小串联模拟靶标(Short tandem target mimic,STTM)技术是一种新开发的miRNA功能研究方法。Tae-miR9677作为一种新发现的在小麦穗部特异性高表达的miRNA,其功能至今未知。为了进一步探索Tae-miR9677的功能,构建了Ubiqutin(UBI)启动子启动...小串联模拟靶标(Short tandem target mimic,STTM)技术是一种新开发的miRNA功能研究方法。Tae-miR9677作为一种新发现的在小麦穗部特异性高表达的miRNA,其功能至今未知。为了进一步探索Tae-miR9677的功能,构建了Ubiqutin(UBI)启动子启动的Tae-miR9677 STTM过表达载体,并通过基因枪介导法对小麦品种绵阳19幼胚愈伤组织进行转化。结果表明,3 683个愈伤组织经过PPT(Phosphinothricin)筛选,最终分化获得42株再生植株;利用特异性引物进行PCR检测,鉴定出8株T0代阳性植株。展开更多
As a critical food crop,sweetpotato(Ipomoea batatas(L.)Lam.)is widely planted all over the world,but it is deeply affected by Sweetpotato Virus Disease(SPVD).The present study utilized short tandem target mimic(STTM)t...As a critical food crop,sweetpotato(Ipomoea batatas(L.)Lam.)is widely planted all over the world,but it is deeply affected by Sweetpotato Virus Disease(SPVD).The present study utilized short tandem target mimic(STTM)technology to effectively up-regulate the expression of laccase(Ib LACs)by successfully inhibiting the expression of mi R397.The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation,including phenylalanine ammonialyase(PAL),4-coumarate-Co Aligase(4 CL),hydroxycinnamoyl Co A:shikimatetransferase(HTC),caffeicacid O-methyltransferase(COMT),and cinnamyl alcohol dehydrogenase(CAD).Meanwhile,the activities of PAL and LAC increased significantly,finally leading to increased lignin content.Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants,reduced the accumulation of SPVD transmitted by Bemisia tabaci(Gennadius),and promoted healthy sweetpotato growth.The results provide new insights for disease resistance breeding and green production of sweetpotato.展开更多
【目的】利用短串联靶标模拟(short tandem target mimic, STTM)技术获得了miR161.1敲减的转基因突变体,且突变体具有明显表型变异,能够为miR161功能研究奠定基础,进而应用于作物杂交种的不育化制种。【方法】通过构建拟南芥pFGC5941-ST...【目的】利用短串联靶标模拟(short tandem target mimic, STTM)技术获得了miR161.1敲减的转基因突变体,且突变体具有明显表型变异,能够为miR161功能研究奠定基础,进而应用于作物杂交种的不育化制种。【方法】通过构建拟南芥pFGC5941-STTM161.1双元表达载体及遗传转化,获得STTM161.1转基因T0代种子。利用基因型分析和表型分析得到STTM161.1突变体。通过转录组分析对miR161.1参与的基因表达调控和生物途径进行研究。【结果】通过实时荧光定量,在拟南芥STTM161.1株系中miR161.1的表达量显著下降,而靶基因则显著上调表达。与野生型相比,STTM161.1突变体植株表现生育期提前、叶片变大、部分不育、果荚出现种子败育的表型。转录组分析结果表明,数个质体表达基因表现显著上调,miR161.1的差异表达基因主要参与的代谢途径包括:次生代谢物生物合成、苯丙烷生物合成、嘌呤代谢、α-亚麻酸代谢。STTM161.1转录组筛选到的差异表达关键基因包括花器官发育关键调控因子MADS编码基因,植物雌雄蕊发育和花粉粒发育相关的转录因子bHLH编码基因。【结论】miR161.1参与了拟南芥雄性不育性、生育期、种子发育等方面的调控。验证17个上调差异表达基因,6个下调差异表达基因可能与miR161.1的功能相关。展开更多
为了阐明Stu-miR156在马铃薯侧根发育中的生物学功能,以马铃薯栽培品种‘Desiree’为试验材料,利用短串联靶标模拟物(Short tandem target mimic,STTM)技术,成功构建了Stu-miR156沉默表达载体,通过农杆菌介导法转化马铃薯获得了转化植株...为了阐明Stu-miR156在马铃薯侧根发育中的生物学功能,以马铃薯栽培品种‘Desiree’为试验材料,利用短串联靶标模拟物(Short tandem target mimic,STTM)技术,成功构建了Stu-miR156沉默表达载体,通过农杆菌介导法转化马铃薯获得了转化植株,qRT-PCR技术检测Stu-miR156及其靶基因StSPL9在转基因植株中的表达量。并通过构建pCAM-GFP-StSPL9融合蛋白表达载体转化烟草,发现靶基因StSPL9位于细胞质和细胞核中。q RT-PCR分析结果表明:在马铃薯转基因株系L1和L2中,Stu-miR156表达量受到严重抑制,在根、茎和叶中均不同程度地下调,其靶基因StSPL9均上调表达。表型分析表明:与野生型对照相比,转基因植株的侧根数量显著减少,生长受到抑制。展开更多
MicroRNA(miRNA)作为一类大小长约21-24个核苷酸的内源非编码RNA,通过影响靶基因mRNA的稳定性或者翻译过程调控基因的表达。随着现代分子生物学技术的发展,越来越多的研究表明miRNA在植物的生长和发育过程中发挥着重要的作用。但是,miRN...MicroRNA(miRNA)作为一类大小长约21-24个核苷酸的内源非编码RNA,通过影响靶基因mRNA的稳定性或者翻译过程调控基因的表达。随着现代分子生物学技术的发展,越来越多的研究表明miRNA在植物的生长和发育过程中发挥着重要的作用。但是,miRNA功能研究缺乏有效的、适用性广的方法。介绍了一种可以特异地靶标miRNA方法,即短串联靶标模拟(Short tandem target mimic,STTM),并对STTM的基本特点和作用机制、与其他类似技术的比较以及其在植物中miRNA功能研究中的应用等方面进行了总结,有望为今后植物miRNA的功能研究提供技术参考。展开更多
