CN117024541A - 烟草铁氧化还原蛋白基因Ntab0899580及其应用 - Google Patents
烟草铁氧化还原蛋白基因Ntab0899580及其应用 Download PDFInfo
- Publication number
- CN117024541A CN117024541A CN202310836794.4A CN202310836794A CN117024541A CN 117024541 A CN117024541 A CN 117024541A CN 202310836794 A CN202310836794 A CN 202310836794A CN 117024541 A CN117024541 A CN 117024541A
- Authority
- CN
- China
- Prior art keywords
- gene
- tobacco
- plant
- iron redox
- redox protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 58
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 41
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 21
- 244000061176 Nicotiana tabacum Species 0.000 title claims description 4
- 241000196324 Embryophyta Species 0.000 claims abstract description 46
- 241000208125 Nicotiana Species 0.000 claims abstract description 37
- 235000000346 sugar Nutrition 0.000 claims abstract description 20
- 238000010362 genome editing Methods 0.000 claims abstract description 11
- 230000001276 controlling effect Effects 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 4
- 230000014509 gene expression Effects 0.000 claims abstract description 3
- 108091033409 CRISPR Proteins 0.000 claims description 18
- 238000010354 CRISPR gene editing Methods 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000005516 engineering process Methods 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 239000000463 material Substances 0.000 description 27
- 238000012258 culturing Methods 0.000 description 11
- 238000000034 method Methods 0.000 description 8
- 230000004069 differentiation Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000030279 gene silencing Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 241000589158 Agrobacterium Species 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000012226 gene silencing method Methods 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000219194 Arabidopsis Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 2
- 244000020518 Carthamus tinctorius Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 2
