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菜心BcDof3;1启动子的克隆及活性分析

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分子植物育种 2024年

作者：朱云娜沈雪晴符质赵明锋汤婉君刘建国宋世威韶关学院生物与农业学院广东省粤北食药资源利用与保护重点实验室华南农业大学园艺学院

以菜心(Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)为材料，利用同源克隆法获得了菜心BcDof3;1启动子序列，长度为1 815 bp。生物信息分析表明，该启动子中包含多个光响应、激素信号、逆境应答、组织特异表达等... 详细信息

以菜心(Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)为材料，利用同源克隆法获得了菜心BcDof3;1启动子序列，长度为1 815 bp。生物信息分析表明，该启动子中包含多个光响应、激素信号、逆境应答、组织特异表达等顺式作用元件。构建菜心BcDof3;1启动子与gus的融合表达载体，并用农杆菌介导法转化拟南芥。通过对T3代转基因拟南芥植株gus活性检测分析发现，该启动子gus活性受植株生长发育时期影响，gus染色在植株地上部和根系均有分布，在植株根部、薹叶、花蕾表现出较深的gus染色，而茎部染色颜色较浅。此外，不同浓度NH4NO3处理也明显影响BcDof3;1启动子活性，随着NH4NO3浓度增加，其表达活性逐渐降低。研究结果表明，菜心BcDof3;1可能在低氮浓度下对氮吸收、代谢方面具有重要调控作用。

关键词：菜心 BcDof3 1启动子 gus 氮浓度

GhKLCR1, a kinesin light chain-related gene, induces drought-stress sensitivity in Arabidopsis

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Science China(Life Sciences) 2019年第1期62卷 63-75页

作者： Jie Li Daoqian Yu Ghulam Qanmber Lili Lu Lingling Wang Lei Zheng Zhao Liu Huanhuan Wu Xiaodong Liu Quanjia Chen Fuguang Li Zuoren Yang Xinjiang Research Base State Key Laboratory of Cotton Biology Xinjiang Agricultural University State Key Laboratory of Cotton Biology Key Laboratory of Biological and Genetic Breeding of Cotton Institute of Cotton ResearchChinese Academy of Agricultural Sciences

Drought stress results in significant losses in agricultural production, and especially that of cotton. The molecular mechanisms that coordinate drought tolerance remain elusive in cotton. Here, we isolated a drought-response gene GhKLCR1, which is a close homolog of AtKLCR1, which encodes a kinesin light chain-related protein enriched with a tetratrico peptide-repeat region.A subcellular localization assay showed that GhKLCR1 is associated with the cell membrane. A tissue-specific expression profile analysis demonstrated that GhKLCR1 is a cotton root-specific gene. Further abiotic and hormonal stress treatments showed that GhKLCR1 was upregulated during abiotic stresses, especially after polyethylene glycol treatments. In addition, the glucuronidase(gus) staining activity increased as the increment of mannitol concentration in transgenic Arabidopsis plants harboring the fusion construct PGhKLCR1::gus. The root lengths of 35 S::GhKLCR1 lines were significantly reduced compared with that of wild type. Additionally, seed germination was strongly inhibited in 35 S::GhKLCR1 lines after 300-mmol L^(-1) mannitol treatments as compared with Columbia-0, indicating the sensitivity of GhKLCR1 to drought. These findings provide a better understanding of the structural, physiological and functional mechanisms of kinesin light chain-related proteins.

关键词： GhKLCR1 kinesin Arabidopsis mannitol seed germination drought sensitivity gus cis-elements

ZYGOTE-ARREST 3 that encodes the tRNA ligase is essential for zygote division in Arabidopsis

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Journal of Integrative Plant Biology 2017年第9期59卷 680-692页

作者： Ke-Jin Yang Lei Guo Xiu-Li Hou Hua-Qin Gong Chun-Ming Liu Key Laboratory of Plant Molecular Physiology Institute of BotanyChinese Academy of SciencesBeijing 100093China University of Chinese Academy of Sciences Beijing 100049China School of Life Science and Technology Nanyang Normal UniversityNanyang 473061China Institute of Crop Sciences Chinese Academy of Agricultural SciencesBeijing 100081China

In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST 3 （ZYG3） in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG3 encodes the transfer RNA （tRNA） ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL：：cAtRNL-sYFP- complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.

关键词： NL ZYGOTE-ARREST 3 that encodes the tRNA ligase is essential for zygote division in Arabidopsis gus Figure

Phylogenetically Distinct Cellulose Synthase Genes Support Secondary Wall Thickening in Arabidopsis Shoot Trichomes and Cotton Fiber

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Journal of Integrative Plant Biology 2010年第2期52卷 205-220页

作者： Lissete Betancur Bir Singh Ryan A.Rapp Jonathan F.Wendel M. David Marks Alison W.Roberts Candace H.Haigler Department of Plant Biology North Carolina State University Department of Crop Science North Carolina State University EEOB Department Iowa State University Department of Plant Biology University of Minnesota Department of Biological Sciences CBLSUniversity of Rhode Island

Through exploring potential analogies between cotton seed trichomes （or cotton fiber） and arabidopsis shoot trichomes we discovered that CesAs from either the primary or secondary wall phylogenetic clades can support secondary wall thickening. CesA genes that typically support primary wall synthesis, AtCesA 1,2,3,5, and 6, underpin expansion and secondary wall thickening of arabidopsis shoot trichomes. In contrast, apparent orthologs of CesA genes that support secondary wall synthesis in arabidopsis xylem, AtCesA4,7, and 8, are up-regulated for cotton fiber secondary wall deposition. These conclusions arose from：（a） analyzing the expression of CesA genes in arabidopsis shoot trichomes; （b） observing birefringent secondary walls in arabidopsis shoot trichomes with mutations in AtCesA4, 7, or 8; （c） assaying up-regulated genes during different stages of cotton fiber development; and （d） comparing genes that were co.expressed with primary or secondary wall CesAs in arabidopsis with genes up- regulated in arabidopsis trichomes, arabidopsis secondary xylem, or cotton fiber during primary or secondary wall deposition. Cumulatively, the data show that：（a） the xylem of arabidopsis provides the best model for secondary wall cellulose synthesis in cotton fiber; and （b） CesA genes within a ＂cell wall toolbox＂ are used in diverse ways for the construction of particular specialized cell walls.

关键词： Phylogenetically Distinct Cellulose Synthase Genes Support Secondary Wall Thickening in Arabidopsis Shoot Trichomes and Cotton Fiber gus

烟草NtCycB2启动子的克隆和组织驱动特性分析

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中国烟草学报 2023年第3期29卷 131-138页

作者：睢雅琳易梦博周超毅苏鹏飞原慧扬闫筱筱王召军崔红张洪映河南农业大学烟草学院郑州市450046

【背景和目的】烟草B型细胞周期蛋白CycB2可以负调控腺毛的发生,为分析烟草Nt CycB2启动子的组织驱动特性。【方法】通过实时定量PCR分析普通烟草NtCycB2-1和Nt CycB2-2两种序列的组织表达差异,克隆NtCycB2-1和NtCycB2-2启动子序列,对... 详细信息

