作者： 杨慧;胡信畅;次央;阮颖;刘春林

期刊： 分子植物育种,2020年18(4):1144-1151 ISSN：1672-416X

作者机构： [刘春林; 杨慧; 次央; 胡信畅; 阮颖] 湖南农业大学生物科学技术学院,作物表观遗传调控与发育湖南省重点实验室,长沙,410128;[杨慧] 湖南文理学院国际学院,常德,415000

关键词： 甘蓝型油菜;遗传转化

摘要： ABI4 (Abscisic acid-insensitive 4)是ABA响应途径中重要的转录因子.本研究以甘蓝型油菜全基因组数据库为基础,借助拟南芥ABI4蛋白的氨基酸序列,通过序列比对,获得了甘蓝型油菜的2个BnaA BI4,分别命名为BnaA BI4-A (GenBank登录号为BnaA03g18970D)和BnaABI4-C (GenBank登录号为BnaC03g725-10D).在这两个基因的CDS序列上寻找一段长度为20 bp、序列相同的片段作为基因编辑的目标位点,构建CRISPR/Cas9基因编辑载体pCAMBIA 1300-sgRNA/Cas9-BnaABI4;借助根癌农杆菌介导的油菜下胚轴遗传转化法,成功将BnaA I4的基因编辑表达框转入甘蓝型油菜‘湘油15’中,共获得转基因植株13株,这将为深入研究甘蓝型油菜BnaA BI4的生物学功能提供科学依据.

语种： 中文

作者： Qin, Ling;Chen, Erying;Li, Feifei;Yu, Xiao;Liu, Zhenyu;...

期刊： International Journal of Molecular Sciences,2020年21(22)

通讯作者： Ruan, Ying;Guan, Yan'an

作者机构： [Ruan, Ying; Qin, Ling] Hunan Agr Univ, Coll Biosci & Biotechnol, Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Peoples R China.;[Liu, Bin; Yang, Yanbing; Wang, Hailian; Guan, Yan'an; Chen, Erying; Wang, Runfeng; Qin, Ling; Liu, Zhenyu; Zhang, Huawen; Li, Feifei] Shandong Acad Agr Sci, Crop Res Inst, Featured Crops Engn Lab Shandong Prov, Jinan 250100, Peoples R China.;[Guan, Yan'an; Yu, Xiao] Shandong Normal Univ, Coll Life Sci, Jinan 250014, Peoples R China.

通讯机构： [Ruan, Ying] H;[Guan, Yan'an] S;Hunan Agr Univ, Coll Biosci & Biotechnol, Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Peoples R China.;Shandong Acad Agr Sci, Crop Res Inst, Featured Crops Engn Lab Shandong Prov, Jinan 250100, Peoples R China.;Shandong Normal Univ, Coll Life Sci, Jinan 250014, Peoples R China.

关键词： P5CS genes;RNA sequencing;drought stress;foxtail millet;gene expression

摘要： Foxtail millet (Setaria italica (L.) P. Beauv) is an important food and forage crop because of its health benefits and adaptation to drought stress; however, reports of transcriptomic analysis of genes responding to re-watering after drought stress in foxtail millet are rare. The present study evaluated physiological parameters, such as proline content, p5cs enzyme activity, anti-oxidation enzyme activities, and investigated gene expression patterns using RNA sequencing of the drought-tolerant foxtail millet variety (Jigu 16) treated with drought stress and rehydration. The results indicated that drought stress-responsive genes were related to many multiple metabolic processes, such as photosynthesis, signal transduction, phenylpropanoid biosynthesis, starch and sucrose metabolism, and osmotic adjustment. Furthermore, the Δ1-pyrroline-5-carboxylate synthetase genes, SiP5CS1 and SiP5CS2, were remarkably upregulated in foxtail millet under drought stress conditions. Foxtail millet can also recover well on rehydration after drought stress through gene regulation. Our data demonstrate that recovery on rehydration primarily involves proline metabolism, sugar metabolism, hormone signal transduction, water transport, and detoxification, plus reversal of the expression direction of most drought-responsive genes. Our results provided a detailed description of the comparative transcriptome response of foxtail millet variety Jigu 16 under drought and rehydration environments. Furthermore, we identify SiP5CS2 as an important gene likely involved in the drought tolerance of foxtail millet.

语种： 英文

作者： Huang, Yong;Jiang, Ling;Liu, Bo-Yu;Tan, Cheng-Fang;Chen, Dong-Hong;...