- 229960003669 carbenicillin Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000000243 photosynthetic effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 101001060343 Arabidopsis thaliana Ferredoxin-2, chloroplastic Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241001002356 Valeriana edulis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004136 fatty acid synthesis Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000002165 photosensitisation Effects 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明提供了一种烟草铁氧化还原蛋白基因Ntab0899580在调控烟草株高和叶片数中的应用，降低烟草铁氧化还原蛋白基因Ntab0899580表达后，基因编辑植株株高显著降低，叶片数显著减少，成熟期叶片内的总糖还原糖含量升高，这为进一步阐明烟草铁氧化还原蛋白基因的调控机理提供了理论依据，为培育株高和叶片数显著改变的烟草品种提供了新的遗传材料。
Description
技术领域
本发明属于烟草基因工程领域，具体涉及一种烟草铁氧化还原蛋白基因Ntab0899580及其在调控烟草株高和叶片数中的应用。
背景技术
铁氧化还原蛋白广泛存在于各种植物、动物及微生物体内参与电子传递。在光合过程中，光系统接受光子，经过各个电子传递蛋白，最终将光能转化为化学能，光合型铁氧化还原蛋白作为唯一的可溶性电子受体，可将来自光系统I的电子传输到下游各种铁氧化还原蛋白依赖的代谢过程，涉及碳同化、氮同化、硫同化、叶绿素代谢、光敏色素合成、脂肪酸合成等诸多生物学途径，是光合电子向多种生物学途径分配的中枢元件，对植物的生长发育至关重要。研究发现拟南芥铁氧还蛋白——AtFd2的基因缺失突变体(Fd2-KO突变体)在长日照与短日照培养条件下，较其野生型而言均表现出花期提前的表型，而且显示AtFd2与AtHY2在叶绿体中发生互作，并且Fd2突变体对光敏色素的反应受到抑制。
综上，对烟草中铁氧化还原蛋白的研究具有重要的理论与实践意义。为了解决以上问题，提出本发明。
发明内容
本发明提供了一种烟草铁氧化还原蛋白基因Ntab0899580，其核苷酸序列如SEQID NO.1所示。
优选地，将所述的Ntab0899580基因的核苷酸序列进行翻译后，所得氨基酸序列如SEQ ID NO.2所示。
本发明另一方面提供了一种第一方面所述烟草铁氧化还原蛋白基因Ntab0899580的应用，基因编辑植株成熟期叶片内的总糖、还原糖含量升高，株高显著降低，叶片数显著减少。
优选地，基因编辑是通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除Ntab0899580基因的CRISPR/Cas9编辑载体，经遗传转化后获得了Ntab0899580基因发生编辑的烟草植株。
相对于现有技术，本发明具有以下有益效果：
本发明利用基因沉默技术通过敲除或者沉默Ntab0899580基因后，可以明显增加基因编辑植株新鲜烟叶中总糖还原糖含量，可以明显降低烟草株高，减少叶片数，基于此，这为进一步阐明烟草铁氧化还原蛋白基因的调控机理提供了理论依据，为培育株高和叶片数显著改变的烟草品种提供了新的遗传材料。
附图说明
图1为基因编辑植株自然株高与对照植株比较图；
图2为基因编辑植株打顶株高与对照植株比较图；
图3为基因编辑植株自然叶数与对照植株比较图；
图4为基因编辑植株有效叶数与对照植株比较图；
图5为基因编辑植株烤后烟叶总糖还原糖(左)与对照植株比较图。
具体实施方式
下面结合具体实施例对本发明进行说明，但本发明的实施方式不限于此。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件所用的通用设备、材料、试剂等，如无特殊说明,均可从商业途径得到。
实施例1
本实施例主要就烟草铁氧化还原蛋白基因Ntab0899580的获得过程，简要介绍如下。
以栽培种烟草红花大金元根为实验材料，利用RNA提取试剂盒提取烟草根部总RNA，反转录为cDNA备用：
按照植物RNA提取试剂盒说明书提取烟草总RNA。
1μg从叶片中提取总RNA用于反转录，转录体系如下：
Total RNA 1μg
Oligo(dT)(10μM) 1.5μL
ddH2O up to 15μL
将上述体系混匀后置于PCR中，70℃保温5min，去除后立即置于冰上5min，之后向体系中加入以下试剂：
上述体系放入PCR仪中，42℃下65min，65℃下10min，4℃保温，之后置于-20℃冰箱中保存使用。
通过同源比对的方法，参考拟南芥基因的序列及已知烟草部分基因序列，设计扩增引物序列如下：
F：5’-TGCTCGTTGGGTTCCGTAAA-3’；(SEQ ID No.3)
R：5’-CCAGAAACCAACTTGCCTGC-3’；(SEQ ID No.4)
以上述所制备cDNA为模板，利用上述引物进行PCR扩增：
扩增体系(50μL)：
混匀离心后进行PCR扩增，PCR反应条件为：95℃10sec，52℃30sec，72℃2min，共30个循环；72℃10min；25℃Hold。
对扩增产物进行提纯后测序，获得烟草铁氧化还原蛋白基因Ntab0899580序列，其碱基序列如SEQ ID No.1所示。对该基因序列进行翻译后，其所编码蛋白序列如SEQ IDNo.2所示，进一步对比分析表明，该蛋白含有同源性很高的序列，高度保守。
实施例2
利用实施例1中所获得烟草铁氧化还原蛋白基因Ntab0899580，本发明进一步构建了CRISPR/Cas9载体，以及利用叶盘法转化获得基因编辑植株。
