CN116463355A - 生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 - Google Patents
生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 Download PDFInfo
- Publication number
- CN116463355A CN116463355A CN202310408542.1A CN202310408542A CN116463355A CN 116463355 A CN116463355 A CN 116463355A CN 202310408542 A CN202310408542 A CN 202310408542A CN 116463355 A CN116463355 A CN 116463355A
- Authority
- CN
- China
- Prior art keywords
- lettuce
- gene
- yield
- seq
- lettuce 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.)
- Pending
Links
- 235000003228 Lactuca sativa Nutrition 0.000 title claims abstract description 62
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 58
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 17
- 230000033228 biological regulation Effects 0.000 title claims abstract description 11
- 239000013604 expression vector Substances 0.000 title claims abstract description 7
- 240000008415 Lactuca sativa Species 0.000 title description 48
- 241000196324 Embryophyta Species 0.000 claims abstract description 29
- 230000006872 improvement Effects 0.000 claims abstract description 4
- 235000013311 vegetables Nutrition 0.000 claims abstract description 4
- 241000208822 Lactuca Species 0.000 claims abstract 14
- 230000001105 regulatory effect Effects 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 238000012217 deletion Methods 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 7
- 230000001488 breeding effect Effects 0.000 claims description 7
- 230000009261 transgenic effect Effects 0.000 claims description 7
- 238000003780 insertion Methods 0.000 claims description 6
- 230000037431 insertion Effects 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 238000003259 recombinant expression Methods 0.000 claims description 4
- 230000030279 gene silencing Effects 0.000 claims description 3
- 125000000539 amino acid group Chemical group 0.000 claims description 2
- 230000037429 base substitution Effects 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 108091033409 CRISPR Proteins 0.000 abstract description 2
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 2
- 239000005648 plant growth regulator Substances 0.000 abstract description 2
- 239000007787 solid Substances 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 description 17
- 239000007788 liquid Substances 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 14
