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CRISPR/Cas9-based gene activation and base editing in Populus

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Horticulture Research 2023年第6期10卷 205-213页

作者： Tao Yao Guoliang Yuan Haiwei Lu Yang Liu jin Zhang Gerald A.Tuskan Wellington Muchero jin-gui chen Xiaohan Yang Biosciences Division Oak Ridge National LaboratoryOak RidgeTN 37831USA The Center for Bioenergy Innovation Oak Ridge National LaboratoryOak RidgeTN 37831USA Chemical and Biological Process Development Group Pacific Northwest National Laboratory902 Battelle BoulevardRichlandWA 99352USA Department of Academic Education Central Community College-HastingsHastingsNE 68901USA State Key Laboratory of Subtropical Silviculture College of Forestry and BiotechnologyZhejiang A&F UniversityHangzhou 311300China

The genus Populus has long been used for environmental,agroforestry and industrial applications *** Populus is also recognized as a desirable crop for biofuel production and a model tree for physiological and ecological *** such,various modern biotechnologies,including CRISPR/Cas9-based techniques,have been actively applied to Populus for genetic and genomic improvements for traits such as increased growth rate and tailored lignin ***,CRISPR/Cas9 has been primarily used as the active Cas9 form to create knockouts in the hybrid poplar clone“717-1B4”(*** x *** clone INRA 717-1B4).Alternative CRISPR/Cas9-based technologies,*** involving modified Cas9 for gene activation and base editing,have not been evaluated in most Populus species for their *** we employed a deactivated Cas9(dCas9)-based CRISPR activation(CRISPRa)technique to fine-tune the expression of two target genes,TPX2 and LecRLK-G which play important roles in plant growth and defense response,in hybrid poplar clone“717-1B4”and poplar clone“WV94”(***“WV94”),*** observed that CRISPRa resulted in 1.2-fold to 7.0-fold increase in target gene expression through transient expression in protoplasts and Agrobacterium-mediated stable transformation,demonstrating the effectiveness of dCas9-based CRISPRa system in *** addition,we applied Cas9 nickase(nCas9)-based cytosine base editor(CBE)to precisely introduce premature stop codons via C-to-T conversion,with an efficiency of 13%–14%,in the target gene PLATZ which encodes a transcription factor involved in plant fungal pathogen response in hybrid poplar clone“717-1B4”.Overall,we showcase the successful application of CRISPR/Cas-based technologies in gene expression regulation and precise gene engineering in two Populus species,facilitating the adoption of emerging genome editing tools in woody species.

关键词： CRISPR/Cas9 activation base

The grapevine kinome:annotation,classification and expression patterns in developmental processes and stress responses

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Horticulture Research 2018年第1期5卷 682-697页

作者： Kaikai Zhu Xiaolong Wang jinyi Liu Jun Tang Qunkang cheng jin-gui chen Zong-Ming(Max)cheng College of Horticulture Nanjing Agricultural UniversityNanjingJiangsu210095China Department of Plant Sciences University of TennesseeKnoxvilleTN37996USA Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement Institute of HorticultureJiangsu Academy of Agricultural SciencesNanjingJiangsu210014China Department of Entomology and Plant Pathology University of TennesseeKnoxvilleTN37996USA Biosciences Division Oak Ridge National LaboratoryOak RidgeTN37831USA

Protein kinases(PKs)have evolved as the largest family of molecular switches that regulate protein activities associated with almost all essential cellular *** a fraction of plant PKs,however,have been functionally characterized even in model plant *** the present study,the entire grapevine kinome was identified and annotated using the most recent version of the grapevine genome.A total of 1168 PK-encoding genes were identified and classified into 20 groups and 121 families,with the RLK-Pelle group being the largest,with 872 *** 1168 kinase genes were unevenly distributed over all 19 chromosomes,and both tandem and segmental duplications contributed to the expansion of the grapevine kinome,especially of the RLK-Pelle ***/Ks values indicated that most of the tandem and segmental duplication events were under purifying *** grapevine kinome families exhibited different expression patterns during plant development and in response to various stress treatments,with many being *** comprehensive annotation of grapevine kinase genes,their patterns of expression and coexpression,and the related information facilitate a more complete understanding of the roles of various grapevine kinases in growth and development,responses to abiotic stress,and evolutionary history.

关键词： stress grape treatment

Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants

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Horticulture Research 2021年第1期8卷 3286-3296页

作者： Guoliang Yuan Haiwei Lu Dan Tang Md Mahmudul Hassan Yi Li jin-gui chen Gerald A.Tuskan Xiaohan Yang Bioscie nces Divisi on Oak Ridge Nati onal Lab oratoryOak RidgeTN 37831USA The Center for Bioe nergy Inno vati on Oak Ridge Nati onal Lab oratoryOak RidgeTN 37831USA Department of Plant Science and Landscape Architecture University of ConnecticutStorrsCT 06269USA National Center for Citrus Improvement College of HorticultureHunan Agricultural UniversityChangsha 410128 HunanChina Department of Genetics and Plant Breeding Patuakhali Science and Technology UniversityDumkiPatuakhali 8602Bangladesh

Green fl uorescent protein(GFP)has been widely used for monitoring gene expression and protein localization in diverse ***,highly sensitive imaging equipment,like fl uorescence microscope,is usually required for the visualization of GFP,limitings its application to fi xed locations in samples.A reporter that can be visualized in realtime regardless the shape,size and location of the target samples will increase the fl exibility and ef fi ciency of research ***,we report the application of a GFP-like protein,called eYGFPuv,in both transient expression and stable transformation,in two herbaceous plant species(Arabidopsis and tobacco)and two woody plant species(poplar and citrus).We observed bright fl uorescence under UV light in all of the four plant species without any effects on plant growth or *** was shown to be effective for imaging transient expression in leaf and root *** a focus on in vitro transformation,we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identi fi ed visually during the callus stage and the shoot stage,enabling early and ef fi cient selection of ***,whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic *** addition,our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without *** work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

关键词： transient transformation application

Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research

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BioDesign Research 2020年第1期2020卷 124-137页

作者： Md.Mahmudul Hassan Guoliang Yuan jin-gui chen Gerald A.Tuskan Xiaohan Yang Biosciences Division Oak Ridge National LaboratoryOak Ridge TN 37831USA Center for Bioenergy Innovation Oak Ridge National LaboratoryOak RidgeTN 37831USA Department of Genetics and Plant Breeding Patuakhali Science and Technology UniversityDumkiPatuakhali 8602Bangladesh

Many applications in plant biology requires editing genomes accurately including correcting point mutations,incorporation of single-nucleotide polymorphisms(SNPs),and introduction of multinucleotide insertion/deletions(indels)into a predetermined position in the *** types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems,which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit ***,low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely *** kind of technology known as base editing can perform precise editing;however,only certain types of modifications can be obtained,e.g.,C/G-to-T/A and A/T-to-G/***,a new type of genome-editing technology,referred to as“prime editing,”has been developed,which can achieve various types of editing such as any base-to-base conversion,including both transitions(C→T,G→A,A→G,and T→C)and transversion mutations(C→A,C→G,G→C,G→T,A→C,A→T,T→A,and T→G),as well as small indels without the requirement for inducing double-stranded break in the *** prime editing has wide flexibility to achieve different types of edits in the genome,it holds a great potential for developing superior crops for various purposes,such as increasing yield,providing resistance to various abiotic and biotic stresses,and improving quality of plant *** this review,we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.

关键词： donor holds precise

Reconfiguring Plant Metabolism for Biodegradable Plastic Production

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BioDesign Research 2020年第1期2020卷 111-123页

作者： Haiwei Lu Guoliang Yuan Steven H.Strauss Timothy J.Tschaplinski Gerald A.Tuskan jin-gui chen Xiaohan Yang Biosciences Division Oak Ridge National LaboratoryOak RidgeTN 37831USA The Center for Bioenergy Innovation Oak Ridge National LaboratoryOak RidgeTN 37831USA Department of Forest Ecosystems and Society Oregon State UniversityCorvallisOR 97331USA

For decades,plants have been the subject of genetic engineering to synthesize novel,value-added ***(PHAs),a large class of biodegradable biopolymers naturally synthesized in eubacteria,are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic *** was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic ***,after three decades of effort synthesizing PHAs,and in particular the simplest form polyhydroxybutyrate(PHB),and seeking to improve their production in plants,it has proven very difficult to reach a commercially profitable rate in a normally growing *** seems to be due to the growth defects associated with PHA production and accumulation in plant ***,we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been ***,from the point of view of plant synthetic biology,we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production,with the goal of maximizing PHA yield while minimizing growth ***,we suggest genetic elements that can be considered in genetic circuit design,approaches for nuclear genome and plastome modification,and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways.

关键词： breakthrough seeking Plant

Advances and perspectives in discovery and functional analysis of small secreted proteins in plants

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Horticulture Research 2021年第1期8卷 1618-1631页

作者： Xiao-Li Hu Haiwei Lu Md Mahmudul Hassan jin Zhang Guoliang Yuan Paul E.Abraham Him K.Shrestha Manuel I.Villalobos Solis jin-gui chen Timothy J.Tschaplinski Mitchel J.Doktycz Gerald A.Tuskan Zong-Ming(Max)cheng Xiaohan Yang Department of Plant Sciences University of TennesseeKnoxvilleTNUSA Biosciences Division Oak Ridge National LaboratoryOak RidgeTNUSA State Key Laboratory of Subtropical Silviculture School of Forestry and BiotechnologyZhejiang A&F UniversityHangzhouZhejiangChina The Center for Bioenergy Innovation Oak Ridge National LaboratoryOak RidgeTNUSA Department of Genome Science and Technology University of TennesseeKnoxvilleTNUSA College of Horticulture Nanjing Agricultural UniversityNanjingJiangsuChina

Small secreted proteins(SSPs)are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion *** plants,SSPs have been found to play important roles in various processes,including plant growth and development,plant response to abiotic and biotic stresses,and beneficial plant–microbe *** the past 10 years,substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture,bioenergy,and ***,there are potentially a lot of SSPs that have not been discovered in plant genomes,which is largely due to limitations of existing computational *** advances in genomics,transcriptomics,and proteomics research,as well as the development of new computational algorithms based on machine learning,provide unprecedented capabilities for genome-wide discovery of novel SSPs in *** this review,we summarize known SSPs and their functions in various plant *** we provide an update on the computational and experimental approaches that can be used to discover new ***,we discuss strategies for elucidating the biological functions of SSPs in plants.

关键词： species functional dating

Retrorectal tumors in adults:Magnetic resonance imaging findings

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World Journal of Gastroenterology 2010年第46期16卷 5822-5829页

作者： Bo-Lin Yang,Yun-Fei Gu,Wan-jin Shao,Hong-jin chen,gui-Dong Sun,Department of Coloproctology,The Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China Hei-Ying jin,Department of Coloproctology,The 3rd Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China Xin Zhu,Department of Radiology,The Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China Department of Radiology The Affiliated Hospital of Nanjing University of Chinese Medicine No.155 Hanzhong Road Nanjing 210029 China. 66zhuxin@***

AIM:To retrospectively evaluate the magnetic resonance imaging (MRI) features of adult retrorectal tumors and compare with histopathologic ***:MRI features of 21 patients with preoperative suspicion of retrorectal tumors were analyzed based on the histopathological and clinical ***:Fourteen benign cystic lesions appeared hypointense on T1-weighted images,and hyperintense on T2-weighted images with regular peripheral *** or dermoid cysts were unilocular,and tailgut cysts were *** of intracystic intermediate signal intensity was observed in one case of tailgut cyst with a component of *** solid tumors were malignant lesions and showed heterogeneous intensity on *** adenocarcinomas showed high signal intensity on T2-weighted and mesh-like enhancing areas on fat-suppressed T2-weighted *** was a fistula between the mass and anus with an internal opening in mucinous adenocarcinomas arising from anal *** stromal tumors displayed low signal intensity on T1-weighted images,and intermediate to high signal intensity on T2-weighted *** necrosis could be seen as a high signal on T2-weighted ***:MRI is a helpful technique to define the extent of the retrorectal tumor and its relationship to the surrounding structures,and also to demonstrate possible complications so as to choose the best surgical approach.

关键词： Retrorectal tumor Presacral lesions Magnetic resonance imaging Congenital cyst Malignant tumor Diagnosis

Studies on Growing Edible Fungi on Improved Straw from a Dual Use Rice Cultivar

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Agricultural Sciences in China 2002年第8期1卷 871-877页

作者： ZHENG jin-gui, chen Jun-chen, YANG jing, ZHENG Kai-bin, YE Xin-fu, HUANG Qin-lou, XIE Bao-gui, chen Bing-huan, TU Gai-lin and chen Wang-rui( Fujian Agriculture and Forestry University, Fuzhou 350002, P. R . China Fujian Academy of Agricultural Sciences,Fuzhou 350003, P.R. China) Fujian Agriculture and Forestry University Fuzhou 350002 P.R. China Fujian Academy of Agricultural Sciences Fuzhou 350003 P. R . China

Every year 500 million tons of rice (Oryza sativa L.) straw are generated worldwide, which was almost equal to the production of rice grain. Most straw is not recovered due to the poor quality, high cost or complicated treatment operations. Through genetic improvements, a rice cultivar '201' was developed with both high nutrient content in straw and high grain yield. The results of 15N tracer experiments showed thatls N of urea could be effectively transformed into straw protein of ' 201' and the recovery rate of 15N in the straw of '201' was 104% higher than that of the check 'TY 63'. The straw protein of '201' could be effectively transformed into mushroom protein and the recovery rate of 15N was 21.7% higher than that of 'TY 63'. Using the straw of ' 201' as material to cultivate straw-based edible fungi, improved the yield and protein content of mushrooms. A total of 7.5 mg·ha-1straw of '201' increased the yield and crude protein of the edible mushroom Volvariella volvacea 797 kg yield and 67.9 kg respectively, and increased the yield and crude protein of the edible mushroom Agaricus blazei Murrill 675 kg and 80 kg respectively. The straw powder of '201' could be used to substitute for the same weight of wheat bran to cultivate lignicolous edible fungi, with no significant difference in terms of mushroom yield or the crude protein content. Straw powder could substitute for 20% of total amount of material to grow Auricularia polytricha, Auricularia auricula, Hypsizygus tessulatus, Lentinula edodes and Hericium erinaceus, 8% of that to grow Agrocybe cylindracea and 7.5% of that to grow Flammulina velutipes respectively.

关键词： Rice Oryza sativa Straw quality Breeding Edible fungi

Studies on Feeding Animals with Straw of Grain-Straw-Dual-Use-Rice 201

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Agricultural Sciences in China 2002年第11期1卷 1286-1292页

作者： ZHENG jin-gui, chen Jun-chen, HUANG Qin-lou, ZHENG Kai-bin YE Xin-fu, TU Jie-feng and chen Bing-huan(Fujian University of Agriculture and Forestry , Fuzhou 350002 ,P. R. China Fujian Academy of Agricultural Sciences , Fuzhou 350003 ,P. R. China) Fujian University of Agriculture and Forestry Fuzhou 350002 P.R.China Fujian Academy of Agricultural Sciences Fuzhou 350003 P.R.China

The straw of Grain-Straw-Dual-Use-Rice (GSDUR) variety 201 of which the grain quality and yield were equivalent to that of common rice variety (the grain yield approximately 7.5 t ha-1), but straw protein content was 9.31% (common rice straw i. e. CK was approximately 4.0%), and other eight fodder indexes were better than CK to some extent, was employed to feed animals. 15N tracing result suggested that the protein in 201 straw could be effectively transformed into fish body protein and white mouse body protein. The digestibility of fodder, the 15N recovery rate of animal body and the absorption of fodder protein were 13. 8, 9. 6, 24.49% and 16. 5, 6.0, 47. 2% higher than those of common rice straw respectively when feeding grass carp and white mice with 201 rice straw, whereas the 15N recovery rates of animal manure were 3. 25, 6. 5% lower than those of common rice straw, respectively. The results of feeding animals with 201 straw were as follows: (1)The milk yield of cow was increased by 8.3 % as compared to that of common rice straw. 843. 8 kg milk could be produced with 7. 5 t ha-1 of dry 201 straw. (2)Fish daily weight gain, straw consumption of grass carp, fish weight gain per kg fresh rice straw were increased by 60.0, 16.8 and 37.0 % respectively when 201 fresh straw was used to feed grass carp compared to feeding CK, and fish yield could be increased by 297.5 kg with 25.0 t ha-1 of fresh 201 straw. (3)The goose daily weight gain and the ratio of fodder and meat were increased by 33. 9 and 26.8 % respectively when 201 rice straw was used as the main raw material of the compound fodder to feed white geese compared to feeding CK, and geese weight could be increased by 2 358.0 kg with one hectare of 201 straw. (4)Not only were the feeding results identical when high quality 201 rice straw powder substituting for wheat bran which made up of 5 % compound fodder to feed cross bred pigs compared to CK, but 0.11 kg fine fodder could be saved when 1 kg cross bred pig

关键词： Rice (Oryza saliva L.) Straw Quality improvement Feeding animal