选择Ntab0899580基因中较特异的23nt核苷酸序列TCGACTATAAGTGTTCCTTCAGG(SEQ ID No.5)为CRISPR/Cas9的引导序列，并将该序列片段与CRISPR/Cas9载体(由西南大学提供)进行连接、转化和PCR扩增检测，PCR阳性克隆送测序公司进行测序确认，最后得到CRISPR/Cas9-Ntab0899580编辑载体。
利用上一步所构建的CRISPR/Cas9-Ntab0899580编辑载体质粒，以红花大金元为例，进行遗传转化和组培，以获得烟草铁氧化还原蛋白基因Ntab0899580发生敲除编辑的植株，相关实验过程简要介绍如下。
将烟草种子表面消毒后点种至MS培养基上，待长到4片子叶(15-20d)，移入含MS固体培养基的培养瓶中，于25±1℃、光照强度30-50μmol/(m2·s)，光照时间为16h/d条件继续培养35-40d，备用。
取出-80℃保存的LBA4404电转化感受态农杆菌细胞，置于冰上冻融。待感受态刚刚解冻时，加入CRISPR/Cas9-Ntab0899580编辑载体质粒的2μL，混匀，置于冰上。后将混匀的感受态转移至预冷的电转杯中，将电转杯置于电转仪中进行转化，转化完成后加入1mL的YEB液体培养基与转化液进行混合，后置于摇床28℃，200rpm培养1.5-2h。8000rpm离心菌体弃掉上清培养基，然后用200μL的YEB液体培养基悬浮菌体，涂于含50mg/L利福平、50mg/L链霉素和50mg/L卡那霉素的YEB固体培养基上28℃倒置黑暗培养2-3d。
在超净工作台中制作烟草叶盘成边长为1cm的方形叶盘，用MS液体制备含有CRISPR/Cas9-Ntab0899580编辑载体的农杆菌菌落成悬浮菌液(OD600＝0.6-0.8)。利用悬浮农杆菌菌液浸泡侵染烟草叶盘10min。之后将叶盘置于含2.0mg/LNAA+0.5mg/L 6-BA的MS固体培养基上，28℃，黑暗，共培养3d。之后进行继代培养，放置于含2.0mg/L NAA+0.5mg/L6-BA+250mg/L Cb+50mg/L Kan的MS固体培养基上，培养条件为：28℃光照培养16h/d，光照强度30-50μmol/(m2·s)，25℃黑暗培养8h/d，培养45-60d，直至分化芽形成，每7-10d更换一次分化培养培养基，更换3-4次；培养至分化芽形成；将已有分化芽形成的愈伤组织切下，置于含有500mg/L羧苄青霉素与50mg/L卡那霉素的MS培养基上进行培养，待愈伤组织上分化芽培养长至2-4cm高，培养条件与分化培养条件一致，培养8-14d；再生植株生根培养，将分化芽切下，插入含有500mg/L羧苄青霉素与50mg/L卡那霉素的MS培养基上进行生根培养，培养条件与分化培养条件一致，培养20-30d，再生移栽至花盆后进行培养，后进行转化植株叶片取样，送华大基因进行分子检测，确定获得Ntab0899580基因编辑植株，之后进行收种获得T0代编辑植株种子。T0代种子按23倍进行自交纯合扩繁，待植株长到5-6片叶时，单株的叶片取样，送华大基因进行分子检测，确定获得Ntab0899580基因发生纯合编辑的植株，之后进行收种获得Ntab0899580基因纯合编辑的T1代种子。
本发明利用基因沉默技术通过敲除或者沉默Ntab0899580基因后，可以明显增加基因编辑植株新鲜烟叶中总糖还原糖含量，可以明显降低烟草株高，减少叶片数。现有技术领域内常用的降低基因表达或者基因沉默的方法均适用于本发明。
实施例3
利用实施例2中分子检测确定为Ntab0899580基因纯合敲除的植株，进行收种获得基因纯合编辑素材。随后分别进行Ntab0899580基因纯合敲除素材与对照素材自然株高、打顶株高、自然叶数、有效叶数的比较。
Ntab0899580基因纯合敲除素材自然株高与对照素材(CK)相比结果如图1所示，可以看出，Ntab0899580基因纯合敲除素材和对照素材(CK)比较，自然株高显著降低。
Ntab0899580基因纯合敲除素材打顶株高与对照素材(CK)相比结果如图2所示，可以看出，Ntab0899580基因纯合敲除素材和对照素材(CK)比较，打顶株高显著降低。
Ntab0899580基因纯合敲除素材自然叶数与对照素材(CK)相比结果如图3所示，可以看出，Ntab0899580基因纯合敲除素材和对照素材(CK)比较，自然叶数显著减少。
Ntab0899580基因纯合敲除素材有效叶数与对照素材(CK)相比结果如图3所示，可以看出，Ntab0899580基因纯合敲除素材和对照素材(CK)比较，有效叶数显著减少。
实施例4
利用实施例2中分子检测确定为Ntab0899580基因纯合敲除的植株，进行收种获得基因纯合编辑素材。随后分别进行Ntab0899580基因纯合敲除素材与对照素材成熟期新鲜烟叶总糖还原糖含量比较。
成熟期Ntab0899580基因纯合敲除素材新鲜烟叶总糖、还原糖含量与对照素材(CK)相比结果如图5所示，可以看出，成熟期Ntab0899580基因纯合敲除素材新鲜烟叶和对照素材(CK)比较，总糖、还原糖显著增加27.08％、69.48％。
Claims (4)
1.烟草铁氧化还原蛋白基因Ntab0899580，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的基因，其特征在于，将所述的Ntab0899580基因的核苷酸序列进行翻译后，所得氨基酸序列如SEQ ID NO.2所示。
3.一种权利要求1-2任一所述烟草铁氧化还原蛋白基因Ntab0899580在调控烟草株高和叶片数中的应用，降低烟草铁氧化还原蛋白基因Ntab0899580表达后，基因编辑植株成熟期叶片内的总糖、还原糖含量升高，株高显著降低，叶片数显著减少。
4.根据权利要求3所述的应用，其特征在于，基因编辑是通过CRISPR/Cas9介导的基因编辑技术，构建了用于敲除Ntab0899580基因的CRISPR/Cas9编辑载体，经遗传转化后获得了Ntab0899580基因发生编辑的烟草植株。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310836794.4A CN117024541A (zh) | 2023-07-10 | 2023-07-10 | 烟草铁氧化还原蛋白基因Ntab0899580及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310836794.4A CN117024541A (zh) | 2023-07-10 | 2023-07-10 | 烟草铁氧化还原蛋白基因Ntab0899580及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117024541A true CN117024541A (zh) | 2023-11-10 |
Family
ID=88630747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310836794.4A Pending CN117024541A (zh) | 2023-07-10 | 2023-07-10 | 烟草铁氧化还原蛋白基因Ntab0899580及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117024541A (zh) |
-
2023
- 2023-07-10 CN CN202310836794.4A patent/CN117024541A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110257421B (zh) | 一种甘蓝型油菜基因突变体ptg8的构建方法及其应用 | |
| CN113046371A (zh) | 一种烟草过氧化物酶相关的基因及其应用 | |
| CN113151307B (zh) | 一种烟草乙烯响应转录因子相关的基因及其应用 | |
| CN112921051B (zh) | 一种通过基因编辑技术创制西瓜雄性不育种质的方法 | |
| CN114438105B (zh) | 一种烟草NtMLO6-1基因及其敲除方法与应用 | |
| CN113151296B (zh) | 一种烟草热激蛋白相关的基因及其应用 | |
| CN114507688A (zh) | 一种烟草NtCNGC4基因在制备调控株高和叶片氨基酸含量的烟草突变体材料中的应用 | |
| CN117024541A (zh) | 烟草铁氧化还原蛋白基因Ntab0899580及其应用 | |
| CN114085851B (zh) | 一种调控烟草开花时间相关的基因及应用 | |
| CN114836430B (zh) | 一种烟草ABA受体蛋白基因NtPYL6在调控烟草株高和叶片芸香苷含量中的应用 | |
| CN114807081B (zh) | 一种烟草谷胱甘肽转移相关的基因及其应用 | |
| CN114805513B (zh) | 烟草NtOEE1基因及其在调控茎叶夹角和株高中的应用 | |
| CN114085845B (zh) | 一种烟草烟碱代谢相关的基因及其应用 | |
| CN116144698A (zh) | 一种烟草乙醇脱氢酶相关基因NtADH1在调控烟草腰叶宽度中的应用 | |
| CN117603325A (zh) | 烟草Ntab0167450基因及其编码蛋白在烟叶多酚调控中的应用 | |
| CN115960849B (zh) | 一种水稻抽穗期相关基因OsJMJ718及其编码蛋白的应用 | |
| CN115851754B (zh) | 一种大豆基因GmYSL7及其应用与一种引物对和一种表达载体及其应用 | |
| CN115287290B (zh) | 组蛋白去甲基化酶基因OsJMJ718及其编码蛋白在调控水稻种子活力中的应用 | |
| CN113234730B (zh) | 重组基因片段4IIA1bZIP及其应用 | |
| CN114540367B (zh) | 大豆GmPRR3b基因在调控大豆抗旱性中的应用 | |
| CN113862282B (zh) | 大豆pcl同源基因编辑位点及其应用 | |
| CN117126883A (zh) | 一种烟草生物素合成酶基因NtBIO3-BIO1在调控烟草腰叶长、腰叶宽及多酚含量中的应用 | |
| CN116656696A (zh) | 一种植物铁氧还蛋白PhFd基因及其应用 | |
| CN115948451A (zh) | LsARF3蛋白或其编码基因在调控叶用莴苣的高温抽薹性能中的应用 | |
| CN116217685A (zh) | 一种敲除烟草NtLNP1基因获得叶形改变且低烟碱含量烟草的方法及应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |