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Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

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Horticultural Plant Journal 2024年第2期10卷 450-460页

作者： Huiyu Wang Yushan Zheng Qian Zhou Ying Li Tongkun Liu Xilin Hou State key laboratory of Crop Genetics&germplasm enhancement Key Laboratory of Biology and Genetic Improvement of Horticultural Crops(East China)Ministry of Agriculture and Rural Affairs of ChinaEngineering Research Center of Germplasm Enhancement and Utilization of Horticultural CropsMinistry of Education of ChinaNanjing Agricultural UniversityNanjingJiangsu 210095China Nanjing Suman Plasma Engineering Research Institute Nanjing Agricultural UniversityNanjingJiangsu 210095China

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu *** one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

关键词： Agrobacterium rhizogenes Non-heading Chinese cabbage Transgenic roots Composite plant Transformation efficiency

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Utilizing resequencing big data to facilitate Brassica vegetable breeding:tracing introgression pedigree and developing highly specific markers for clubroot resistance

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Horticultural Plant Journal 2024年第3期10卷 771-783页

作者： Zhiyong Ren Jinquan Li Xingyu Zhang Xingxu Li Junhong Zhang Zhibiao Ye Yuyang Zhang Qijun Nie key laboratory of Vegetable Ecological Cultivation on Highland Ministry of Agriculture and Rural AffairsHubei Key Laboratory of Vegetable Germplasm Enhancement and Genetic ImprovementInstitute of Economic CropsHubei Academy of Agricultural SciencesWuhanHubei 430063China National key laboratory for germplasm Innovation and Utilization of Horticultural Crops Huazhong Agricultural UniversityWuhanHubei 430070China Vegetable Research Institute Wuhan Academy of Agricultural SciencesWuhanHubei 430345China Hubei Hongshan laboratory WuhanHubei 430070China Shenzhen Institute of Nutrition and Health Huazhong Agricultural UniversityWuhanHubei 430070China Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhenGuangdong 518000China

Clubroot caused by Plasmodiophora brassicae is a devastating disease of Cruciferous *** cultivars with clubroot resistance(CR)is the most effective control *** the two major Brassica vegetable species *** and ***,several commercial cultivars with unclear CR pedigrees have been intensively used as CR donors in ***,the continuous occurrence of CR-breaking makes the CR pedigree underlying these cultivars one of the breeders'most urgent *** complex intraspecific diversity of these two major Brassica vegetables has also limited the applicability of CR markers in different breeding *** we first traced the pedigree underlying two kinds of CR that have been widely applied in breeding by linkage and introgression analyses based on public resequencing *** ***,a major locus CRzi8 underlying the CR of the commercial CR donor‘DegaoCR117’was ***8 was further shown to have been introgressed from turnip(*** ***)and that it carried a potential functional allele of *** turnip introgression carried CRb^(c),sharing the same coding sequence with the CRb that was also identified from chromosome C07 of *** CR cultivars with different *** natural populations,variation analysis of linkage intervals of CRzi8,PbBa8.1,CRb,and CRb^(c)yielded easily resolved InDel markers(>20 bp)for these fundamental CR *** specificity of these markers was tested in diverse cultivars panels,and each exhibited high reliability in *** research demonstrates the value of the practice of applying resequencing big data to solve urgent concerns in breeding programs.

关键词： Brassica Clubroot resistance Resequencing Introgression analysis Molecular marker

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Exogenous Spermidine Promotes Somatic Embryogenesis of Cunninghamia lanceolata by Altering the Endogenous Phytohormone Content

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Phyton-International Journal of Experimental Botany 2020年第1期89卷 27-34页

作者： Dandan Wang Yulin Guo Xiaofei Long Yan Pan Dingjie Yang Rui Li Ye Lu Ying Chen Jisen Shi Jinhui Chen key laboratory of Forest Genetics&Biotechnology of Ministry of Education of China Co-Innovation Center for Sustainable Forestry in Southern ChinaNanjing Forestry UniversityNanjing210037China jiangsu key laboratory for poplar germplasm enhancement and variety improvement Nanjing Forestry University Nanjing210037China

In order to study how exogenous hormones in ***(gymnosperm)regulate somatic embryogenesis,we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic *** applying exogenous spermidine,we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone *** on these results,further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic *** research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.