- 238000000034 method Methods 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 239000002699 waste material Substances 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 240000000220 Panda oleosa Species 0.000 description 4
- 235000016496 Panda oleosa Nutrition 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 235000002566 Capsicum Nutrition 0.000 description 3
- 240000008574 Capsicum frutescens Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 108700005075 Regulator Genes Proteins 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000001390 capsicum minimum Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 235000009973 maize Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 2
- 235000006008 Brassica napus var napus Nutrition 0.000 description 2
- 240000000385 Brassica napus var. napus Species 0.000 description 2
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 2
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 2
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 241000219146 Gossypium Species 0.000 description 2
- 235000003222 Helianthus annuus Nutrition 0.000 description 2
- 244000020551 Helianthus annuus Species 0.000 description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241001520808 Panicum virgatum Species 0.000 description 2
- 240000004713 Pisum sativum Species 0.000 description 2
- 235000010582 Pisum sativum Nutrition 0.000 description 2
- 241000209056 Secale Species 0.000 description 2
- 235000007238 Secale cereale Nutrition 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 241001233957 eudicotyledons Species 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101150011812 AADAC gene Proteins 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 101150103518 bar gene Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012881 co-culture medium Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 239000012877 elongation medium Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000012795 verification Methods 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)
- C12N15/8216—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请公开了一种生菜产量调控基因LOC111907799、其编码蛋白、表达载体及其应用;本申请对生菜基因LOC111907799进行CRISPR/Cas9敲除,明确了该基因对生菜叶片数、叶片大小、株高等性状表现的影响;验证了其对生菜产量的提高作用;从而为利用LOC111907799基因提高生菜产量提供了坚实的技术支撑。
Description
技术领域
本发明申请涉及生物基因工程技术领域,具体涉及一种生菜产量调控基因LOC111907799、其编码蛋白、表达载体及其应用。
背景技术
生菜(Lactuca sativa L. var. ramosa Hort)为菊科莴苣属一年生或二年草本植物,其是人们喜食且常见的蔬菜,需求量极大,市场前景广阔。