microRNAs (miRNAs)are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression,leading to gene silencing.We previously developed short tandem target mimic (STTM)...microRNAs (miRNAs)are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression,leading to gene silencing.We previously developed short tandem target mimic (STTM)technology to deactivate endogenous miRNAs in Arabidopsis.Here,we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis,tomato,rice,and maize,providing a resource for the functional interrogation of miRNAs.We not only revealed the functions of several miRNAs in plant development,but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development.RNA-seq and small RNAseq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation,secondary metabolism,and ion-channel activity-associated electrophysiology,demonstrating that STTM technology is an effective approach for studying miRNA functions.To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks,we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTMinduced miRNA knockdown plants.展开更多
MicroRNAs（miRNAs） are a population of highly conserved specific small ribo-regulators that negatively regulate gene expressions in both plants and animals.They play a key role in post-transcriptional gene regulation...MicroRNAs（miRNAs） are a population of highly conserved specific small ribo-regulators that negatively regulate gene expressions in both plants and animals.They play a key role in post-transcriptional gene regulation by destabilizing the target gene transcripts or blocking protein translation from them.Interestingly,these negative regulators are largely compromised by an upstream layer of negative regulators ＂target mimics＂ found in plants or ＂endogenous competing RNAs＂ revealed recently in animals.These endogenous regulatory mechanisms of ＂double negatives making a positive＂ have now been developed into a key strategy in the study of small RNA functions. This review presents some reflections on the long journey to the short tandem target mimic（STTM） for selective destruction/blockage of specific miRNAs in plants and animals,and the potential applications of STTM are discussed.展开更多
基金financially supported by the National Key R&D Program of China (2019YFD1001300 and 2019YFD1001305)the earmarked fund for CARS-10-Sweetpotatothe Jiangsu Postgraduate Scientific Research and Practical Innovation Program Project, China (KYCX19-2207)
文摘As a critical food crop,sweetpotato(Ipomoea batatas(L.)Lam.)is widely planted all over the world,but it is deeply affected by Sweetpotato Virus Disease(SPVD).The present study utilized short tandem target mimic(STTM)technology to effectively up-regulate the expression of laccase(Ib LACs)by successfully inhibiting the expression of mi R397.The upstream genes in the lignin synthesis pathway were widely up-regulated by feedback regulation,including phenylalanine ammonialyase(PAL),4-coumarate-Co Aligase(4 CL),hydroxycinnamoyl Co A:shikimatetransferase(HTC),caffeicacid O-methyltransferase(COMT),and cinnamyl alcohol dehydrogenase(CAD).Meanwhile,the activities of PAL and LAC increased significantly,finally leading to increased lignin content.Lignin deposition in the cell wall increased the physical defence ability of transgenic sweetpotato plants,reduced the accumulation of SPVD transmitted by Bemisia tabaci(Gennadius),and promoted healthy sweetpotato growth.The results provide new insights for disease resistance breeding and green production of sweetpotato.
文摘【目的】利用短串联靶标模拟(short tandem target mimic, STTM)技术获得了miR161.1敲减的转基因突变体,且突变体具有明显表型变异,能够为miR161功能研究奠定基础,进而应用于作物杂交种的不育化制种。【方法】通过构建拟南芥pFGC5941-STTM161.1双元表达载体及遗传转化,获得STTM161.1转基因T0代种子。利用基因型分析和表型分析得到STTM161.1突变体。通过转录组分析对miR161.1参与的基因表达调控和生物途径进行研究。【结果】通过实时荧光定量,在拟南芥STTM161.1株系中miR161.1的表达量显著下降,而靶基因则显著上调表达。与野生型相比,STTM161.1突变体植株表现生育期提前、叶片变大、部分不育、果荚出现种子败育的表型。转录组分析结果表明,数个质体表达基因表现显著上调,miR161.1的差异表达基因主要参与的代谢途径包括:次生代谢物生物合成、苯丙烷生物合成、嘌呤代谢、α-亚麻酸代谢。STTM161.1转录组筛选到的差异表达关键基因包括花器官发育关键调控因子MADS编码基因,植物雌雄蕊发育和花粉粒发育相关的转录因子bHLH编码基因。【结论】miR161.1参与了拟南芥雄性不育性、生育期、种子发育等方面的调控。验证17个上调差异表达基因,6个下调差异表达基因可能与miR161.1的功能相关。
文摘为了阐明Stu-miR156在马铃薯侧根发育中的生物学功能,以马铃薯栽培品种‘Desiree’为试验材料,利用短串联靶标模拟物(Short tandem target mimic,STTM)技术,成功构建了Stu-miR156沉默表达载体,通过农杆菌介导法转化马铃薯获得了转化植株,qRT-PCR技术检测Stu-miR156及其靶基因StSPL9在转基因植株中的表达量。并通过构建pCAM-GFP-StSPL9融合蛋白表达载体转化烟草,发现靶基因StSPL9位于细胞质和细胞核中。q RT-PCR分析结果表明:在马铃薯转基因株系L1和L2中,Stu-miR156表达量受到严重抑制,在根、茎和叶中均不同程度地下调,其靶基因StSPL9均上调表达。表型分析表明:与野生型对照相比,转基因植株的侧根数量显著减少,生长受到抑制。
文摘汞污染已成为威胁全球作物生产的主要重金属污染源之一。microRNA(miRNA)是调控植物生长发育和非生物胁迫响应的重要因子,但其在单子叶植物汞胁迫响应中的作用尚不明确。为挖掘响应汞胁迫的关键miRNA,本研究对B73和郑58(Zheng 58,Z58)自交系幼苗在HgCl_(2)处理后的表型和差异表达的miRNA进行了分析。结果表明,B73对汞胁迫较郑58更敏感,miRNA166l是B73和Z58中共同鉴定到的下调表达的miRNA。为进一步验证miRNA166在汞胁迫中的作用,利用STTM(short tandem target mimic)技术获得了STTM165/166拟南芥转基因稳定株系。STTM166稳定系在HgCl_(2)处理后的表型与玉米幼苗在汞胁迫处理后的表型类似,叶片失绿萎蔫、根长变短。本研究证明miRNA166在玉米汞胁迫响应中有重要调控作用,对其作用机制进行更深入的研究有重要意义。
文摘MicroRNA(miRNA)作为一类大小长约21-24个核苷酸的内源非编码RNA,通过影响靶基因mRNA的稳定性或者翻译过程调控基因的表达。随着现代分子生物学技术的发展,越来越多的研究表明miRNA在植物的生长和发育过程中发挥着重要的作用。但是,miRNA功能研究缺乏有效的、适用性广的方法。介绍了一种可以特异地靶标miRNA方法,即短串联靶标模拟(Short tandem target mimic,STTM),并对STTM的基本特点和作用机制、与其他类似技术的比较以及其在植物中miRNA功能研究中的应用等方面进行了总结,有望为今后植物miRNA的功能研究提供技术参考。
基金the National Science Foundation,USA (IOS-1048216 and IOS-1340001)the National Natural Science Foundation of China (31571679,31501292,31871554)+1 种基金the Major Science and Technology Project of Henan Province (141100110600)the Support Plan of Science and Technology Innovation Team in Universities of Henan Province (171RTSTHN015),and the Key Scientific Research Project in Universities of Henan Province (16A210009).G.T.is also supported by the Guangdong Innovation Research Team Fund (2014ZT058078)and the 111 Project (D16014)to Henan University.S.T.was supported by a post-doctoral fellowship from Henan Agricultural University.F.M.was a visiting scholar supported by the China Scholarship Council (CSC).T.P.,Z.Z.,L.S.,and L.T.were visiting PhD students supported by scholarships from Henan Agricultural University.