关键词： Spermidine C.lanceolata somatic embryogenesis endogenous phytohormones

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EoPHR2,a Phosphate Starvation Response Transcription Factor,Is Involved in Improving Low-Phosphorus Stress Resistance in Eremochloa ophiuroides

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Phyton-International Journal of Experimental Botany 2022年第3期91卷 651-665页

作者： Ying Chen Chuanqiang Liu Qingqing He Jianjian Li Jingjing Wang Ling Li Xiang Yao Shenghao Zhou Haoran Wang poplar germplasm enhancement&variety improvement laboratory Nanjing Forestry UniversityNanjing210037China Institute of Botany Jiangsu Province and Chinese Academy of SciencesNanjing210014China Taixing Rural Energy Technology Development Service Station Taixing225400China

As a macronutrient,Phosphorus(P)takes many roles in plant growth and *** should be significant to explore the molecular mechanism of low-phosphorus stress response of *** starvation response(PHR)transcription factors play important roles in response to phosphorus deficiency stress in *** this study,we isolated a gene related to the plant phosphorus signaling system from the acid-soil-resistant centipedegrass(Eremochloa ophiuroides[Munro]Hack.),termed *** subcellular localization of EoPHR2 protein was observed to be nuclear *** expression patterns of EoPHR2 in different tissues and Al/Pi-stress conditions were analyzed by qRT-PCR,they suggested a potential role in response to the multiple-stress under acid soil *** on the functional identification through transgenic plants,we found that(1)EoPHR2 is involved in the Pi-signaling pathway,and(2)overexpression of EoPHR2 mimics Pi-starvation signalling resulting on enhanced roots whether under Pi-deficiency stress or *** conclusion,EoPHR2 transcription factor plays a role in response to the multiple stresses under acid soil conditions,improving the low-phosphorus stress resistance of Eremochloa ophiuroides.

关键词： Phosphate starvation response transcription factor phosphorus signaling system acid soil adaptability Eremochloa ophiuroides

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Isolation and identification of small species-specific secreted proteins of Marssonina brunnea

Isolation and identification of small species-specific secre...

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2013全国植物生物学大会

作者： CHENG Qiang CAO Youzhi HUANG Minren jiangsu key laboratory for poplar germplasm enhancement and variety improvement Nanjing Forestry University

Fungal plant pathogens produce proteinaceous effectors that can increase their pathogenicity. These fungal effectors are frequently small secreted proteins with limited phylogenetic *** a proteomics approach for analyzing the secretome is a promising strategy for the identification of effector proteins of phytopathogenic ***,the primary limitation of this strategy is the low identification rate of spectrometry-based proteomic methods that rely heavily on the accuracy and integrity of the reference sequence database. Marssonina brunnea is an important fungal pathogen of the Populus *** further our understanding of the pathogenesis of ***,we initiated a proteome-level study of the fungal *** de novo peptide sequencing by MS/MS,we obtained peptide sequences for 32 protein *** proteins were identified by sequence homology to conserved proteins in public databases using MS-driven *** identify additional protein spots,we combined a degenerate PCR method,based on the Consensus-Degenerate Hybrid Oligonucleotide Primer(CODEHOP) method,and a rapid amplification of cDNA ends method to clone the full-length cDNA fragments encoding the proteins identified in the gel. Using this method,we cloned the full-length cDNA fragments encoding 9 putative Lysm effector *** method provides an efficient approach to identification of species specific effector proteins of non-sequenced fungal organisms.

关键词： effector Consensus-Degenerate Hybrid Oligonucleotide Primer degenerate PCR

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PbrRALF2-elicited reactive oxygen species signaling is mediated by the PbrCrRLK1L13-PbrMPK18 module in pear pollen tubes

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Horticulture Research 2021年第1期8卷 3042-3054页

作者： Xiaobing Kou Jiangmei Sun Peng Wang Danqi Wang Peng Cao Jing Lin Youhong Chang Shaoling Zhang Juyou Wu Center of Pear Engineering Technology Research State Key Laboratory of Crop Genetics and Germplasm EnhancementCollege of HorticultureNanjing Agricultural University210095 NanjingChina jiangsu key laboratory for Horticultural Crop Genetic improvement 210014 NanjingChina

Rapid alkalinization factors(RALFs)are cysteine-rich peptides that play important roles in a variety of biological processes,such as cell elongation and immune *** studies in Arabidopsis have shown that RALFs regulate pollen tube growth via plasma membrane receptor-like kinases(RLKs).However,the downstream signal transduction mechanisms of RLKs in pollen tubes are ***,we identified PbrRALF2,a pear(Pyrus bretschneideri)pollen RALF peptide that inhibits pollen tube *** found that PbrRALF2 interacts with a malectin-like domain-containing RLK,*** relative affinity between PbrRALF2 and PbrCrRLK1L13 was at the submicromolar level,which is consistent with the values of ligand–receptor kinase pairs and the physiological concentration for PbrRALF2-mediated inhibition of pollen tube *** binding to its extracellular domain,PbrRALF2 activated the phosphorylation of PbrCrRLK1L13 in a dose-dependent *** further showed that the MAP kinase PbrMPK18 is a downstream target of PbrCrRLK1L13 that mediates PbrRALF2-elicited reactive oxygen species(ROS)*** excessive accumulation of ROS inhibits pollen tube *** show that MPK acts as a mediator for CrRLK1L to stimulate ROS production,which might represent a general mechanism by which RALF and CrRLK1L function in signaling pathways.

关键词： RALF reactive alkali

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Molecular Characterization and Expression Profiles of Myrosinase Gene(RsMyr2) in Radish(Raphanus sativus L.)

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Journal of Integrative Agriculture 2014年第9期13卷 1877-1888页

作者： PAN Yan XU Yuan-yuan ZHU Xian-wen LIU Zhe GONG Yi-qin XU Liang GONG Mao-yong LIU Li-wang National key laboratory of Crop Genetics and germplasm enhancement/Engineering Research Center of Horticultural Crop germplasm enhancement and Utilization Ministry of Education/Key Laboratory of Horticultural Crop Biology and Genetic Improvement (East China)Ministry of Agriculture/College of HorticultureNanjing Agricultural University Department of Plant Sciences North Dakota State UniversityFargoND 58105USA

Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish（Raphanus sativus）. In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5′-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages（e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage）. Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid（ABA）, methyl jasmonate（MeJA） and hydrogen peroxide（H2O2）, whereas it was down-regulated by wounding（WO） treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.

关键词： radish RsMyr2 promoter heterologous expression qRT-RCR

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Insights into ionic association boosting water oxidation activity and dynamic stability

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Journal of Energy Chemistry 2024年第2期89卷 99-109,I0004页

作者： Zanling Huang Shuqi Zhu Yuan Duan Chaoran Pi Xuming Zhang Abebe Reda Woldu Jing-Xin Jian Paul K.Chu Qing-Xiao Tong Liangsheng Hu Xiangdong Yao Department of Chemistry and key laboratory for Preparation and Application of Ordered Structural Materials of Guangdong Province Shantou UniversityShantou 515063GuangdongChina Chemistry and Chemical Engineering Guangdong laboratory Shantou 515063GuangdongChina State key laboratory of Refractories and Metallurgy and Institute of Advanced Materials and Nanotechnology Wuhan University of Science and TechnologyWuhan 430081HubeiChina Department of Physics Department of Materials Science and Engineeringand Department of Biomedical EngineeringCity University of Hong KongTat Chee AvenueKowloonHong KongChina School of Advanced Energy Sun Yat-sen University(Shenzhen)Shenzhen 518107GuangdongChina

There have been reports about Fe ions boosting oxygen evolution reaction(OER)activity of Ni-based catalysts in alkaline conditions,while the origin and reason for the enhancement remains ***,we attempt to identify the activity improvement and discover that Ni sites act as a host to attract Fe(Ⅲ)to form Fe(Ni)(Ⅲ)binary centres,which serve as the dynamic sites to promote OER activity and stability by cyclical formation of intermediates(Fe(Ⅲ)→Fe(Ni)(Ⅲ)→Fe(Ni)-OH→Fe(Ni)-O→Fe(Ni)OOH→Fe(Ⅲ))at the electrode/electrolyte interface to emit O_(2).Additionally,some ions(Co(Ⅱ),Ni(Ⅱ),and Cr(Ⅲ))can also be the active sites to catalyze the OER process on a variety of *** Fe(Ⅲ)-catalyzed overall water-splitting electrolyzer comprising bare Ni foam as the anode and Pt/Ni-Mo as the cathode demonstrates robust stability for 1600 h at 1000 mA cm^(-2)@~1.75 *** results provide insights into the ioncatalyzed effects boosting OER performance.

关键词： Oxygen evolution reaction Fe(Ⅲ)-catalysis Ni-Fe binary active centers Ion-catalyzed effects Robust stability

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ESCRT-Ⅲ component OsSNF7.2 modulates leaf rolling by trafficking and endosomal degradation of auxin biosynthetic enzyme OsYUC8 in rice

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Journal of Integrative Plant Biology 2023年第6期65卷 1408-1422页

作者： Liang Zhou Saihua Chen Maohong Cai Song Cui Yulong Ren Xinyue Zhang Tianzhen Liu Chunlei Zhou Xin Jin Limin Zhang Minxi Wu Shuyi Zhang Zhijun Cheng Xin Zhang Cailin Lei Qibing Lin Xiuping Guo Jie Wang Zhichao Zhao Ling Jiang Shanshan Zhu Jianmin Wan State key laboratory for Crop Genetics and germplasm enhancement Jiangsu Plant Gene Engineering Research CenterNanjing Agricultural UniversityNanjing 210095China National key Facility for Crop Gene Resources and Genetic improvement Institute of Crop SciencesChinese Academy of Agricultural SciencesBeijing 100081China jiangsu key laboratory of Crop Genomics and Molecular Breeding/key laboratory of Plant Functional Genomics of the Ministry of Education/jiangsu key laboratory of Crop Genetics and Physiology Agricultural College of Yangzhou UniversityYangzhou 225009China

The endosomal sorting complex required for transport(ESCRT)is highly conserved in eukaryotic cells and plays an essential role in the biogenesis of multivesicular bodies and cargo degradation to the plant vacuole or *** ESCRT components affect a variety of plant growth and development processes,their impact on leaf development is rarely ***,we found that OsSNF7.2,an ESCRT-Ⅲ component,controls leaf rolling in rice(Oryza sativa).The Ossnf7.2 mutant rolled leaf 17(rl17)has adaxially rolled leaves due to the decreased number and size of the bulliform ***7.2is expressed ubiquitously in all tissues,and its protein is localized in the endosomal ***7.2 homologs,including OsSNF7,OsSNF7.3,and OsSNF7.4,can physically interact with OsSNF7.2,but their single mutation did not result in leaf *** ESCRT complex subunits,namely OsVPS20,OsVPS24,and OsBRO1,also interact with *** assays revealed that OsSNF7.2 interacts with OsYUC8 and aids its vacuolar *** Osyuc8and rl17 Osyuc8 showed rolled leaves,indicating that OsYUC8 and OsSNF7.2 function in the same pathway,conferring leaf *** study reveals a new biological function for the ESCRT-Ⅲcomponents,and provides new insights into the molecular mechanisms underlying leaf rolling.

关键词： bulliform cell leaf rolling OsSNF7.2 OsYUC8 rice(Oryza sativa) vacuolar degradation

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A chromosome-scale genome assembly of Dasypyrum villosum provides insights into its application as a broad-spectrum disease resistance resource for wheat improvement

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Molecular Plant 2023年第2期16卷 432-451页

作者： Xu Zhang Haiyan Wang Haojie Sun Yingbo Li Yilong Feng Chengzhi Jiao Mengli Li Xinying Song Tong Wang Zongkuan Wang Chunxia Yuan Li Sun Ruju Lu Wenli Zhang Jin Xiao Xiue Wang State key laboratory of Crop Genetics and germplasm enhancement Cytogenetics InstituteNanjing Agricultural University/JCIC-MCPNanjingJiangsu 210095China Biotech Research Institute Shanghai Academy of Agricultural SciencesShanghai 201106China

Dasypyrum villosum is one of the most valuable gene resources in wheat improvement,especially for disease *** mining of favorable genes from *** is frustrated by the lack of a whole genome *** this study,we generated a doubled-haploid line,91C43^(DH),using microspore culture and obtained a 4.05-GB high-quality,chromosome-scale genome assembly for *** assembly contains39727 high-confidence genes,and 85.31% of the sequences are *** reciprocal translocation events were detected,and 7VS-4VL is a unique translocation in *** prolamin seed storage protein-coding genes were found to be duplicated;in particular,the genes encoding low-molecular-weight glutenin at the Glu-V3 locus were significantly *** sequencing(RNA-seq)analysis indicated that,after Blumeria graminearum *** tritici(Bgt)inoculation,there were more upregulated genes involved in the pattern-triggered immunity and effector-triggered immunity defense pathways in *** than in Triticum *** hypersensitive sequencing(MH-seq)identified two Bgt-inducible MH sites(MHSs),one in the promoter and one in the 3'terminal region of the powdery mildew resistance(Pm)gene *** site had two subpeaks and they were termed MHS1(MHS1.1/1.2)and MHS2(MHS2.1/2.2).Bgt-inducible MHS2.2 was uniquely present in ***,and MHS1.1 was more inducible in *** than in wheat,suggesting that MHSs may be critical for regulation of NLR1-V expression and plant *** summary,this study provides a valuable genome resource for functional genomics studies and *** introgression *** identified regulatory mechanisms may also be exploited to develop new strategies for enhancing Pm resistance by optimizing gene expression in wheat.

关键词： D.villosum genome crop wild relative genome evolution disease resistance RNA-seq MNase hy-persensitivityassay

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