期刊： BMC Genomics,2019年20(1):1-18 ISSN：1471-2164

通讯作者： Ruan, Ying

作者机构： [Huang, Yong; Jiang, Ling; Liu, Bo-Yu; Tan, Cheng-Fang; Shen, Wen-Hui; Ruan, Ying] Hunan Agr Univ, Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Hunan, Peoples R China;[Huang, Yong; Jiang, Ling; Liu, Bo-Yu; Tan, Cheng-Fang; Shen, Wen-Hui; Ruan, Ying] Hunan Agr Univ, Int Associated Lab CNRS FU HAU Plant Epigenome Re, Changsha 410128, Hunan, Peoples R China;[Huang, Yong; Jiang, Ling; Liu, Bo-Yu; Tan, Cheng-Fang; Ruan, Ying] Hunan Agr Univ, Key Lab, Plant Genet & Mol Biol Educ Dept Hunan Prov, Changsha 410128, Hunan, Peoples R China;[Chen, Dong-Hong] Zhejiang A&F Univ, State Key Lab Subtrop Silviculture, SFGA Engn Res Ctr Dendrobium Catenatum D Officina, Hangzhou 311300, Zhejiang, Peoples R China;[Shen, Wen-Hui] Univ Strasbourg, Inst Biol Mol Plantes, CNRS, 12 Rue Gen Zimmer, F-67084 Strasbourg, France

通讯机构： [Ruan, Ying] H;Hunan Agr Univ, Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Hunan, Peoples R China. Hunan Agr Univ, Int Associated Lab CNRS FU HAU Plant Epigenome Re, Changsha 410128, Hunan, Peoples R China.

关键词： Polycomb;PRC1;Phylogenetic analysis;Domain organization;Evolution

摘要： Background: Polycomb group (PcG) proteins play important roles in animal and plant development and stress response. Polycomb repressive complex 1 (PRC1) and PRC2 are the key epigenetic regulators of gene expression, and are involved in almost all developmental stages. PRC1 catalyzes H2A monoubiquitination resulting in transcriptional silencing or activation. The PRC1 components in the green lineage were identified and evolution and conservation was analyzed by bioinformatics techniques. RING Finger Protein 1 (RING1), B lymphoma Mo-MLV insertion region 1 homolog (BMI1), Like Heterochromatin Protein 1 (LHP1) and Embryonic Flower 1 (EMF1) are the PRC1 core components and Vernalization 1 (VRN1), VP1/ABI3-Like 1/2/3 (VAL1/2/3), Alfin-like 1-7 (AL1-7), Inhibitor of growth 1/2 (ING1/2), and Early Bolting in Short Days (EBS) / Short Life (SHL) are the associated factors. Results: Each PRC1 subunit possesses special domain organizations, such as RING and the ring finger and WD40-associated ubiquitin-like (RAWUL) domains for RING1 and BMI1, chromatin organization modifier (CHROMO) and chromo shadow (ChSh) domains for LHP1, one or two B3 DNA binding domain(s) for VRN1, B3 and zf-CW domains for VAL1/2/3, Alfin and Plant HomeoDomain (PHD) domains for AL1-7, ING and PHD domains for ING1/2, Bromoadjacent homology (BAT) and PHD domains for EBS/SHL. Six new motifs are uncovered in EMF1. The PRC1 core components RING1 and BMI1, and the associated factors VAL1/2/3, AL1-7, ING1/2, and EBS/SHL exist from alga to higher plants, whereas LHP1 only occurs in higher plants. EMF1 and VRN1 are present only in eudicots. PRC1 components undergo duplication in the plant evolution. Most of plants carry the homologous core component LHP1, the associated factor EMF1, and several homologs in RING1, BMI1, VRN1, AL1-7, ING1/2/3, and EBS/SHL. Cabbage, cotton, poplar, orange and maize often exhibit more gene copies than other species. Domain organization analysis shows that duplicated gene functions may be of diverse. Conclusions: The PRC1 core components RING1 and BMI1, and the associated factors VAL1/2/3, AL1-7, ING1/2, and EBS/SHL originate from algae. The core component LHP1 is from moss and the associated factors EMF1 and VRN1 are from dicotyledon. PRC1 components are of functional redundancy and diversity in evolution. © 2019 The Author(s).

语种： 英文

作者： 吕晶森;张斌;刘春林;阮颖

期刊： 分子植物育种,2019年17(1):92-98 ISSN：1672-416X

作者机构： 湖南农业大学生物科学技术学院,长沙,410128;湖南农业大学农学院,长沙,410128;[刘春林; 吕晶森; 阮颖; 张斌] 湖南农业大学

关键词： 拟南芥;sb10突变体;NaCl胁迫

摘要： 为了研究拟南芥SB10基因在耐盐中的功能,我们利用CRISPR/Cas9技术构建载体敲除SB10基因,对转基因植株进行筛选成功获得SB10基因沉默的突变体材料。本研究通过观察突变体根长及萌发率等表型,测定突变体植株脯氨酸含量相关的抗逆生理指标,研究拟南芥SB10基因在耐盐中的功能。结果显示:NaCl胁迫下,sb10突变体的根长和萌发率明显高于野生型拟南芥,sb10突变体拟南芥植物体内脯氨酸含量明显高于野生型拟南芥。说明SB10基因功能的缺失可提高拟南芥植株的耐盐能力。该研究为SB10基因参与拟南芥植株耐盐中的分子作用途径及分析提供线索。

