CN117402877B - 长链非编码rna natal57在调节水稻产量相关性状中的应用 - Google Patents
长链非编码rna natal57在调节水稻产量相关性状中的应用 Download PDFInfo
- Publication number
- CN117402877B CN117402877B CN202311352479.0A CN202311352479A CN117402877B CN 117402877 B CN117402877 B CN 117402877B CN 202311352479 A CN202311352479 A CN 202311352479A CN 117402877 B CN117402877 B CN 117402877B
- Authority
- CN
- China
- Prior art keywords
- rice
- natal
- long
- chain non
- yield
- 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.)
- Active
Links
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 83
- 235000009566 rice Nutrition 0.000 title claims abstract description 83
- 230000033228 biological regulation Effects 0.000 title claims abstract description 10
- 108091027963 non-coding RNA Proteins 0.000 title abstract description 16
- 102000042567 non-coding RNA Human genes 0.000 title abstract description 16
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 91
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 239000002773 nucleotide Substances 0.000 claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 13
- 230000001105 regulatory effect Effects 0.000 claims abstract description 8
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 241000196324 Embryophyta Species 0.000 claims description 23
- 108091030071 RNAI Proteins 0.000 claims description 21
- 230000009368 gene silencing by RNA Effects 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 8
- 108091081021 Sense strand Proteins 0.000 claims description 7
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 210000005069 ears Anatomy 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 230000001276 controlling effect Effects 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 10
- 108091046869 Telomeric non-coding RNA Proteins 0.000 abstract description 8
- 230000006872 improvement Effects 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 3
- 238000010362 genome editing Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 235000013339 cereals Nutrition 0.000 description 19
- 108020005198 Long Noncoding RNA Proteins 0.000 description 13
- 238000010586 diagram Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- 102100034225 Armadillo repeat-containing X-linked protein 1 Human genes 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 101000925943 Homo sapiens Armadillo repeat-containing X-linked protein 1 Proteins 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000021759 endosperm development Effects 0.000 description 2
