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Efficient and precise genomic deletion in rice using enhanced prime editing

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aBIOTECH 2024年第2期5卷 214-218页

作者： Mengyuan Liu Xiang Zhang Wen Xu Guiting Kang Ya Liu Xinxiang Liu Wen Ren jiuran zhao Jinxiao Yang Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Beijing Academy of Agriculture and Forestry SciencesBeijing 100097China

Efficient and precise genomic deletion shows promise for investigating the function of proteins in plant research and enhancing agricultural *** this study,we tested the PRIME-Del(PDel)strategy using a pair of prime editing guide RNAs(pegRNAs)that targeted opposite DNA strands and achieved an average deletion efficiency of 55.8%for 60 bp fragment deletions at six endogenous ***,as high as 84.2%precise deletion efficiency was obtained for a 2000 bp deletion at the OsGS1 site in transgenic rice *** add the bases that were unintentionally deleted between the two nicking sequences,we used the PDel/Syn strategy,which introduced multiple synonymous base mutations in the region that had to be patched in the RT *** PDel/Syn strategy achieved an average of 58.1%deletion efficiency at six endogenous targets,which was higher than the PDel *** strategies presented in this study contribute to achieving more accurate and flexible deletions in transgenic rice plants.

关键词： Prime editing Precise genomic deletion CRISPR Cas9

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玉米南方锈病抗病育种研究进展

中国农业科学 2024年第14期57卷 2732-2743页

作者：王帅张如养王荣焕宋伟赵久然北京市农林科学院玉米研究所/玉米 DNA 指纹及分子育种北京市重点实验室

玉米是全世界也是我国种植范围最广、用途最多、总产量最高的作物，玉米生产对保障我国粮食安全具有重要意义。玉米南方锈病是由多堆柄锈菌（Puccinia polysora Underw.）引起的一种气传性真菌病害，主要发生在热带、亚热带玉米种植区... 详细信息

玉米是全世界也是我国种植范围最广、用途最多、总产量最高的作物，玉米生产对保障我国粮食安全具有重要意义。玉米南方锈病是由多堆柄锈菌（Puccinia polysora Underw.）引起的一种气传性真菌病害，主要发生在热带、亚热带玉米种植区。近年来，由于气候变化，该病害开始向高纬度地区蔓延，已逐渐成为我国黄淮海夏玉米区的主要病害，直接导致玉米籽粒品质变差、产量降低，对玉米生产造成严重威胁。目前，我国玉米生产上推广种植的玉米品种大多不抗玉米南方锈病，一旦南方锈病发生和流行，将会导致在短时间内大面积蔓延，而常规化学防治难以控制。因此，挖掘和利用玉米种质资源中的南方锈病抗性基因，进一步选育抗病品种是应对南方锈病为害最经济有效的技术途径。玉米资源中高抗南方锈病的种质较为匮乏，主要来自热带、亚热带，可直接利用的温带种质极少。与国外玉米种质相比，我国玉米种质中的高抗材料较少，主要来自农家种或含有热带血缘的P群材料，遗传基础狭窄。玉米南方锈病抗性基因的鉴定与克隆，对开展分子标记辅助育种、加快育种进程及抗病新品种选育具有重要促进作用，目前，已有多个南方锈病抗性基因被鉴定和克隆，为分子标记辅助选择育种奠定了基础。多年来，我国育种家利用有限的抗性种质资源，选育了多个抗南方锈病的优良玉米自交系，并成功育成抗病杂交种。近期，南方锈病病原菌基因组方面的研究揭示我国多堆柄锈菌群体已分化出高毒谱系，从而逃逸抗病基因的识别。因此，挖掘和利用抗性种质中蕴涵的丰富基因资源仍需进一步加强。本文概述了南方锈病的生物学特性及危害，系统梳理了南方锈病抗性种质资源鉴定、抗性基因定位与克隆，以及抗性品种选育的研究进展，并展望了玉米南方锈病未来研究方向，以期为玉米南方锈病防治和抗病品种选育提供参考。

关键词：玉米南方锈病种质资源抗病基因抗病育种

Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

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The Crop Journal 2020年第3期8卷 408-417页

作者： Feipeng Wang Chengwei Zhang Wen Xu Shuang Yuan Jinling Song Lu Li jiuran zhao Jinxiao Yang Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Beijing Academy of Agriculture&Forestry SciencesBeijing 100097China

The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.