语种： 中文

作者： Jin, Lu;Ouyang, Ning;Huang, Yong;Liu, Chunlin;Ruan, Ying*

期刊： PLOS ONE,2019年14(8):e0221422 ISSN：1932-6203

通讯作者： Ruan, Ying

作者机构： [Jin, Lu; Ouyang, Ning; Ruan, Ying; Huang, Yong] Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha, Hunan, Peoples R China.;[Jin, Lu; Ouyang, Ning; Ruan, Ying; Huang, Yong] Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha, Hunan, Peoples R China.;[Jin, Lu; Ouyang, Ning; Ruan, Ying; Huang, Yong] Educ Dept Hunan Prov, Key Lab Plant Genet & Mol Biol, Changsha, Hunan, Peoples R China.;[Liu, Chunlin] Hunan Agr Univ, Agr Coll, Changsha, Hunan, Peoples R China.

通讯机构： [Ruan, Ying] H;[Ruan, Ying] K;[Ruan, Ying] E;Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha, Hunan, Peoples R China.;Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha, Hunan, Peoples R China.

摘要： Sulfotransferases (SOTs; EC 2.8.2.-), which are widespread from prokaryotes to eukaryotes, constitute a multi-protein family that plays crucial roles in plant growth, development and stress adaptation. However, this family has not been systemically investigated in Brassica rapa. Here, a genome-wide systemic analysis of SOT genes in B. rapa subsp. pekinensis, a globally cultivated vegetable, were conducted. We identified 56 SOT genes from the whole B. rapa genome using Arabidopsis SOT sequences as queries and classified them into nine groups, rather than the eight groups of previous research. 56 B. rapa SOT genes (BraSOTs) were distributed on all 10 chromosomes except for chromosome 5. Of these, 27 BraSOTs were distributed in seven clusters on five chromosomes (ChrA01, ChrA02, Chr03, ChrA07, and Chr09). Among the BraSOT proteins, 48 had only one SOT_1 domain and 6 had two, while 2 had one SOT_3 domain. Additionally, 47 BraSOT proteins contained only known SOT domains. The remaining nine proteins, five in group-VIII and two in group-IX, contained additional transmembrane domains. Specific motif regions I and IV for 3'-phosphoadenosine 5'-phosphosulfate binding were found in 41 BraSOT proteins. Introns were present in only 18 BraSOT genes, and all seven BraSOT genes in groups VIII and IX had more than three introns. To identify crucial SOTs mediating the response to abiotic stress in B. rapa, expression changes in 56 BraSOT genes were determined by quantitative RT-PCR after drought, salinity, and ABA treatments, and some BraSOT genes were associated with NaCl, drought and ABA stress, e.g. Bra017370, Bra009300, Bra027880.

语种： 英文

作者： Xiao, Mu;Liu, Rong;Ruan, Ying;Liu, Chunlin*

期刊： PLANT SIGNALING & BEHAVIOR,2019年14(7):1607466 ISSN：1559-2316

通讯作者： Liu, Chunlin

作者机构： [Ruan, Ying; Liu, Rong; Liu, Chunlin; Xiao, Mu] Hunan Agr Univ, Key Lab Hunan Prov Crop Epigenet Regulat & Dev, Changsha, Hunan, Peoples R China.;[Ruan, Ying; Liu, Rong; Xiao, Mu] Hunan Agr Univ, Coll Biosci & Biotechnol, Dept Hunan Prov Plant Genet & Mol Biol, Key Lab Educ, Changsha, Hunan, Peoples R China.;[Liu, Chunlin] Hunan Agr Univ, Coll Agron, Changsha, Hunan, Peoples R China.

通讯机构： [Liu, Chunlin] H;Hunan Agr Univ, Key Lab Hunan Prov Crop Epigenet Regulat & Dev, Changsha, Hunan, Peoples R China.

关键词： Sodium chloride;defense priming;herbivory;jasmonic acid pathway

摘要： Priming for better defense performance is an important strategy in acclimation to the ever-changing environment. In the present study, defense priming induced by sodium chloride at the seedling stage significantly increased the expression of defense gene VSP2, the content of total glucosinolates and the level of the reactive oxygen species in mature Arabidopsis thaliana plants after transferred into the stress-free environment. The previously primed plants could effectively resist the feeding of Spodoptera litura (Fabricius) larvae. Salt-priming enhanced defense of Arabidopsis plants in the absence of either MYC2 or AOS, which encodes a critical transcription factor in JA-signaling and an important enzyme in JA biosynthesis, respectively. Our results supported the JA-independent defense primed by sodium chloride, as well as the elevated ROS and glucosinolate level in primed plants. In addition, the feasibility of using mild salt-priming to improve crop performance in field was proposed.