- 230000001973 epigenetic effect Effects 0.000 description 2
- 230000004049 epigenetic modification Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000012214 genetic breeding Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 101150116497 sacm1l gene Proteins 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101001036576 Oryza sativa subsp. japonica MADS-box transcription factor 50 Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000408 embryogenic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000014075 nitrogen utilization Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000003234 polygenic effect Effects 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于分子育种技术领域，具体涉及长链非编码RNA NATAL57在调节水稻产量相关性状中的应用。本发明从水稻中克隆得到长链非编码RNA NATAL57基因，其核苷酸序列如SEQ ID NO：1所示，利用基因编辑技术在水稻中抑制lncRNA NATAL57的表达能使水稻株高增高、稻穗变大、种子变大，说明NATAL57在调节水稻产量相关性状上具有重要的作用，将为水稻的分子育种提供新的思路和方向，在作物遗传改良上具有重要的意义。同时，RNA NATAL57将为水稻产量性状的遗传改良提供宝贵的资源，具有一定的研究价值和社会效益，并有望应用于实际生产中。
Description
技术领域
本发明属于分子育种技术领域，具体涉及长链非编码RNA NATAL57在调节水稻产量相关性状中的应用。
背景技术
水稻是最重要的粮食作物之一，其高产、稳产对粮食安全具有举足轻重的作用。水稻产量是一个复杂的农艺性状，主要由每株穗数、每穗粒数和粒重三个要素决定。这三个要素是水稻产量提高的关键调控因素，往往表现为多基因控制的复杂数量性状。因此，探索水稻高产的基因资源，对产量性状的优化和作物遗传改良具有重要意义。
长链非编码RNA(long noncoding RNAs，lncRNAs)是一类长度超过200nt，不具备编码蛋白功能的内源性RNA分子。研究发现，lncRNAs在调节水稻生长发育、应对生物胁迫及非生物胁迫过程中发挥着重要功能(Gao CX,et al.Roles of lncRNAs in Rice:Advancesand Challenges.Rice Science(2020):384-395.)。例如，lncRNA LAIR过表达可增加水稻籽粒产量，并上调多个LRK基因的表达(Wang Y,et al.Overexpressing lncRNA LAIRincreases grain yield and regulates neighbouring gene cluster expression inrice.Nat Commun(2018):1-9.)。Ef-cd是从开花激活因子OsSOC1基因座的反义链转录而来的lncRNA，可以促进氮素利用和提高光合作用速率，参与调控水稻成熟(Fang J,et al.Ef-cd locus shortens rice maturity duration without yield penalty.Proc Natl AcadSci USA(2019):18717-18722.)。lncRNA ALEX1在水稻中过表达，能够激活JA途径，对白叶枯病具有显著的抗性(Yu Y,et al.Transcriptional landscape of pathogen-responsive lncRNAs in rice unveils the role of ALEX1 in jasmonate pathway anddisease resistance.Plant Biotechnol J(2020):679-690.)。母本来源、调控胚乳发育的lncRNA MISSEN，MISSEN RNAi可以导致种子出现明显的“大肚子”表型(Zhou YF,et al.Theparent-of-origin lncRNA MISSEN regulates rice endosperm development.NatCommun(2021):6525.)。水稻ESP(Epigenetic short panicle,Os01g0356951)的功能获得性表观等位基因，编码一个假定的lncRNA，其表观遗传修饰与水稻穗结构的调节有关(LuanX,et al.Epigenetic modification of ESP,encoding a putative long noncodingRNA,affects panicle architecture in rice.Rice(2019):1-8.)。
