF-α)、黄嘌呤氧化酶(XOD)和尿素氮(BUN)水平以及小鼠肾组织匀浆上清液中环氧化酶2(COX-2)活性的影响,分析其对痛风性肾... 详细信息

目的:探讨痛风宁胶囊(TFN)对痛风性肾病模型小鼠血清中尿酸(UA)、肌酐(Cr)、单核趋化蛋白(MCP-1)、肿瘤坏死因子(TNF-α)、黄嘌呤氧化酶(XOD)和尿素氮(BUN)水平以及小鼠肾组织匀浆上清液中环氧化酶2(COX-2)活性的影响,分析其对痛风性肾病小鼠肾脏、脾脏和胸腺病理变化的改善作用。方法:采用腺嘌呤联合乙胺丁醇片制备小鼠痛风性肾病模型。将70只小鼠随机分为对照组,模型组,低(200mg·kg-1)、中(400mg·kg-1)和高(800mg·kg-1)剂量TFN组,别嘌醇片(50mg·kg-1)和痛风舒片(TFS)(600mg·kg-1)阳性药对照组,每组10只。实验结束后检测各组小鼠血清中UA、Cr、MCP-1、TNF-α、XOD和BUN水平及小鼠肾组织匀浆上清液中COX-2活性,HE染色观察小鼠肾脏、脾脏和胸腺组织病理形态表现。结果:与对照组比较,模型组小鼠血清中UA、Cr、BUN、MCP-1、TNF-α和XOD水平明显升高(P0.05)。与对照组比较,模型组小鼠肾小球萎缩,肾小管和肾间质出现大量的尿酸盐结晶;胸腺皮质和髓质分界不清晰,细胞稀疏;脾脏红髓与白髓分界不清晰,脾小结不同程度缩小,脾窦出现充血现象。与模型组比较,阳性药对照组和各剂量TFN组小鼠肾脏肾小球萎缩、脾脏脾窦充血和胸腺皮质与髓质分界不清等病理变化均有一定程度改善。结论:TFN对于痛风性肾病引起的小鼠脏器病变及各生化指标异常有一定的改善作用。

关键词：痛风宁胶囊痛风性肾病腺嘌呤乙胺丁醇片

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