语种： 英文

作者： 皮博艺;阮颖;黄勇

期刊： 分子植物育种,2019年17(7):2171-2177 ISSN：1672-416X

作者机构： 湖南农业大学生物科学技术学院,长沙,410128;[黄勇; 阮颖; 皮博艺] 湖南农业大学

关键词： 串联CCCH锌指蛋白;RR-TZF蛋白;进化;研究进展

摘要： 典型的串联CCCH型锌指结构蛋白(tandem CCCH zinc finger proteins, TZFs)含有由18个氨基酸分隔的二个串联CCCH motifs(C-X_(7-8) -C-X_5 -C-X_3 -H、C-X_5 -C-X_4 -C-X_3 -H)。富含精氨酸的RR-TZF蛋白是植物特有,研究表明RR-TZF蛋白通过介导RNA的降解来调节基因的表达水平,影响植物的生长发育以及胁迫反应。本研究主要综述了植物RR-TZF蛋白在生长发育及胁迫反应中的功能及作用机制等;同时利用生物信息学手段对RR-TZF蛋白进行聚类分析,系统地分析了RR-TZF蛋白从低等植物到高等植物中的进化与保守性,并展望了未来可能的研究方向。为探究CCCH型锌指结构蛋白家族基因在作物中的具体功能及其应对非生物胁迫的分子机制研究提供了重要的理论依据。

语种： 中文

作者： 王金彪;何星辉;高谢旺;覃诗琪;邓淞月;...

期刊： 分子植物育种,2019年17(20):6673-6679 ISSN：1672-416X

作者机构： 湖南农业大学生物科学技术学院,长沙,410128;湖南农业大学农学院,长沙,410128;[刘春林; 王金彪; 邓淞月; 覃诗琪; 高谢旺; 何星辉; 阮颖] 湖南农业大学

关键词： 甘蓝型油菜;分泌信号肽;BnaLCR78基因;生物信息学;基因编辑

摘要： 分泌信号肽是生物体内的一类重要功能分子。通过生物信息学分析,揭示甘蓝型油菜BnaLCR78基因编码一个由78个氨基酸组成、N端包含分泌信号肽且C端富含半胱氨酸的蛋白,属于富含半胱氨酸的分泌信号肽。为进一步研究该基因的功能,借助农杆菌介导的甘蓝型油菜下胚轴遗传转化方法,利用CRISPR/Cas9系统对甘蓝型油菜BnaLCR78基因进行编辑,实现定向敲除。本实验共计使用1 200个外植体,经除草剂草铵膦的抗性筛选及对213株抗性苗基因组DNA的PCR检测,获得了26株转基因油菜,转化率约为2.17%,但在T0代并未获得发生了目标基因被编辑的植株。幸运地是从T0自交获得的T1植株中获得基因被编辑了的植株。通过扩增T1代27株转基因油菜靶位点并测序,发现有1株在靶位点2有一个T碱基的插入,其基因型为+1/WT的杂合突变体,说明在T1植株中,基因的编辑效率达到了3.7%。这对后续研究甘蓝型油菜BnaLCR78基因功能的研究提供了帮助。

语种： 中文

作者： Zhang, Bin;Qiu, Han-Lin;Qu, Dong-Hai;Ruan, Ying*;Chen, Dong-Hong*

期刊： GENOME,2018年61(6):405-415 ISSN：0831-2796

通讯作者： Ruan, Ying;Chen, Dong-Hong

作者机构： [Ruan, Ying; Zhang, Bin; Qu, Dong-Hai] Hunan Agr Univ, Key Lab Educ, Dept Hunan Prov Plant Genet & Mol Biol, Coll Biosci & Biotechnol, Changsha 410128, Hunan, Peoples R China.;[Chen, Dong-Hong; Qiu, Han-Lin] Zhejiang A&F Univ, State Key Lab Subtrop Silviculture, Hangzhou 311300, Zhejiang, Peoples R China.

通讯机构： [Ruan, Ying] H;[Chen, Dong-Hong] Z;Hunan Agr Univ, Key Lab Educ, Dept Hunan Prov Plant Genet & Mol Biol, Coll Biosci & Biotechnol, Changsha 410128, Hunan, Peoples R China.;Zhejiang A&F Univ, State Key Lab Subtrop Silviculture, Hangzhou 311300, Zhejiang, Peoples R China.

关键词： J-proteins;Arabidopsis;Brassica oleracea;phylogenetic analysis;domain organization;gene structure;protéines J;Arabidopsis;Brassica oleracea;analyse phylogénétique;organisation des domaines;structure génique

摘要： Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants. © 2018 Published by NRC Research Press.