然而，尽管水稻lncRNAs的研究取得了一定的进展，但对其研究作物产量的研究知之甚少。此外，lncRNAs数量众多，绝大部分lncRNAs确切的基因组注释和功能都还是未知的。因此，挖掘具有调控水稻产量性状的lncRNAs具有重要的意义。
发明内容
为了克服上述现有技术的不足，本发明提供了一种长链非编码RNA，命名为NATAL57，该RNA可以在水稻株型、穗型和粒型等目标性状上改良水稻的产量，为水稻遗传育种提供新的遗传资源。
为了实现上述目的，本发明所采用的技术方案是：
本发明第一方面提供了长链非编码RNA NATAL57在调节水稻产量相关性状中的应用，所述长链非编码RNA NATAL57的核苷酸序列如SEQ ID No.1所示。
应当理解，考虑到密码子的简并性，在不改变氨基酸序列的前提下，对上述编码基因的核苷酸序列进行修改，也属于本发明的保护范围内。
优选地，所述水稻产量相关性状包括株型、穗型和粒型。
优选地，所述调节水稻产量相关性状为通过抑制或降低NATAL57在水稻中的表达，进而改善水稻产量相关性状。所述改善水稻产量性相关性状包括使稻株增高、使稻穗增大，使稻粒长度增加。
本发明前期从水稻中克隆得到一个长链非编码RNA NATAL57，其核苷酸序列如SEQID No.1所示，SEQ ID No.1所示的核苷酸序列由2368个脱氧核糖核苷酸组成。经研究发现，通过抑制该基因后，会导致水稻出现株高增高、稻穗变大、种子变大的表型，有望为水稻株型、穗型和粒型的调节提供一种新的方法，对水稻遗传改良具有重要的理论意义和应用价值。
优选地，所述长链非编码RNA NATAL57还包括与其相关的生物材料，所述相关的生物材料包括编码NATAL57的蛋白，含有NATAL57的重组载体、表达盒、转基因细胞系或重组菌。
优选地，所述水稻的品种包括中花11号。
本发明第二方面提供了一种培育高产水稻的方法，具体为：利用基因编辑技术抑制长链非编码RNA NATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量，进而培育得到水稻产量性状改善的水稻植株，所述长链非编码RNA NATAL57的核苷酸序列如SEQ ID No.1所示。
优选地，利用RNAi技术，通过构建NATAL57 RNAi载体并转化水稻植株，进而抑制长链非编码RNA NATAL57在水稻中的表达，或者降低长链非编码RNA NATAL57在水稻中的表达量。
更优选地，用于构建NATAL57 RNAi载体的引物包括如SEQ ID No.4、5所示的正义链上下游引物和如SEQ ID No.6、7所示的反义链上下游引物。
与现有技术相比，本发明的有益效果是：
本发明首次公开了NATAL57的生物学功能，经研究发现，利用基因编辑技术在水稻中抑制NATAL57的表达能使水稻株高增高、稻穗变大、种子变大，说明NATAL57在调节水稻产量相关性状上具有重要的作用，将为水稻的分子育种提供新的思路和方向，在作物遗传改良上具有重要的意义。同时，lncRNA NATAL57将为水稻产量性状的遗传改良提供宝贵的资源，具有一定的研究价值和社会效益，并有望应用于实际生产中。
附图说明
图1为NATAL57 RNAi载体的构建示意图；
图2为NATAL57 RNAi植株的基因表达抑制效果；
图3为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的整体株型，比例尺为20厘米；
图4为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的穗型和粒型示意图；其中，A为穗型示意图，比例尺为10厘米；B为粒型示意图，比例尺为1厘米；
图5为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的粒长和粒宽示意图，比例尺为1厘米；其中，A为粒长示意图；B为粒宽示意图；
图6为野生型与两个RNAi植株iNATAL57-1和iNATAL57-2的产量相关性状统计，数据所示为平均值±标准偏差，数据分析采用t检验确定，星号表示与野生型相比在统计学上有显著差异(*P<0.05，**P<0.01；n＝3)；其中，A为一级分枝；B为穗粒数；C为千粒重。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买得到。
长链非编码RNA NATAL57调控水稻产量相关性状的作用研究
1、NATAL57 RNAi载体的构建
根据NATAL57的核苷酸序列(SEQ ID No.1所示的核苷酸序列)，设计589bp的正义链(SEQ ID No.2)和反义链(SEQ ID No.3)。同时，根据正义链和反义链的序列设计相应的引物用于构建NATAL57的RNAi载体(图1)，具体引物序列如下：
RNAi-sense589-BamH1-F：agaggatccGGTTTGCTATGTATCCTTGTTGTTC(SEQ IDNo.4)；