文摘microRNAs (miRNAs)are endogenous small non-coding RNAs that bind to mRNAs and target them for cleavage and/or translational repression,leading to gene silencing.We previously developed short tandem target mimic (STTM)technology to deactivate endogenous miRNAs in Arabidopsis.Here,we created hundreds of STTMs that target both conserved and species-specific miRNAs in Arabidopsis,tomato,rice,and maize,providing a resource for the functional interrogation of miRNAs.We not only revealed the functions of several miRNAs in plant development,but also demonstrated that tissue-specific inactivation of a few miRNAs in rice leads to an increase in grain size without adversely affecting overall plant growth and development.RNA-seq and small RNAseq analyses of STTM156/157 and STTM165/166 transgenic plants revealed the roles of these miRNAs in plant hormone biosynthesis and activation,secondary metabolism,and ion-channel activity-associated electrophysiology,demonstrating that STTM technology is an effective approach for studying miRNA functions.To facilitate the study and application of STTM transgenic plants and to provide a useful platform for storing and sharing of information about miRNA-regulated gene networks,we have established an online Genome Browser (https://blossom.ffr.mtu.edu/designindex2.php) to display the transcriptomic and miRNAomic changes in STTMinduced miRNA knockdown plants.
基金Work done in Guiliang Tang's lab was supported,in whole or in part,by USDA National Research Initiative grants(2006- 35301-17115 and 2006-35100-17433)the National Science Foundation grants(MCB-0718029:Subaward No.S-00000260 and IOS-1048216/IOS-1219316)+1 种基金the startup of Michigan Technological UniversityWork done in Xiaoqing Tang's lab was supported in part by the National Institutes of Health(K01 DK078648 and R03 DK084166)
文摘MicroRNAs（miRNAs） are a population of highly conserved specific small ribo-regulators that negatively regulate gene expressions in both plants and animals.They play a key role in post-transcriptional gene regulation by destabilizing the target gene transcripts or blocking protein translation from them.Interestingly,these negative regulators are largely compromised by an upstream layer of negative regulators ＂target mimics＂ found in plants or ＂endogenous competing RNAs＂ revealed recently in animals.These endogenous regulatory mechanisms of ＂double negatives making a positive＂ have now been developed into a key strategy in the study of small RNA functions. This review presents some reflections on the long journey to the short tandem target mimic（STTM） for selective destruction/blockage of specific miRNAs in plants and animals,and the potential applications of STTM are discussed.