生菜的生物产量构成因子主要包括株高、叶面积、叶片数、叶片大小、株径等;近年来随着基因组学和分子生物学技术的不断发展,一些研究已经鉴定出一些与生菜产量相关的基因,如LsAP2、LsMYB、LsNAC、LsWRKY等。这些基因可能参与了生菜产量的调控过程,为生菜产量的遗传改良提供了新的基因资源。但生菜的产量性状是由多个基因控制的复杂性状,当前对生菜产量基因的研究还相对较少,目前获得的功能基因对全面阐明产量形成的分子机制仍十分有限。因此,需进一步挖掘更多的生菜生物产量相关性状的基因并解析其利用途径,从而丰富产量形成的遗传基础,并为生菜新品种选育提供优良的基因资源。
公开于该背景技术部分的信息仅仅旨在加深对本发明总体背景技术的理解,而不应当被视为承认或以任何形式暗示该信息构成本领域技术人员所公知的现有技术。
发明内容
发明人在生菜中确定了与玉米基因KWE2同源的基因LOC111907799,并创制相应的敲除突变进而明确了其在辣椒叶片数及其大小性状上的表现和对辣椒产量的调控作用,从而为利用LOC111907799基因提高生菜产量奠定了坚实的技术基础。
本申请的目的之一在于提供一种生菜产量调控基因LOC111907799,其核苷酸序列为下组中的至少一种:
(1)如SEQ ID NO.1所示;
(2)在SEQ ID NO.1的基础之上经过一至数个碱基替换和/或一至数个碱基的插入和/或缺失以及大片段的核苷酸序列插入/缺失/移位/倒位所形成的能影响产量相关性状表型的DNA分子。
本申请公开的另一个方面,提供一种生菜产量调控基因LOC111907799的编码蛋白,其氨基酸序列为下组中的至少一种:
(1)由SEQ ID NO.1所编码的氨基酸序列组成的蛋白质;
(2)如SEQ ID NO.2所示;
(3)由SEQ ID NO.2经过一个或数个氨基酸残基的取代、缺失和/或添加且具有影响产量相关性状的蛋白质。
本申请公开的第三个方面,提供一种含所述生菜产量调控基因LOC111907799的重组表达载体、表达盒、转基因细胞系或者重组菌。
本申请公开的第四个方面,提供一种鉴定生菜调控基因LOC111907799突变的引物对,其序列如下:
JLS4080-A1-F1 5’-TCCAGCTTACTTTTTATTTGAGCAC- 3’,
JLS4080-A1-R1 5’-GGTTGCAAACGACAAGCGAT- 3’。
本申请公开的第五个方面,将所述生菜产量调控基因LOC111907799、所述引物应用在生菜产量性状改良及优势品种/品系选育当中。
例如,沉默/下调表达所述生菜产量调控基因LOC111907799,以增加生菜的叶面积、叶片数、株高或/和株径。构建生菜产量调控基因LOC111907799的敲除载体或沉默载体并转化植物,培养并筛选出对应的纯合突变体,并进行杂交育种。
本申请可转化的植物可以是单子叶和双子叶植物,包括但不限于生菜、油菜、向日葵、高粱、玉米、小麦、大麦、黑麦、棉花、甘薯、马铃薯、大豆、豌豆、苜蓿、柳枝稷、拟南芥属等。
本申请实施例中提供的一个或多个技术方案,至少具有如下任一技术效果或优点:
1.对生菜产量调控基因LOC111907799进行CRISPR/Cas9敲除,获得纯合的CRISPR-Cas9突变体,明确了LOC111907799基因对生菜的叶面积、叶片数、株高或/和株径等性状表现的影响;确证了其对生菜生物产量的调控作用;从而为利用LOC111907799基因提高生菜产量提供了技术支撑。
2.利用本申请公开的基因及方法,可方便生菜、玉米、小麦等农作物高产育种材料创制,缩短农作物新品种的选育周期,降低选育成本,提高育种效率。
附图说明
图1为本申请实施实例中生菜LOC111907799基因的进化分析树;邻接法构建系统进化树,每个分支上的数值代表1000次模拟运算中的支持度,比例尺为每个位点的平均氨基酸替代数。
图2为本申请实施实例中 生菜LOC111907799基因编辑骨架载体示意图。
图3为本发明实施实例中生菜LOC111907799编辑的中间模板载体示意图。
图4为本申请实施实例中生菜LOC111907799双靶标的构建片段PCR电泳示意图。
图5为本申请实施实例中生菜LOC111907799基因编辑的终载体示意图。
图6为本申请实施实例中生菜LOC111907799基因编辑终载体的检测示意图。
图7为本申请实施实例中生菜LOC111907799基因编辑苗的阳性筛选过程示意图。
图8为本申请实施实例中生菜的表型性状对比图,其中,左图为LOC111907799基因的敲除系,右图为转基因对照株。
具体实施方式
相关术语定义及说明:
术语“生菜生物量调控基因”指一段具有编码蛋白能力的核苷酸序列,该序列特异编码具有调控生物产量功能的蛋白活性多肽。
所述“生菜产量调控基因”,还包括能编码具有天然的调控生物产量的基因KWE2相同功能蛋白的、开放阅读框序列的变异形式;这些变异形式包括但并不限于:一个或多个核苷酸的缺失、插入和/或取代,以及5’或3’端添加几个(通常为60个以内,较佳的为30个以内,更佳的为10个以内,最佳的为5个以内)的核苷酸。
所述“生菜产量调控基因”,还包括能翻译一类具备调控生菜株高、叶面积、叶片数、叶片大小、株径和生物产量功能的氨基酸序列,如SEQ ID NO.2的氨基酸序列。该类氨基酸序列还包括具有天然调控生菜生物产量蛋白相同功能的变异形式。这些变异形式包括但并不限于:1个或几个氨基酸的缺失、插入和/或取代,以及C末端和/或N末端添加1个或几个(通常为20个以内,较佳的为10个以内,更佳的为5个以内)的氨基酸。在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能;在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。
另外,所述“生菜产量调控基因”的核苷酸全长序列或其片段通常可以用PCR 扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本实施例公开的有关核苷酸序列,尤其是开放阅读框序列来设计对应引物,并用市售的cDNA文库或按本领域技术人员已知的常规方法所制备的cDNA文库作为模板,扩增到有关序列。当序列较长时,通常需要两次或者多次的巢式PCR扩增,然后将各次PCR扩增产物按正确的顺序拼接在一起。