RNAi-sense589-Sac1-R：aggtgagctcGCTTGCAGAGTATTGTATAGGCAGG(SEQ IDNo.5)；
RNAi-antisense589-Kpn1-F：gctcggtaccGCTTGCAGAGTATTGTATAGGCAGG(SEQ IDNo.6)；
RNAi-antisense589-Not1-R：actagtgcggccgcGGTTTGCTATGTATCCTTGTTGTTC(SEQID No.7)。
首先，以水稻cDNA为模板，将引物对RNAi-sense589-BamH1-F/RNAi-sense589-Sac1-R加入KOD FX Neo DNA聚合酶(TOYOBO BIOTECH，Shanghai)反应体系中，扩增获得片段，相应的PCR程序为：98℃2min；98℃15s、65℃45s，35个循环；68℃5min延伸。将获得的片段纯化后进行BamH1和Sac1双酶切反应，并用T4 DNA连接酶连接到经过BamH1和Sac1双酶切的载体(pRNAi43)中，获得插入正义链的重组载体。
通过相同方法，以水稻cDNA为模板，将引物对RNAi-antisense589-Kpn1-F/RNAi-antisense589-Not1-R加入KOD FX Neo DNA聚合酶扩增获得片段。将获得的片段纯化后进行Kpn1和Not1双酶切反应，并用T4 DNA连接酶连接到经过Kpn1和Not1双酶切的含正义链的重组载体中，得到NATAL57 RNAi载体(NATAL57-pRNAi43)(图1)。
NATAL57的核苷酸序列(lncRNA sequence)(2368bp，SEQ ID NO：1)：
ATCGCTCGCGGTCGCAGTCGCAGTCGCCCACACCCTTGCCGCACCATGGCTGGAACCCGCGGCGGATTCGAGTTATTGCACATGGTGGCACCAAGCATGAGGCAACAACTGCATTACTTGCAAGAAAGGCACAAGTCTCTAACACCTAGGCCAGAAAGCTCCTCACCCATCAGTTGCCTACAACCATGCAAAAGAGTAATCTCAGCAGTTCAAGAAGTCCCAATCTATATGCCACTACAGCTCTCTTCTGTCGACATGGTTTGAGACAGGCCAAACTTATTTGTTAAAGAAACTTAGGAATACTATTAAAATAAATCTGACAGTGAATATTCTAATTATATTCCTAAAATTTTCCTTCAAGTATTTAAGCATCTTTTGGTGGTTCTTATACAATTCCATGTGACATTTAAATTGTTGATAAACTACTTTCTTGATAAATCTGACAGTGAATATTCTGATTATATTCCTAAAGTTTTCCTTCAAGTATTTAAGCATCTTTTGGTGGTTCTTATACAATTCCATGTGGCATTTAAATTGTTGATAAACTACTTTCTTGATGAATTTTATATTAATGTATGATCAATTTCCATTGCAGAATCATTAGATGATTACTTGTGGCTTTCTTGCAAGTTGTGCAGTTGCTGCCTCAAGCTTGCTGCCTCCCTCTGCCAAATCTATCAGCAAAAAAAGAAAATCATGTCAGTTTCCTTGAACTATATACATATGATTAATCTAGAAACGCAGTATGTTGCCAAAAATCACACACAACTCAATCACATATGCATTTGAGATGAGGTAAAATGCTATCTGATTCACATAGTTCCAATATCTCAAGATGTGGGATATGCGTAGCACAAGTGCATCAGAGTGTTATAGTTGTTCAGTCTTCAGTACTTATTATTCTAGATTAATTTGGACCAACTTGTCAATTCAGCTGACACTTGTTTTTGTCTAGTGCTCCAGTAGCTTGGTTGTTTCATCATCATTAGACTTTTAAACCTTTGGTCCTAAACTCCTAGCTGAAAAATTTGCACACTGAACTTCCCTGTGTTCTAAGGAAAAAAAAAATCTAGACATAACCTTGCTGATCTGTATTATGGACTGGCCATTGGAGTGCATAATGCATAACTGCTGATCACTTTGTTGGCACAAAACTTACGCATAGACATAGGAGGTACTTATGTATAATTGTATCTTTAATCAGGACTTTGTTTTCAACCATTTTGTTGATATAACAGTTATACTGTAGTAATGATGAGTACAGTCTCCAATGTAAACTCAGGGCAAATCAGATTTATCCCTTTTTGGTGCTTGGTATCTTTTTGTATTTTTCATGAGCGATGGCATTTTTGAACCCAAAATAGAGGGAAAGAGAAGGATCTCTGTGGGGGCATGCTATGTACTACAGTTCTTTGAGACCATGTAGGTGTGGATGCTTGTTTTGACCTTTTCCTTAACATATAGAAAGAATAAACATTAAATAAATGATGACATGGAAATGAAATTCATAAAGGAGTACATAAGCTAGTACTCCTGAAAATATCACTTCATATTCCCCCATGATTCAGTAACATGAATATCATACCATCAGCAGCAGTGAAATTTTTATGCGTAGACGGTTTGTTTCTTAAGGTATATATTTTTTATGTTTACAATTGTCTGTCGAGTTTACTGGTCAATTGAGCAACAACGGTACTTTTAACTGTTAATATCACTTGGTTTGCTATGTATCCTTGTTGTTCTGGCTTTGTTGCTTTACAGCCATTCTGCTTACGCTATTGGCATCTGTTATTTAGAAAACCATTTTCTAACAGTTTTTAGATTTCCTTTCGAGTCTCCAACTGTTTTTAACCTCCAAGTTGCCACATAATGCATAAATATACCGAGTAAGTTTGCCCCCCTCTGAAATATGGATGTTAGATACTCAGACAGGTTCAGTATGTGTTGGGTGTTGTTTTCTGAGTTTGTGGTTGTCATAAGATGCAGCGAAATTACTGCGCTGTATCTTGGACCAATGCAATCAGCGAAGTCCTTATCGTGCCAAAAGGATGTGCATGGATTTTCCATCATTTGCAAACTTGAGGATGGCCTGCCCTGATGCCCTCAGATGCTGATGAAGTGTGTGGTTCATGAAAACGTGAGAAGCTAAGTAGTGGAGTAACGGGAGTGAGTACCAGTGTGGCCACTATGAAGAAAGTTAATTACTGAGAAATTAAGACTTGAGCATGTTATTTTCAAGCAACTTGTACAGCATTATATTGCACTACCATCAAATGCTTCTCCTGCCTATACAATACTCTGCAAGCTCTTGTATTGTACACTGTTTTATCTTGTTTAAAGTATGCTTGTTTTTGCTTATGTTGAGATT。
正义链核苷酸序列(589bp，SEQ ID No.2)
GGTTTGCTATGTATCCTTGTTGTTCTGGCTTTGTTGCTTTACAGCCATTCTGCTTACGCTATTGGCATCTGTTATTTAGAAAACCATTTTCTAACAGTTTTTAGATTTCCTTTCGAGTCTCCAACTGTTTTTAACCTCCAAGTTGCCACATAATGCATAAATATACCGAGTAAGTTTGCCCCCCTCTGAAATATGGATGTTAGATACTCAGACAGGTTCAGTATGTGTTGGGTGTTGTTTTCTGAGTTTGTGGTTGTCATAAGATGCAGCGAAATTACTGCGCTGTATCTTGGACCAATGCAATCAGCGAAGTCCTTATCGTGCCAAAAGGATGTGCATGGATTTTCCATCATTTGCAAACTTGAGGATGGCCTGCCCTGATGCCCTCAGATGCTGATGAAGTGTGTGGTTCATGAAAACGTGAGAAGCTAAGTAGTGGAGTAACGGGAGTGAGTACCAGTGTGGCCACTATGAAGAAAGTTAATTACTGAGAAATTAAGACTTGAGCATGTTATTTTCAAGCAACTTGTACAGCATTATATTGCACTACCATCAAATGCTTCTCCTGCCTATACAATACTCTGCAAGC。
反义链核苷酸序列(589bp，SEQ ID No.3)
GCTTGCAGAGTATTGTATAGGCAGGAGAAGCATTTGATGGTAGTGCAATATAATGCTGTACAAGTTGCTTGAAAATAACATGCTCAAGTCTTAATTTCTCAGTAATTAACTTTCTTCATAGTGGCCACACTGGTACTCACTCCCGTTACTCCACTACTTAGCTTCTCACGTTTTCATGAACCACACACTTCATCAGCATCTGAGGGCATCAGGGCAGGCCATCCTCAAGTTTGCAAATGATGGAAAATCCATGCACATCCTTTTGGCACGATAAGGACTTCGCTGATTGCATTGGTCCAAGATACAGCGCAGTAATTTCGCTGCATCTTATGACAACCACAAACTCAGAAAACAACACCCAACACATACTGAACCTGTCTGAGTATCTAACATCCATATTTCAGAGGGGGGCAAACTTACTCGGTATATTTATGCATTATGTGGCAACTTGGAGGTTAAAAACAGTTGGAGACTCGAAAGGAAATCTAAAAACTGTTAGAAAATGGTTTTCTAAATAACAGATGCCAATAGCGTAAGCAGAATGGCTGTAAAGCAACAAAGCCAGAACAACAAGGATACATAGCAAACC。
2、NATAL57 RNAi载体的遗传转化和转基因植株表达量检测
将第一部分获得的NATAL57 RNAi载体(NATAL57-pRNAi43)通过农杆菌EHA105介导的遗传转化方法侵染水稻中花11的胚性愈伤组织，经过共培养、阳性愈伤筛选、再分化、生根、移栽等步骤后得到潮霉素抗性的遗传转化植株(T0代转基因材料)。具体操作方法参照文献“Nishimura,A.,Aichi,I.,&Matsuoka,M.(2006).A protocol for Agrobacterium-mediated transformation in rice.Nature protocols,1(6),2796-2802.”。