语种： 英文

作者： Jiang, Ling;Li, Donghao;Jin, Lu;Ruan, Ying;Shen, Wen-Hui*;...

期刊： PLANT JOURNAL,2018年95(4):672-685 ISSN：0960-7412

通讯作者： Liu, Chunlin;Shen, Wen-Hui

作者机构： [Jin, Lu; Ruan, Ying; Liu, Chunlin; Li, Donghao; Jiang, Ling] Hunan Agr Univ, Hunan Prov Key Lab Crop Germplasm Innovat & Utili, Changsha 410128, Hunan, Peoples R China.;[Jin, Lu; Ruan, Ying; Liu, Chunlin; Li, Donghao; Jiang, Ling] Hunan Agr Univ, Coll Biosci & Biotechnol, Dept Hunan Prov Plant Genet & Mol Biol, Key Lab Educ, Changsha 410128, Hunan, Peoples R China.;[Shen, Wen-Hui] Univ Strasbourg, CNRS, IBMP, UPR2357, 12 Rue Gen Zimmer, F-67084 Strasbourg, France.

通讯机构： [Liu, Chunlin] H;[Shen, Wen-Hui] U;Hunan Agr Univ, Hunan Prov Key Lab Crop Germplasm Innovat & Utili, Changsha 410128, Hunan, Peoples R China.;Hunan Agr Univ, Coll Biosci & Biotechnol, Dept Hunan Prov Plant Genet & Mol Biol, Key Lab Educ, Changsha 410128, Hunan, Peoples R China.;Univ Strasbourg, CNRS, IBMP, UPR2357, 12 Rue Gen Zimmer, F-67084 Strasbourg, France.

关键词： Brassica napus;BnaSDG8;gene silence;floral transition;H3K36 methylation

摘要： Although increasing experimental evidence demonstrates that histone methylations play important roles in Arabidopsis plant growth and development, little information is available regarding Brassica napus. In this study, we characterized two genes encoding homologues of the Arabidopsis histone 3 lysine 36 (H3K36) methyltransferase SDG8, namely, BnaSDG8.A and BnaSDG8.C. Although no duplication of SDG8 homologous genes had been previously reported to occur during the evolution of any sequenced species, a domain-duplication was uncovered in BnaSDG8.C. This duplication led to the identification of a previously unknown NNH domain in the SDG8 homologues, providing a useful reference for future studies and revealing the finer mechanism of SDG8 function. One NNH domain is present in BnaSDG8.A, while two adjacent NNH domains are present in BnaSDG8.C. Reverse transcriptase-quantitative polymerase chain reaction analysis revealed similar patterns but with varied levels of expression of BnaSDG8.A/C in different plant organs/tissues. To directly investigate their function, BnaSDG8.A/C cDNA was ectopically expressed to complement the Arabidopsis mutant. We observed that the expression of either BnaSDG8.A or BnaSDG8.C could rescue the Arabidopsis sdg8 mutant to the wild-type phenotype. Using RNAi and CRISPR/Cas9-mediated gene editing, we obtained BnaSDG8.A/C knockdown and knockout mutants with the early flowering phenotype as compared with the control. Further analysis of two types of the mutants revealed that BnaSDG8.A/C are required for H3K36 m2/3 deposition and prevent the floral transition of B. napus by directly enhancing the H3K36 m2/3 levels at the BnaFLC chromatin loci. This observation on the floral transition by epigenetic modification in B. napus provides useful information for breeding early-flowering varieties. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd

语种： 英文

作者： 张斌;阮颖

期刊： 基因组学与应用生物学,2018年37(3):1308-1314 ISSN：1674-568X

作者机构： [张斌; 阮颖] 湖南农业大学,生物科学技术学院, 湖南省作物种质资源创新和利用重点实验室, 长沙, 410128

关键词： 纯合子鉴定;转基因水稻;三引物检测法;旁侧序列;事件特异性检测

摘要： 对于研究转基因农作物来说,建立其检测方法至关重要。本研究利用TAIL-PCR法,获得了转基因水稻U41的右旁侧序列,结果显示T-DNA插入位点在水稻1号染色体第22 013~22 014之间。通过设计引物扩增左旁侧序列,建立了U41转化事件特异性检测的方法,可以从含0.1%目的基因中扩增出529 bp的特异性条带;还建立了三引物检测的方法,能快速和准确地鉴定U41的纯合株系。这些方法的建立,缩短了获得U41纯合体的时间,对U41的检测、研究和利用具有实用价值。