【背景和目的】烟草B型细胞周期蛋白CycB2可以负调控腺毛的发生,为分析烟草Nt CycB2启动子的组织驱动特性。【方法】通过实时定量PCR分析普通烟草NtCycB2-1和Nt CycB2-2两种序列的组织表达差异,克隆NtCycB2-1和NtCycB2-2启动子序列,对其进行进化分析、顺式元件预测和组织驱动特性分析。【结果】(1)烟草NtCycB2-1和NtCycB2-2均为腺毛优势表达基因,NtCycB2-1的表达量显著高于NtCycB2-2;(2)普通烟草中存在两种NtCycB2启动子序列类型(ProNtCycB2-1和ProNtCycB2-2),其中ProNtCycB2-1来自于林烟草,ProNtCycB2-2来自于绒毛烟草,这些CycB2启动子中普遍存在ABRE等激素应答元件,以及MYB-motif、MYC-motif和TC-rich repeats等逆境响应元件;(3)以gus为报告基因,研究NtCycB2-1和NtCycB2-2两个启动子的组织驱动特性发现,NtCycB2-1和NtCycB2-2启动子均能驱动gus基因在叶片和茎部腺毛中特异表达,其中NtCycB2-1启动子的驱动能力高于NtCycB2-2。【结论】Nt CycB2启动子可驱动下游基因在腺毛中特异表达。本研究为寻找腺毛特异表达顺式元件、构建腺毛人工启动子、研究腺毛发生机制奠定基础。

关键词：烟草 NtCycB2 启动子腺毛 gus

Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition

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Journal of Integrative Plant Biology 2018年第10期60卷 1000-1014页

作者： Huiyan Guo Liuqiang Wang Chuanping Yang Yiming Zhang Chunrui Zhang Chao Wang State Key Laboratory of Tree Genetics and Breeding Northeast Forestry University State Key Laboratory of Tree Genetics and Breeding Research Institute of ForestryChinese Academy of Forestry

Transcription factors （TFs） play vital roles in various biological processes by binding to cis-acting elements to control expressions of their target genes. The MYB TF BplMYB46, from Betula platyphylla, is involved in abiotic stress responses and secondary wall deposition. In the present study, we used a TF-centered yeast onehybrid technology （TF-centered YIH） to identify the cis- acting elements bound by BplMYB46. We screened a shortinsert random library and identified three cis-elements bound by BplMYB46： an E-box （CA（A/T/C）（A/G/C）TG） and two novel motifs, a TO-box （T（GIA）TCG（C/G）） and a GT-box （A（G/T）T（AIC）GT（T/G）C）. Chromatin immunoprecipitation （CHIP） and effector-reporter coexpression assays inNicotiana tabacum confirmed binding of BplMYB46 to the TC-box, GT-box, and E-box motifs in the promoters of the phenylalanine ammonia lyase （PAL）, peroxidase （POD）, and superoxide dismutase （SOD） genes, which function in abiotic stress tolerance and secondary wall biosynthesis. This finding improves our understanding of potential regulatory mechanisms in the response to abiotic stress and secondary wall deposition of BplMYB46 in B. platyphylla.

关键词： POD SOD Identification of novel cis-elements bound by BplMYB46 involved in abiotic stress responses and secondary wall deposition cis TCG gus Figure

ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis:A disconnect between somatic and germinal cells

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Journal of Genetics and Genomics 2018年第12期45卷 681-684页

作者： Qiwei Shan Nicholas J.Baltes Paul Atkins Elida R. Kirkland Yong Zhang Joshua A.Ballet Levi G.Lowder Aimee A.Malzahn John C.Haugner Ⅲ Burckhard Seelig Daniel F. Voytas Yiping Qi Department of Genetics Cell Biology & Development and Center for Genome EngineeringUniversity of MinnesotaMinneapolisMN 55455USA Department of Biology East Carolina UniversityGreenvilleNC 27858USA Department of Biotechnology School of Life Sciences and Technology University of Electronic Science and Technology of ChinaChengdu 610054China Minnesota Supercomputing Institute University of Minnesota MinneapolisMN 55455USA Department of Plant Science and Landscape Architecture University of MarylandCollege ParkMD 20742USA Department of Biochemistry Molecular Biology and Biophysics University of MinnesotaMinneapolisMN 55455USA BioTechnology Institute University of MinnesotaSt.PaulMN 55108 USA Institute for Bioscience and Biotechnology Research University of MarylandRockvilleMD 20850USA

Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break

关键词： ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis gus A disconnect between somatic and germinal cells

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枳PtMLP1启动子的克隆和表达分析

中国农业科学 2023年第24期56卷 4906-4915页

作者：姚利晓苏娟郭兴茹李凤龙何永睿邹修平陈善春西南大学柑桔研究所/国家柑桔工程技术中心/国家柑桔品种改良中心

【目的】基因工程是柑橘品种改良的一种重要手段。本研究基于枳根消减文库中主要乳胶蛋白基因PtMLP1片段，克隆根特异启动子序列，为研究外源基因在柑橘根组织的特异表达奠定基础。【方法】同源克隆PtMLP1及启动子序列。利用ExPASy、PSI... 详细信息

【目的】基因工程是柑橘品种改良的一种重要手段。本研究基于枳根消减文库中主要乳胶蛋白基因PtMLP1片段，克隆根特异启动子序列，为研究外源基因在柑橘根组织的特异表达奠定基础。【方法】同源克隆PtMLP1及启动子序列。利用ExPASy、PSIPRED、SWISS-MODEL等在线软件对PtMLP1编码蛋白的理化特征、二级结构和三级结构进行生物信息学分析，利用PlantCARE数据库对PtMLP1启动子的顺式作用元件进行预测。实时荧光定量PCR法对PtMLP1在不同树龄枳根和叶中的表达进行分析。构建PtMLP1启动子与gus标记基因的融合载体，利用根癌农杆菌转化法转化枳上胚轴，gus染色观察标记基因的表达部位。【结果】枳PtMLP1含2个外显子和1个内含子，开放阅读框长471 bp。PtMLP1蛋白由156个氨基酸组成，分子量17.63 kDa，等电点5.49，含Bet v I功能域。其二级结构含3个α-螺旋和7个β-折叠，三级结构包含一个保守疏水基结合位点和一个富含甘氨酸的回环结构。5′端1 666 bp的上游调控序列不仅有TATA-box、CAAT-box等启动子结构的核心元件，还具有多个根组织特异表达元件，以及TGACG-motif、P-box和ABRE等激素应答相关的顺式作用元件。3′端非翻译区具有加尾信号AATAAA。该基因在1月龄苗、6月龄苗、20年生成年枳根中的表达量分别是叶中的46.34、74.82、110.25倍。构建启动子的融合表达载体pBI121-ProPtMLP1::gus，获得枳转基因植株。PtMLP1启动子驱动gus在转基因枳幼苗根中特异表达，gus在3个转基因枳株系的根中表达量分别为叶中表达量的124.78、11.53和7.76倍。【结论】获得柑橘主要乳胶蛋白PtMLP1及启动子序列，该启动子可驱动标记基因在柑橘根组织特异表达。

关键词：枳主要乳胶蛋白根特异性启动子 gus

gus-property for Lorentz cone linear complementarity problems on Hilbert spaces

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Science China Mathematics 2011年第6期54卷 1259-1268页

作者： MIAO XinHe HUANG ZhengHai Department of Mathematics Tianjin University Tianjin 300072 China

Given a real(finite-dimensional or infinite-dimensional) Hilbert space H with a Jordan product,we consider the Lorentz cone linear complementarity problem,denoted by LCP(T,Ω,q),where T is a continuous linear operator on H,ΩH is a Lorentz cone,and q ∈ H.We investigate some conditions for which the problem concerned has a unique solution for all q ∈ H(i.e.,T has the gus-property).Several sufficient conditions and several necessary conditions are given.In particular,we provide two suficient and necessary conditions of T having the gus-property.Our approach is based on properties of the Jordan product and the technique from functional analysis,which is different from the pioneer works given by Gowda and Sznajder(2007) in the case of finite-dimensional spaces.

关键词：线性互补问题 Hilbert空间洛伦兹 gus 财产有限维空间希尔伯特空间线性算子