关键词： CBE ABE NLS Developing high-efficiency base editors by combining optimized synergistic core components with new types of nuclear localization signal peptide

Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

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Molecular Plant 2021年第2期14卷 191-194页

作者： Chengwei Zhang Yao Wang Feipeng Wang Si zhao Jinling Song Feng Feng jiuran zhao Jinxiao Yang Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Beijing Academy of Agriculture&Forestry SciencesShuguang Garden Middle Road No.9Haidian DistrictBeijingChina

Dear Editor,Base editors(BEs),including cytosine base editor(CBE)and adenine base editor(ABE),have been widely used to generate irreversible nucleotide substitution in plants and ***,their wide applications are largely hindered by the strict NG protospacer adjacent motif(PAM)sequences recognized by Streptococcus pyogenes Cas9(SpCas9)and its engineered variants,such as SpCas9-NG and xCas9(Hua et al.,2019;Ren et al.,2019;Wu et al.,2019;Zhong et al.,2019;Zhang et al.,2020).Most recently,it was reported that three new SpCas9 variants,SpCas9-NRRH,SpCas9-NRTH,and SpCas9-NRCH,could recognize non-G PAMs(NRNH,where R is A or G and H is A,C,or T)in human cells(Miller et al.,2020).Meanwhile,SPRY,another new SpCas9 variant,was developed to greatly expand the editing scope of BEs to nearly PAMless(Walton et al.,2020).In this study,we generated a series of efficient BE toolkits and almost achieved C-to-T mutation without PAM restriction except for NTG PAM,and largely expanded A-to-G mutation scope in stable transformed rice,providing a reference for application in other plants.

关键词： base irreversible transformed

Trash to treasure:lactate and protein lactylation in maize root impacts response to drought

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Science China(Life Sciences) 2023年第8期66卷 1903-1914页

作者： Zi Shi Miaoyi Zhou Wei Song Ya Liu Ronghuan Wang Yuandong Wang Ruyang Zhang jiuran zhao Wen Ren Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Beijing Academy of Agriculture and Forestry SciencesBeijing 100097China

Lactate,protein lactylation(Kla),and specifically histone lactylation have recently been shown to regulate antipathogenic immune responses in ***,after we confirmed the presence and accumulation of lactate in maize roots under drought conditions,a lactylome profiling analysis revealed that Kla modifications were invariably present in maize roots,that there were obvious differences in the lactylomes of drought-sensitive(B73)***-tolerant(Jing2416)lines,and that growing Jing2416 under drought conditions caused significant decreases in the lactylation of multiple enzymes responsible for fatty acid *** by findings of histone-Kla based epigenetic regulation of immune functions in animals,we initially discovered 37 Kla sites on 16 histones in the maize genome,and again detected obvious differential histone Klamediated trends between two lines by Ch ***,only 2.7%of genes with differential histone Kla peaks detected during drought stress were commonly present in both lines,a finding demonstrating that abiotic stress triggers distinct epigenetic activities in diverse germplasm while also strongly supporting that a histone Kla layer of regulation is associated with physiological responses to drought ***,exogenous application of spermidine improved the drought tolerance of B73and substantially altered the levels of lactate,protein lactylation,and histone Kla ***,beyond extending the known domain of Kla-based biochemical and epigenetic regulation from animal immunity to plant stress physiology,our study suggests the physiological,biochemical,and genetic function of“the best-known metabolic waste”,lactate.

关键词： lactate lactylome drought spermidine maize

Activation of Mitochondrial orf355 Gene Expression by a Nuclear-Encoded DREB Transcription Factor Causes Cytoplasmic Male Sterility in Maize

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Molecular Plant 2020年第9期13卷 1270-1283页

作者： Senlin Xiao Jie Zang Yuanrong Pei Jie Liu Juan Liu Wei Song Zi Shi Aiguo Su jiuran zhao Huabang Chen State Key Laboratory of Plant Cell and Chromosome Engineer ngInnovative Academy of Seed DesignInstitute of Genetics and Developmental BiologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100864China Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Maize Research CenterBeijing Academy of Agriculture and Forestry SciencesBeijing100097China

Coordination between mitochondria and the nucleus is crucial for fertility determination in plants with cytoplasmic male sterility(CMS).Using yeast one-hybrid screening,we identified a transcription factor,ZmDREB1.7,that is highly expressed in sterile microspores at the large vacuole stage and activates the expression of mitochondria-encoded CMS gene orf355.Δpro,a weak allele of ZmDREB1.7 with the loss of a key unfolded protein response(UPR)motif in the promoter,partially restores male fertility of CMS-S ***1.7 expression increases rapidly in response to antimycin A treatment,but this response is attenuated in theΔpro ***,we found that expression of orf355 in mitochondria activates mitochondrial retrograde signaling,which in turn induces ZmDREB1.7 *** together,these findings demonstrate that positive-feedback transcriptional regulation between a nuclear regulator and a mitochondrial CMS gene determines male sterility in maize,providing new insights into nucleus-mitochondria communication in plants.

关键词： maize cytoplasmic male sterility transcription factor nucleus-mitochondria communication

ORF355 confers enhanced salinity stress adaptability to S-type cytoplasmic male sterility maize by modulating the mitochondrial metabolic homeostasis

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Journal of Integrative Plant Biology 2023年第3期65卷 656-673页

作者： Senlin Xiao Wei Song Jinfeng Xing Aiguo Su Yanxin zhao Chunhui Li Zi Shi Zhiyong Li Shuai Wang Ruyang Zhang Yuanrong Pei Huabang Chen jiuran zhao Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Maize Research InstituteBeijing Academy of Agriculture and Forestry SciencesBeijing 100097China State Key Laboratory of Plant Cell and Chromosome Engineering Innovative Academy of Seed DesignInstitute of Genetics and Developmental Biologythe Chinese Academy of SciencesBeijing 100101China

Moderate stimuli in mitochondria improve wideranging stress adaptability in animals, but whether mitochondria play similar roles in plants is largely unknown. Here, we report the enhanced stress adaptability of S-type cytoplasmic male sterility(CMS-S) maize and its association with mild expression of sterilizing gene ORF355. A CMS-S maize line exhibited superior growth potential and higher yield than those of the near-isogenic N-type line in saline fields. Moderate expression of ORF355 induced the accumulation of reactive oxygen species and activated the cellular antioxidative defense system. This adaptive response was mediated by elevation of the nicotinamide adenine dinucleotide concentration and associated metabolic homeostasis. Metabolome analysis revealed broad metabolic changes in CMS-S lines, even in the absence of salinity stress. Metabolic products associated with amino acid metabolism and galactose metabolism were substantially changed, which underpinned the alteration of the antioxidative defense system in CMS-S plants. The results reveal the ORF355-mediated superior stress adaptability in CMS-S maize and might provide an important route to developing salt-tolerant maize varieties.

关键词： cytoplasmic male sterility metabolic homeostasis mitochondria salinity stress adaptability Zea mays L.

Gene-lndexed Mutations in Maize

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Molecular Plant 2018年第3期11卷 496-504页

作者： Xiaoduo Lu Jisheng Liu Wen Ren Qun Yang Zhenguang Chai Rumei Chen Lei Wang Jun zhao Zhihong Lang Haiyang Wang Yunliu Fan jiuran zhao Chunyi Zhang Institute of Molecular Breeding for Maize Qilu Normal University Jinan 250200 China Biotechnology Research Institute Chinese Academy of Agricultural Sciences Beijing 100081 China Maize Research Center Beijing Academy of Agriculture & Forestry Sciences Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding Beijing100097 China These authors contributed equally to this article.

The availability of the B73 inbred reference genome sets the stage for high-throughput functional charac- terization of maize genes on a whole-genome scale. Among the 39 324 protein-coding genes predicted, the vast majority are untapped due to the lack of suitable high-throughput reverse genetic resources. We have generated a gene-indexed maize mutant collection through ethyl methanesulfonate mutagenesis and de- tected the mutations by combining exome capture and next-generation sequencing. A total of 1086 mutated MI plants were sequenced, and 195 268 CG〉TA-type point mutations, including stop gain/loss, missplice, start gain/loss, and various non-synonymous protein mutations as well as 4610 InDel mutations, were identified. These mutations were distributed on 32 069 genes, representing 82% of the predicted protein-coding genes in the maize genome. We detected an average of 180 mutations per mutant line and 6.1 mutations per gene. As many as 27 214 mutations of start codons, stop codons, or missplice sites were identified in 14 101 genes, among which 6232 individual genes harbored more than two such muta- tions. Application of this mutant collection is exemplified by the identification of the ent-kaurene synthase gene, which encodes a key enzyme in the gibberellin biosynthesis pathway. This gene-indexed genome- wide mutation collection provides an important resource for functional analysis of maize genes and may bring desirable allelic variants for genetic breeding in maize.

关键词： maize ethyl methanesulfonate mutation exome capture next-generation sequencing functional genomics

高温胁迫对玉米杂交种及其亲本自交系光合特性、保护酶活性和产量的影响

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中国生态农业学报（中英文） 2024年第9期32卷 1470-1480页

作者：徐田军吕天放张勇刘宏伟刘月娥蔡万涛张如养宋伟邢锦丰赵久然王荣焕北京市农林科学院玉米研究所/玉米DNA指纹及分子育种北京市重点实验室北京100097

研究并明确高温胁迫对玉米杂交种及其亲本自交系叶片光合性能、产量性状等的影响,以期为耐高温玉米品种选育和推广提供理论依据。以‘京农科728’‘MC812’‘先玉335’和‘郑单958’及其亲本自交系为试验材料,于花期前后(大喇叭口期—... 详细信息

研究并明确高温胁迫对玉米杂交种及其亲本自交系叶片光合性能、产量性状等的影响,以期为耐高温玉米品种选育和推广提供理论依据。以‘京农科728’‘MC812’‘先玉335’和‘郑单958’及其亲本自交系为试验材料,于花期前后(大喇叭口期—吐丝后7 d)对其进行高温胁迫(高温胁迫15 d)处理,研究高温胁迫对不同基因型玉米杂交种及其亲本自交系叶绿素相对含量、光合特性、最大光化学效率(F_(v)/F_(m))、保护酶活性、产量及其产量构成要素的影响。结果表明:1)与大田对照相比,花期前后高温胁迫下不同杂交种及其亲本自交系的叶绿素相对含量、净光合速率、气孔导度和F_(v)/F_(m)均呈降低趋势,杂交种以‘京农科728’降幅最小,亲本自交系以‘京2416’降幅最小。胞间CO_(2)浓度和蒸腾速率则均呈升高趋势,杂交种以‘京农科728’增幅最小,亲本自交系以‘京2416’增幅最小。2)花期前后高温胁迫导致玉米杂交种及其亲本自交系的穗位叶磷酸烯醇式丙酮酸羧化酶(PEPCase)、核酮糖-1,5-二磷酸羧化酶(RuBPCase)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性呈降低趋势。3)花期前后高温胁迫降低了玉米杂交种及其亲本自交系的穗行数和行粒数,进而导致产量下降,杂交种‘京农科728’‘MC812’‘先玉335’和‘郑单958’产量分别降低3.18%、5.00%、50.61%和9.50%;亲本自交系中,‘京2416’的穗行数、行粒数和产量降幅最小,而‘PH6WC’产量降幅最大。由此可见,花期前后高温胁迫下‘京农科728’‘MC812’及其父本自交‘京2416’具有较高的叶绿素相对含量、光合能力和保护酶活性,产量受高温影响较小,具有较好的耐热性。

关键词：玉米杂交种亲本自交系高温胁迫光合特性保护酶活性产量

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