特别优选在高等植物中表达的至少一种本申请公开的生菜产量调控基因LOC111907799的蛋白,一旦将期望的核苷酸序列转化进入特定植物物种中,可以在该物种中繁殖它或用常规育种技术将它转移进入相同物种的其它品种(特别包括商业品种)中。可将本申请公开的各生菜产量调控基因LOC111907799的核苷酸序列插入到表达盒中,或将其包含在非致病自我复制的病毒中,然后优选地,将该表达盒稳定整合在所述植物基因组中。本申请转化的受体可以是单子叶和双子叶植物,包括但不限于玉米、小麦、大麦、黑麦、稻、油菜、棉花、向日葵、马铃薯、豆、豌豆、柳枝稷、拟南芥属等。通过在转基因植物中表达本发明公开的核苷酸序列,由此在转基因植物中促进能够增强相应杂种优势表现的功能蛋白的生物合成。以这种方式,可产生增强杂种优势表现的转基因植物。为了在转基因植物中表达本发明核苷酸序列,本发明公开的核苷酸序列可能需要修饰和优化,可以在保持本发明所述核苷酸序列编码的氨基酸的同时改变其密码子以符合植物偏爱性。而且,从有至少约35%,优选多于约45%,更优选多于50%,最优选多于约60%GC含量的编码序列可以最好地实现植物中高水平的表达。
下面对本发明的一些具体实施方式进行详细描述,以下的实施例更便于更好的理解本发明,但应当理解本发明的保护范围并不受具体实施方式的限制。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其它实施例,都属于本发明保护的范围。
本申请各实施例中所述实验方法,如无特殊说明,均为常规方法;本领域技术人员应该理解,下述实施例中所用的试剂、酶类等除特别说明外,均为可从试剂公司商购的分析纯级别的试剂或酶类。材料、方法和实施例仅作阐述用,而非加以限制。
实施例一、生菜LOC111907799基因的功能验证
研究发现生菜LOC111907799基因与玉米KWE2基因的亲缘关系最近(图1),两者为同源基因;进一步的研究证实生菜LOC111907799基因与玉米KWE2基因具有相似的负调控产量和杂种优势的基因功能。
为进一步验证生菜LOC111907799基因对生菜生物产量的调控作用,对该基因进行了CRISPR-Cas9敲除。
具体操作如下:
1. 载体构建:采用酶切T4连接酶构建(图2)
依据基因序列设计如下靶标并构建敲除载体,载体构建完成后进入转化。
表1靶标设计
。
2. 载体处理
用BsaI酶切1µg载体,37℃,1h左右。
50µL 体系:
WMC028载体 1µg(10µL)
BsaI酶 1µL
BsaI酶的buffer 5µL
ddH2O 34µL。
时间到后直接原液回收。
使用DC301试剂盒(PCR凝胶回收试剂盒):
①将50µL酶切载体加入到回收柱,同时加入300µL的GDP,离心12000g,1min;②将废液重新加入回收柱中,再过柱一次,离心12000g,1min;③弃废液,加入600µL GW至回收柱中,离心12000g,1min;④重复步骤③;⑤弃废液,空甩,离心12000g,2min;⑥将回收柱放入1.5mL ep灭菌离心管,等酒精挥发数分钟后,加入30µL的洗脱液(ddH2O,或者EB洗脱液),静置2min,12000g,2min后,弃回收柱,得到酶切载体产物。
2. 引物设计及PCR扩增(图3)
用以上载体做模板进行PCR扩增:
上游引物F:保护碱基+BsaI+靶标1+模板16bp左右;
上游引物R:保护碱基+BsaI+靶标2+模板16bp左右。
溶解引物:12000rpm 5min 按照引物管壁提示的水量×10,加入到EP管中(400500)配成10µM浓度的引物,加完水后,盖严管盖,震荡1min,置4℃冰箱中备用。
PCR扩增:
50µL 体系
F/R引物 各1µL
模板 1µL
Pfu酶 (高保真酶) 20µL
ddH2O 27µL。
PCR仪运行以下系列反应:
95℃,3min;(95℃,30s;58℃,30s)*30个循环;72℃,1min;72℃,5min;4℃下保存温度。
反应后PCR产物取3µL点胶看条带是否正确(图4),正确大小600bp左右,即对原液回收。
使用DC301试剂盒(PCR凝胶回收试剂盒):
①将50µL PCR产物加入到回收柱,同时加入300µL的GDP,离心12000g,1min;②将废液重新加入回收柱中,再过柱一次,离心12000g,1min;③弃废液,加入600µL GW至回收柱中,离心12000g,1min;④重复步骤③;⑤弃废液,空甩,离心12000g,2min;⑥将回收柱放入1.5mL ep灭菌离心管,等酒精挥发数分钟后,加入30µL的洗脱液(ddH2O,或者EB洗脱液),静置2分钟,12000g,2min后,弃回收柱,得到所对应PCR产物。
3. PCR产物酶切处理
用BsaⅠ酶切以上PCR产物,37℃ 1小时左右。
50µL 体系
PCR产物 30µL
BsaⅠ酶 1µL
BsaⅠ酶的buffer 5µL
ddH2O 14µL。
使用DC301试剂盒(PCR凝胶回收试剂盒):
①将50µL PCR酶切产物加入到回收柱,同时加入300µL的GDP,离心12000g,1min;②将废液重新加入回收柱中,再过柱一次,离心12000g,1min;③弃废液,加入600µL GW至回收柱中,离心12000g,1min;④重复步骤③;⑤弃废液,空甩,离心12000g,2min;⑥将回收柱放入1.5mL ep灭菌离心管,等酒精挥发数分钟后,加入30µL的洗脱液(H2O,或者EB洗脱液),静置2min,12000g,2min后,弃回收柱,得到所对应PCR酶切产物。
4. 连接体系(冰上混合)-得到所要载体(图5)
PCR酶切产物(上图阴影部分) 6µL
酶切载体产物(上图其余部分) 1µL
T4酶 1µL
T4酶buffer 1µL
ddH2O 1µL
总计 10µL。
PCR仪运行 Ligase程序 24℃、1h 后冰箱放置。
5. 转化DH5α
10µL以上连接产物+100µL大肠杆菌感受态:
感受态DH5α冰箱取出后,迅速放入冰上,5min后待菌块溶解加入连接产物,冰上静置25min,42℃热击45s,冰上放置2min(勿晃动),加入无抗生素的LB 100µL,37℃,200rpm,摇菌1h,涂板,LB+Kana,37℃培养一天。
6. 载体质控
菌落PCR:平板随机挑取12个单克隆放入2mL无菌EP管(提前加入200µL Kana+LB)中,标记1,2,3,4,5,6,7~12,摇菌6h,每一个样品吸取5µL菌液PCR。
PCR Mix 10µL
引物(QC1+QC10) 1/1µL
菌液 5µL
ddH2O 3µL
总计 20µL。
目的片段约1200/600 bp(QC1+QC10)左右,PCR跑胶为双带的且条带大小正确(图6);挑选正确菌液送测序,如1,2等即进行测序,测序引物信息如下:
QC1: CTGGCGAAAGGGGGATGTGCTGCAA;
QC10:GCCATTTGTCTGCAGAATTG 。
7. 测序结果正确的质粒转化农杆菌:
①20µl农杆菌(EHA105-WM)+1µL质粒,冰浴5min,液氮速冻5min,37℃水浴5min,冰浴5min。加入100µL无抗的LB,28℃ 200 rpm 摇菌2h,直接涂板YP+Kana+rif ,28℃培养两天。
②挑取单克隆摇菌:两天后挑取一个单克隆放入5mL无菌EP管中,提前加入2mL YP+Kana+rif,过夜摇菌。
③保存甘油菌:400µL菌液+100µL 75%无菌甘油,做好标记,放入-70度冰箱保存。剩余菌液提取质粒转化大肠杆菌(1µL质粒+20µL大肠杆菌感受态 ,冰上静置30min,42℃热击35s,冰上放置2min,加入无抗生素的LB 100µL,37℃,200rpm,摇菌1h,涂板,LB+Kana,37℃培养一天)。
④挑一个单克隆早上摇菌,晚上直接菌液送测序,测序引物QC1。
⑤测序反馈正确,验证农杆菌没有问题,所制备农杆菌即可以进行后面转化实验。将载体通过电击法转入农杆菌EHA105中,PCR 进行鉴定。以萌发5天的子叶为材料,放入含有50mL农杆菌悬浮液的培养皿中,2h内大约处理300个外植体,室温放置30min进行侵染。侵染结束后弃去农杆菌菌液,外植体放于共培养基中,23℃黑暗共培养2天。共培养后,将外植体转移到相应抗性的筛选培养基上,于25℃光照培养4周,诱导抗性芽。然后转到相应抗性的伸长培养基上培养4~6周,25℃,5000lx,光照培养直到生根(图7)。
移栽穴盘后对T0植株进行aada+bar基因及靶标位点进行检测,引物信息如下:AADA-F2 CAGGGTGAGGACCACATTCC;AADA-R2 TCCGACATCGATCTCCTGGT。扩增大小435bp。
⑥对应突变位点的鉴定方法(引物、扩增、电泳等程序)
引物:JLS4080-A1-F1 TCCAGCTTACTTTTTATTTGAGCAC;JLS4080-A1-R1GGTTGCAAACGACAAGCGAT。扩增程序如表2所示。扩增大小486bp。
表2 扩增程序
Temp | go to | cycle | 时间 | ||
1 | 95 | 5min | |||
2 | 95 | 30s | |||
3 | 64 | -0.5/cycle | 30s | ||
4 | 72 | 2 | 20 | 30s | |
5 | 95 | 30s | |||
6 | 54 | 30s | |||
7 | 72 | 5 | 20 | 30s | |
8 | 72 | 5min | |||
9 | 16 | 9 | 99 | 1h | |
扩增体系 | |||||
Taq Mix | 10µL | ||||
Primer | 1/1µL | ||||
DNA Templet | 1µL | ||||
H2O | 7µL |
实施例二、生菜LOC111907799基因敲除后的表型
将生菜LOC111907799敲除后的转基因系和对照朝品种种植在相同的环境条件下,在生长期进行表型调查。结果表明(图8),LOC111907799基因敲除后的生菜株系与野生型对照相比,其叶片数、叶片宽度(增加了67.4%)和株高(增加了10.3%)显著增加。说明玉米KWE2基因的生菜同源基因LOC111907799对生菜产量和株高性状具有保守的负调控功能。
尽管已描述了本发明的一些优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本申请权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (8)
1.一种生菜产量调控基因LOC111907799,其核苷酸序列为下组中的至少一种:
(1)如SEQ ID NO.1所示;
(2)在SEQ ID NO.1的基础之上经过一至数个碱基替换和/或一至数个碱基的插入和/或缺失以及大片段的核苷酸序列插入/缺失/移位/倒位所形成的能影响产量相关性状表型的DNA分子。
2.一种生菜产量调控基因LOC111907799的编码蛋白,其氨基酸序列为下组中的至少一种:
(1)由SEQ ID NO.1所编码的氨基酸序列组成的蛋白质;
(2)如SEQ ID NO.2所示;
(3)由SEQ ID NO.2经过一个或数个氨基酸残基的取代、缺失和/或添加且具有影响产量相关性状的蛋白质。
3.一种含有权利要求1所述生菜产量调控基因LOC111907799的重组表达载体、表达盒、转基因细胞系或者重组菌。
4.一种鉴定生菜调控基因LOC111907799突变的引物对,其序列如下:
JLS4080-A1-F1 5’-TCCAGCTTACTTTTTATTTGAGCAC- 3’,
JLS4080-A1-R1 5’-GGTTGCAAACGACAAGCGAT- 3’。
5.权利要求1所述生菜产量调控基因LOC111907799、权利要求3所述重组表达载体或权利要求4所述引物对在调控生菜的叶面积、叶片数、株高或/和株径中的应用。
6.权利要求1所述生菜产量调控基因LOC111907799、权利要求3所述重组表达或权利要求4所述引物对在生菜产量性状改良及优势品种/品系选育中的应用。
7.根据权利要求6所述的应用,其特征在于,沉默/下调表达所述生菜产量调控基因LOC111907799,以增加生菜的叶面积、叶片数、株高或/和株径。
8.根据权利要求7所述的应用,其特征在于,包括如下步骤:
构建生菜产量调控基因LOC111907799的敲除载体或沉默载体并转化植物,培养并筛选出对应的突变体,与对应的自交系进行杂交育种。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310408542.1A CN116463355A (zh) | 2023-04-17 | 2023-04-17 | 生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310408542.1A CN116463355A (zh) | 2023-04-17 | 2023-04-17 | 生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116463355A true CN116463355A (zh) | 2023-07-21 |
Family
ID=87173079
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310408542.1A Pending CN116463355A (zh) | 2023-04-17 | 2023-04-17 | 生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116463355A (zh) |
-
2023
- 2023-04-17 CN CN202310408542.1A patent/CN116463355A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2014144094A1 (en) | Tal-mediated transfer dna insertion | |
CN110904071B (zh) | Raf49蛋白及其编码基因在调控植物抗旱性中的应用 | |
CN105753953B (zh) | 小麦抗病蛋白与编码基因及其在调控植物抗病性中的应用 | |
CN109750047B (zh) | 茶树己糖转运体基因CsSWEET17及其在调控植物营养生长和种子大小中的应用 | |
CN105755022A (zh) | 一种水稻耐镉基因OsGSTU5及其编码蛋白与应用 | |
CN116445507A (zh) | 小麦有效分蘖数及产量调控基因TraesCS2A02G577100、其编码蛋白、表达载体及其应用 | |
CN105585623B (zh) | 抗病转TaMYB-KW基因小麦的培育方法及相关生物材料与应用 | |
CN106632627B (zh) | Lnsm蛋白及其编码基因在植物转基因中的应用 | |
CN116463355A (zh) | 生菜产量调控基因loc111907799、其编码蛋白、表达载体及其应用 | |
CN104805100B (zh) | 水稻基因OsSμBP‑2在延缓植物叶片衰老中的应用 | |
CN114106121A (zh) | FvGR3蛋白及其编码基因和用途 | |
CN106434692A (zh) | 水稻OsPCF7基因在培育高分蘖水稻品种中的应用 | |
CN112662687A (zh) | 推迟玉米花期的方法、试剂盒、基因 | |
CN116622723A (zh) | 大豆结荚数及产量调控基因glyma_02g083400、其编码蛋白、表达载体及其应用 | |
CN115927237B (zh) | 一种油菜海藻糖-6-磷酸合成酶基因在调控含油量与脂肪酸组成中的用途 | |
CN116121292B (zh) | 一种水稻myb转录因子及其编码蛋白的应用 | |
CN116064653B (zh) | 番茄SlBBX17基因在促进番茄低温抗性中的应用 | |
CN116445506A (zh) | 柳枝稷分蘖数及生物量调控基因ent、其编码蛋白、表达载体及其应用 | |
CN116606864A (zh) | 油菜产量调控基因skp1-ip15、其编码蛋白、表达载体及其应用 | |
CN116574739A (zh) | 辣椒产量调控基因loc107867680、其编码蛋白、表达载体及其应用 | |
CN116790620A (zh) | 水稻产量调控基因Os04t0686700、其编码蛋白、表达载体及其应用 | |
CN115960948A (zh) | 一种玉米ZmCP03基因的应用 | |
CN116590305A (zh) | 棉花果枝数及产量调控基因gh_d05g2737、其编码蛋白、表达载体及其应用 | |
CN116590270A (zh) | 一种控制水稻谷粒大小的基因及其应用 | |
CN117447569A (zh) | 水稻OsERF52蛋白及其编码基因在提高植物低温耐受性中的应用 |
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 |