语种： 中文

作者： 李东昊;姜玲;刘春林;阮颖

期刊： 湖南农业大学学报(自然科学版),2018年44(4):346-352 ISSN：1007-1032

作者机构： 湖南农业大学生物科学技术学院, 湖南, 长沙, 410128;湖南农业大学农学院, 湖南, 长沙, 410128;[李东昊; 姜玲; 阮颖] 湖南农业大学生物科学技术学院, 湖南, 长沙, 410128;[刘春林] 湖南农业大学农学院, 湖南, 长沙, 410128

关键词： 甘蓝型油菜;开花调控

摘要： 以甘蓝型油菜‘湘油15号’为试材,通过生物信息学分析,运用CRISPR/Cas9系统对Bna SDG8第1个外显子上的Bna SDG8–A和Bna SDG8–C保守序列区域进行CRISPR/Cas9敲除位点设计,构建了CRISPR/Cas9敲除载体p CAMBIA1300–sg RNA/Cas9–Bna SDG8;通过遗传转化,获得转化植株19株;经测序比对,4株植株的Bna SDG8被成功编辑;通过对定向敲除植株开花时间的统计分析,初步确定Bna SDG8参与甘蓝型油菜开花时间的调控。

语种： 中文

作者： 庞奥运;金璐;陈吉杨;刘春林;阮颖

期刊： 分子植物育种,2018年16(13):4301-4307 ISSN：1672-416X

作者机构： 湖南农业大学生物科学技术学院, 长沙, 410128;湖南农业大学农学院, 长沙, 410128;[庞奥运; 金璐; 陈吉杨; 阮颖] 湖南农业大学生物科学技术学院, 长沙, 410128;[刘春林] 湖南农业大学农学院, 长沙, 410128

关键词： 拟南芥;紧密连锁;多MATH结构域基因;定向敲除

摘要： 为了研究含有多MATH结构域基因的功能,本研究利用CRISPR/Cas9基因编辑系统对拟南芥中两个紧密连锁的功能冗余基因M320 (AT2G25320)和M330 (AT2G25330)进行共敲除,获得了之前通过杂交无法获得的多MATH结构域基因双突变体材料。通过构建针对M320和M330基因的CRISPR/Cas9共敲除质粒,借助农杆菌介导的浸花法成功转化野生型拟南芥Col-0。最终测序验证显示多MATH结构域基因被成功敲除。M320、M330以及这两个基因同时被敲除的成功率分别是5.13%、17.95%和2.56%;通过测序分析发现突变体共产生了3种可造成移码突变的杂合突变类型:分别为在靶位点插入一个A或T以及缺失一个G。本研究为我们解析多MATH结构域基因的功能提供了材料基础,同时为紧密连锁基因的功能研究提供了创制突变体的有效方法。

语种： 中文

作者： 屈东海;仇汉林;黄勇;阮颖;陈东红

期刊： 植物生理与分子生物学学报,2018年54(9):1441-1450 ISSN：1671-3877

通讯作者： Chen, D.-H.

作者机构： 湖南农业大学生物科学技术学院, 长沙, 410128;浙江农林大学, 亚热带森林培育国家重点实验室, 浙江, 临安, 311300;[屈东海; 黄勇; 阮颖] 湖南农业大学生物科学技术学院, 长沙, 410128;[仇汉林; 陈东红] 浙江农林大学, 亚热带森林培育国家重点实验室, 浙江, 临安, 311300

通讯机构： State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Zhejiang, China

关键词： 锯缘落地生根;器官发育;无性苗;多梳蛋白

摘要： 在拟南芥中,多梳蛋白抑制复合体1 （PRC1）的核心组分AtRING1作为重要的表观遗传因子,其功能缺失导致植物在营养阶段产生胚性愈伤结构,这与落地生根叶生无性苗的发生相类似。本研究首先系统观察了锯缘落地生根主要器官的结构特征,探讨了锯缘落地生根整体发育模式。然后通过同源克隆技术获得2个AtRING1在锯缘落地生根的同源基因并命名为KsRNG1a和KsRING1b,GenBank登录号分别为MH144360和MH144361。实时定量PCR结果表明, KsRING1a/b基因广泛表达于锯缘落地生根的不同组织器官中,表明其功能贯穿于植物生长发育的整个时期。特别是KsRING1a/b的表达量在叶缘无性苗中的表达最高,这与拟南芥同源基因AtRING1a/b在种子胚中的高表达一致,不同于AtRING1突变导致的体细胞胚性结构发生,这表明锯缘落地生根叶缘无性苗的发生过程更接近于常规的种子胚发生方式。

语种： 中文

作者： 秦岭;陈二影;张艳亭;杨延兵;孔清华;...

期刊： 分子植物育种,2018年16(22):7460-7465 ISSN：1672-416X

作者机构： 湖南农业大学生物科学技术学院, 长沙, 410128;山东省农业科学院作物研究所, 济南, 250100;[秦岭] 湖南农业大学生物科学技术学院, 长沙, 410128 山东省农业科学院作物研究所, 济南, 250100;[陈二影; 张艳亭; 杨延兵; 孔清华; 张华文; 王海莲; 王润丰; 管延安] 山东省农业科学院作物研究所, 济南, 250100;[阮颖] 湖南农业大学生物科学技术学院, 长沙, 410128

关键词： 脯氨酸;谷子;SiP5CR基因;干旱

摘要： 脯氨酸在植物对干旱胁迫应答中起重要的渗透调节作用,增加植物体内脯氨酸含量可以提高植物抗旱性,吡咯啉-5-羧酸还原酶(P5CR)是合成脯氨酸的重要还原酶。为探讨干旱对谷子脯氨酸含量及脯氨酸代谢关键基因的影响,本研究以抗旱性突出的谷子品种‘济谷16'为材料,测定了其地上部分在干旱和复水条件下脯氨酸含量,结果表明脯氨酸含量随胁迫程度的增强而增加,复水后下降。经PCR扩增获得SiP5CR基因全长cDNA序列,序列分析表明SiP5CR基因长度为852 bp,编码283个氨基酸(GenBank登录号: MG833228),含有P5CR保守域,与玉米和高粱亲缘关系最近,亲缘关系最远的是大豆和豇豆。利用荧光定量RT-qPCR分析了该基因在干旱胁迫和复水不同时期的表达情况。结果表明干旱胁迫诱导SiP5CR在谷子地上部分上调表达,复水后相对表达量开始下降。因此,推测SiP5CR基因对干旱条件脯氨酸的积累起到重要的作用。

语种： 中文

作者： Pi, Boyi;He, Xinghui;Ruan, Ying;Jang, Jyan-Chyun;Huang, Yong*

期刊： BMC Plant Biology,2018年18(1):1-15 ISSN：1471-2229

通讯作者： Huang, Yong

作者机构： [Ruan, Ying; He, Xinghui; Huang, Yong; Pi, Boyi] Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha 410128, Hunan, Peoples R China.;[Ruan, Ying; He, Xinghui; Huang, Yong; Pi, Boyi] Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Hunan, Peoples R China.;[Ruan, Ying; He, Xinghui; Huang, Yong; Pi, Boyi] Educ Dept Hunan Prov, Key Lab Plant Genet & Mol Biol, Changsha 410128, Hunan, Peoples R China.;[Jang, Jyan-Chyun] Ohio State Univ, Dept Hort & Crop Sci, Mol Genet, Columbus, OH 43210 USA.;[Jang, Jyan-Chyun] Ohio State Univ, Ctr Appl Plant Sci, Columbus, OH 43210 USA.

通讯机构： [Huang, Yong] H;[Huang, Yong] K;Hunan Agr Univ, Coll Biosci & Biotechnol, Changsha 410128, Hunan, Peoples R China.;Key Lab Crop Epigenet Regulat & Dev Hunan Prov, Changsha 410128, Hunan, Peoples R China.

关键词： CCCH zinc finger family;Tandem CCCH zinc finger;Evolution;Abiotic stress;Brassica rapa

摘要： Background: Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear. Results: In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X 7/8 -C-X 5 -C 3 -H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses. Conclusion: This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops. © 2018 The Author(s).

语种： 英文

作者： 吕晶森;潭成方;刘春林;阮颖

期刊： 中国农学通报,2018年34(8):27-36 ISSN：1000-6850

作者机构： 湖南农业大学生物科学技术学院,长沙,410128;湖南农业大学农学院,长沙,410128

关键词： 拟南芥;双MATH结构域基因;紧密连锁;定点双敲除

摘要： 为了研究含双MATH结构域基因sb3与sb6的功能,需要创制这2个紧密连锁基因的双突变体。利用CRISPR/Cas9基因编辑技术对拟南芥Col-0中的sb3、sb6基因进行特异性定点双敲除。通过PCR和Singer测序验证,共获得10株T0代转基因植株,其中5株在目标片段上没有发生编辑;另5株在sb3基因上的目标序列都发生了编辑,在sb6基因的目标序列上只有2株发生了编辑。至此成功获得了sb3与sb6基因双敲除的突变体,且获得该突变的类型为核苷酸缺失突变体。这个双突变体的获得为进一步研究双MATH结构域基因在拟南芥中的功能奠定了良好的基础。

语种： 中文

作者： 张斌;阮颖

期刊： 基因组学与应用生物学,2018年37(1):386-392 ISSN：1674-568X

作者机构： [张斌; 阮颖] 湖南农业大学生物科学技术学院, 湖南省作物种植资源创新和利用重点实验室, 长沙, 410128

关键词： 水稻;日本晴;基因沉默;遗传转化

摘要： 多胺(腐胺,亚精胺和精胺)不仅参与植物的生长发育调控,还与生物胁迫和非生物胁迫的抗性密切相关.由于多胺具有多重功能,细胞内多胺含量受到严格控制,例如受转运的调节.因此,在水稻中研究水稻多胺转运蛋白OsPUT1的功能至关重要.本研究以OsPUT1为研究对象,借助生物信息学分析,发现OsPUT1蛋白是一种疏水性蛋白,具有12个跨膜结构,定位于细胞核内.通过构建OsPUT1基因过表达载体,借助根癌农杆菌将目的基因转入到水稻愈伤组织的基因组中,以潮霉素进行筛选,通过PCR鉴定,成功获得了20株转基因植株.对其中的4株转基因水稻进一步作实时荧光定量PCR检测,显示全部转基因苗OsPUT1基因表达明显上调,实现了设计目标.这些水稻为进一步研究OsPUT1的功能提供了良好的材料.

语种： 中文

作者： Song, C.;Liu, Z.;Xie, Q.;Wang, H.;Huang, Y.;...

期刊： Applied Biochemistry and Microbiology,2017年53(5):524-532 ISSN：0003-6838

通讯作者： Chen, D.

作者机构： [Xie, Q.; Wang, H.; Ruan, Y.; Huang, Y.; Song, C.; Chen, D.] Hunan Agr Univ, Coll Biosci & Biotechnol, Dept Hunan Prov Plant Genet & Mol Biol, Key Lab Educ, Changsha 410128, Hunan, Peoples R China.;[Liu, Z.] Hunan Agr Univ, Coll Agr, Changsha 410128, Hunan, Peoples R China.

通讯机构： [Chen, D.] H;Hunan Agr Univ, Coll Biosci & Biotechnol, Dept Hunan Prov Plant Genet & Mol Biol, Key Lab Educ, Changsha 410128, Hunan, Peoples R China.

关键词： Pseudomonas putida;alkaline lipase;thermostability;lipolytic enzyme;alpha/beta-hydrolase

摘要： A novel lipolytic enzyme-producing endophytic strain PC2 was successfully isolated from the seeds of an ideal bioenergy plant Pistacia chinensis Bunge. Based on the analysis of morphology and 16S rRNA sequence, bacterial strain PC2 was identified as a subspecies of Pseudomonas putida, therefore named as P. putida PC2. Whole-genome sequencing showed PC2 contained a 1224-nucleotide lipase gene (named lip-PC2) predicted to encode a 407-amino-acid protein. Purified lipases from both the original PC2 strain and heterologously expressed Escherichia coli were nearly 50 kD with specific activity of 9.48 U/mL. LIP-PC2 displayed the maximal activity at 50°C or pH 8.0, and maintained above 80% relative activity in the range of from 40 to 60°C or pH in the range of from 6.0 to 8.0, indicating thermostable and alkaline properties. Enzyme activity was enhanced by Mg2+, Na+ and Mn2+, but strongly inhibited by Cu2+, Zn2+ Co2+, EDTA as well as organic solvents and surfactants. Additionally, the analysis of amino acid sequence and structure indicated that LIP-PC2 was a novel member belonging to family I.3 of bacterial lipolytic enzymes and its catalytic triad was consisted of Ser-200, Asp-342 and His-374. © 2017, Pleiades Publishing, Inc.

语种： 英文

作者： 张斌;刘询;耿雅楠;阮颖

期刊： 分子植物育种,2017年15(2):559-564 ISSN：1672-416X

作者机构： 湖南农业大学,生物科学技术学院, 湖南省作物种植资源创新和利用重点实验室, 长沙, 410128;湖南农业大学农学院, 长沙, 410128;[张斌; 刘询; 阮颖] 湖南农业大学,生物科学技术学院, 湖南省作物种植资源创新和利用重点实验室, 长沙, 410128;[耿雅楠] 湖南农业大学农学院, 长沙, 410128

关键词： 日本晴;基因沉默

摘要： RNA干扰是一种双链RNA引发的转录后基因沉默技术,具有特异性和高效性,已成为一种基因功能研究的有效手段。在RNA干扰技术中,干扰片段的选取方式会影响基因沉默的效果。但是,目前并无选取干扰片段的固定标准。本研究以水稻多胺转运蛋白基因OsPUT1为对象,在CDS前部、中部和后部分别设计了三种不同干扰片段,构建基因三个RNAi载体,借助农杆菌EHA105介导水稻遗传转化,得到三种转基因苗;通过RT-PCR和Q-PCR技术检测目的基因的表达,结果显示三个干扰部位都具有一定的沉默效果,且在CDS的中间位置干扰效果最好。说明OsPUT1在进行RNAi载体构建时,应考虑在CDS的中间位置进行干扰。

语种： 中文