将T0代转基因材料种于温室获得T1代种子。T1代种子消毒后于1/2MS培养基(含50mg/L Hyg)中培养7天，洗掉培养基后置于自来水中继续培养水稻幼苗(光暗周期12h/12h，温度28℃，相对湿度75％)。将3周龄秧苗移栽至大田(广东省农业科学院实验基地)，株距为20厘米×20厘米。待植株成熟后收取T2代种子，消毒后于1/2MS培养基(含50mg/L Hyg)中培养7天，洗掉培养基后置于自来水中继续培养7天，取叶片进行NATAL57表达量检测(图2)。具体检测方法为：用trizol提取RNA，反转录试剂使用PrimeScriptTMRT reagent Kitwith gDNA Eraser(Takara)，qPCR使用试剂为TBPremix Ex TaqTMII(Tli RNaseHPlus)(Takara)，所有操作均按照说明书操作规范进行。qPCR所用引物的序列为：NATAL57(F:TGGCTTTCTTGCAAGTTGTGC，SEQ ID No.8；R:GTGATTTTTGGCAACATACTGCG,SEQ ID No.9)；FhaB(F:GAGAAGCAGAAGCAGAAGGA，SEQ ID No.10；R:CTTGAATAGGACCATCAGGC,SEQ IDNo.11)。反应程序为95℃30s；95℃10s、60℃30s，40个循环。相对表达量用2-ΔΔct计算。以FhaB(NCBI Reference Sequence:XM_015788020.2)作为内参，以野生型中花11号水稻样品作为对照组，最终得到NATAL57表达明显受抑制的植株。
3、NATAL57 RNAi植株的产量相关性状观察
待第2部分中移栽至大田的植株成熟后进行产量相关性状的观察和统计。植株表型分析表明，NATAL57 RNAi植株表现出株高增加(图3)，稻穗增高增大(图4A)，籽粒增大(图4B)，籽粒长度明显增加(图5)的表型，从统计数据来看，NATAL57的表达抑制增加了水稻的千粒重(图6)。上述结果说明，NATAL57可以在水稻株型、穗型和粒型等目标性状上改良水稻的产量，为水稻遗传育种提供新的遗传资源，具有重要的潜在应用价值。
综上可见，通过抑制或降低NATAL57在水稻中的表达，可以使水稻表现出株高增加，稻穗增大，籽粒长度增加的表型，在调节水稻产量相关性状方面具有重要的前景。
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
Claims (4)
1.长链非编码RNANATAL57在调节水稻产量相关性状中的应用，其特征在于，通过RNAi降低长链非编码RNANATAL57在水稻中的表达，进而使水稻的株高增加，稻穗和籽粒增大，并增加水稻的千粒重；所述长链非编码RNANATAL57的核苷酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的应用，其特征在于，所述水稻的品种包括中花11号。
3.一种调控水稻产量相关性状的方法，其特征在于，利用RNAi技术，通过构建NATAL57RNAi载体并转化水稻植株，降低长链非编码RNANATAL57在水稻中的表达量，进而使水稻的株高增加，稻穗和籽粒增大，并增加水稻的千粒重；所述长链非编码RNANATAL57的核苷酸序列如SEQ ID No.1所示。
4.根据权利要求3所述的一种调控水稻产量相关性状的方法，其特征在于，用于构建NATAL57 RNAi载体的引物包括如SEQ ID No.4、5所示的正义链上下游引物和如SEQIDNo.6、7所示的反义链上下游引物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311352479.0A CN117402877B (zh) | 2023-10-19 | 2023-10-19 | 长链非编码rna natal57在调节水稻产量相关性状中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311352479.0A CN117402877B (zh) | 2023-10-19 | 2023-10-19 | 长链非编码rna natal57在调节水稻产量相关性状中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117402877A CN117402877A (zh) | 2024-01-16 |
| CN117402877B true CN117402877B (zh) | 2024-04-30 |
Family
ID=89499394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311352479.0A Active CN117402877B (zh) | 2023-10-19 | 2023-10-19 | 长链非编码rna natal57在调节水稻产量相关性状中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117402877B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640770A (zh) * | 2023-05-17 | 2023-08-25 | 广东省农业科学院农业生物基因研究中心 | OsBRY1基因在改良水稻产量性状中的应用 |
-
2023
- 2023-10-19 CN CN202311352479.0A patent/CN117402877B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640770A (zh) * | 2023-05-17 | 2023-08-25 | 广东省农业科学院农业生物基因研究中心 | OsBRY1基因在改良水稻产量性状中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| Emerging roles of long non-coding RNAs in regulating agriculturally important seed traits;Pinky et al.;Plant PhysiologyandBiochemistry;20230907;全文 * |
| Juan Zhao et al..Genome-Wide Identification of lncRNAs During Rice Seed Development.2020,全文. * |
| PREDICTED: Oryza sativa Japonica Group uncharacterized LOC9272559 (LOC9272559), transcript variant X4, ncRNA.NCBI Reference Sequence: XR_003240523.1.2018,全文. * |
| RiceLncPedia: a comprehensive database of rice long non-coding RNAs;Zhengfeng Zhang et al.;Plant Biotechnology Journal;20211231;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117402877A (zh) | 2024-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Multi-gene co-expression can improve comprehensive resistance to multiple abiotic stresses in Brassica napus L. | |
| CN113337520B (zh) | 陆地棉GhA0749和GhD0744转录因子及其调控开花方面的应用 | |
| WO2023065966A1 (zh) | Bfne基因在番茄株型改良和生物产量提高中的应用 | |
| CN105985954B (zh) | 水稻miR160b基因在调控分蘖角度中的应用 | |
| CN112126655A (zh) | 一种亚洲棉GaNCED3基因在提高植物抗旱性中的应用 | |
| CN101412751A (zh) | 一种与植物耐冷性相关的蛋白及其编码基因与应用 | |
| CN116694661A (zh) | 一种调控植物萌发速率的ShN/AINV5-4D基因及其应用 | |
| CN117402877B (zh) | 长链非编码rna natal57在调节水稻产量相关性状中的应用 | |
| CN113913440B (zh) | GhD1119基因调控陆地棉开花方面的应用 | |
| CN108218967B (zh) | 水稻抽穗期相关蛋白及其编码基因与应用 | |
| CN113024645B (zh) | 小麦转录因子wrky70基因在调控植物生长发育中的应用 | |
| CN115094070A (zh) | 一种玉米耐盐基因Zm00001d033878的发现方法及其应用 | |
| US20220251589A1 (en) | RHIZOBIAL tRNA-DERIVED SMALL RNAs AND USES THEREOF FOR REGULATING PLANT NODULATION | |
| CN116376964B (zh) | 一种调控水稻低温发芽的基因及其应用 | |
| CN114516906B (zh) | 玉米与菌根真菌共生相关蛋白及其编码基因与应用 | |
| CN115724934B (zh) | 拟南芥AtFLZ13基因在植物抗旱育种中的应用 | |
| CN116789785B (zh) | 长雄蕊野生稻高产、高光效基因FarL1及其应用 | |
| CN114672492B (zh) | 一种调控水稻株型的基因及其应用 | |
| CN116004672B (zh) | 提高植物生物量和产量的磷酸甘油酸激酶基因及其应用 | |
| CN117946232A (zh) | 长雄蕊野生稻大穗高产基因OlGn8.2及其应用 | |
| CN117660485A (zh) | 拟南芥erf012基因在调控种子萌发中的应用 | |
| CN114807129A (zh) | 一种基于lncRNA测序的玉米耐盐基因发现方法及其应用 | |
| CN117025626A (zh) | 烟草硝酸盐转运蛋白NtNPF7.4及其编码基因、基因编辑载体和应用 | |
| CN117683809A (zh) | 产量相关蛋白OsSOE及其生物材料和应用 | |
| CN115960913A (zh) | 一种茎瘤芥BjuA017664基因在提高植物抗干旱胁迫中的应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |