CN113604451B - Cipk6蛋白激酶在调控植物角果长度中的应用 - Google Patents
Cipk6蛋白激酶在调控植物角果长度中的应用 Download PDFInfo
- Publication number
- CN113604451B CN113604451B CN202111062538.1A CN202111062538A CN113604451B CN 113604451 B CN113604451 B CN 113604451B CN 202111062538 A CN202111062538 A CN 202111062538A CN 113604451 B CN113604451 B CN 113604451B
- Authority
- CN
- China
- Prior art keywords
- cipk6
- protein kinase
- plant
- fruits
- regulating
- 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
- 101150023850 CIPK6 gene Proteins 0.000 title claims abstract description 57
- 102000001253 Protein Kinase Human genes 0.000 title claims abstract description 30
- 108060006633 protein kinase Proteins 0.000 title claims abstract description 30
- 230000001105 regulatory effect Effects 0.000 title abstract description 14
- 230000001276 controlling effect Effects 0.000 title abstract description 9
- 241000196324 Embryophyta Species 0.000 claims description 47
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 abstract description 27
- 108090000623 proteins and genes Proteins 0.000 abstract description 24
- 235000013399 edible fruits Nutrition 0.000 abstract description 17
- 230000002018 overexpression Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 210000001519 tissue Anatomy 0.000 abstract description 4
- 210000000170 cell membrane Anatomy 0.000 abstract description 3
- 230000035772 mutation Effects 0.000 abstract description 3
- 210000003855 cell nucleus Anatomy 0.000 abstract 1
- 241000219194 Arabidopsis Species 0.000 description 26
- 230000009261 transgenic effect Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 9
- 230000037361 pathway Effects 0.000 description 8
- 240000002791 Brassica napus Species 0.000 description 7
- 235000011293 Brassica napus Nutrition 0.000 description 7
- 102000043136 MAP kinase family Human genes 0.000 description 7
- 108091054455 MAP kinase family Proteins 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 241000707216 Scopelarchidae Species 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 description 4
- 229930192334 Auxin Natural products 0.000 description 4
- 239000002363 auxin Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 241000219198 Brassica Species 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000001744 histochemical effect Effects 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000011331 Brassica Nutrition 0.000 description 2
- 240000007124 Brassica oleracea Species 0.000 description 2
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 2
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 2
- 101150042565 CIPK14 gene Proteins 0.000 description 2
- 229930191978 Gibberellin Natural products 0.000 description 2
- 244000299507 Gossypium hirsutum Species 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 230000009418 agronomic effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 2
- 239000004062 cytokinin Substances 0.000 description 2
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 229930182830 galactose Natural products 0.000 description 2
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 2
- 239000003448 gibberellin Substances 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 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 2
- 238000005259 measurement Methods 0.000 description 2
- 229930195732 phytohormone Natural products 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000003375 plant hormone Substances 0.000 description 2
- 244000000003 plant pathogen Species 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 230000004960 subcellular localization Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 230000009105 vegetative growth Effects 0.000 description 2
- 102100021908 3-mercaptopyruvate sulfurtransferase Human genes 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 240000000385 Brassica napus var. napus Species 0.000 description 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 108091007381 CBL proteins Proteins 0.000 description 1
- 101150109235 CBL4 gene Proteins 0.000 description 1
- 101150106973 CIPK19 gene Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102100035813 E3 ubiquitin-protein ligase CBL Human genes 0.000 description 1
- 102100030011 Endoribonuclease Human genes 0.000 description 1
- 101710199605 Endoribonuclease Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 235000009429 Gossypium barbadense Nutrition 0.000 description 1
- 101000753843 Homo sapiens 3-mercaptopyruvate sulfurtransferase Proteins 0.000 description 1
- 101000715390 Homo sapiens E3 ubiquitin-protein ligase CBL Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101710113029 Serine/threonine-protein kinase Proteins 0.000 description 1
- 101000640206 Tityus serrulatus Alpha-mammal toxin Ts2 Proteins 0.000 description 1
- 101150050727 WHY1 gene Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010201 enrichment analysis Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000004345 fruit ripening Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- ZNJFBWYDHIGLCU-UHFFFAOYSA-N jasmonic acid Natural products CCC=CCC1C(CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-UHFFFAOYSA-N 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008638 plant developmental process Effects 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011222 transcriptome analysis Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 235000018322 upland cotton Nutrition 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- 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
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01037—Protein kinase (2.7.1.37)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明公开了CIPK6蛋白激酶在调控植物角果长度中的应用，CIPK6基因在在各个组织中都有表达，但在角果及花中表达量最高，在种子中表达量最低；定位于细胞质膜及细胞核中，研究发现在植物中过表达CIPK6蛋白激酶编码基因使角果变短，CIPK6突变植株能使角果变长，因此可将CIPK6蛋白激酶用于调控植物角果长度，将植物中突变或干扰CIPK6蛋白激酶编码基因使CIPK6蛋白激酶无活性或活性降低获得角果变长的植物，对提高农作物产量具有重要意义。
Description
技术领域
本发明涉及生物技术领域，具体涉及CIPK6蛋白激酶在调控植物角果长度中的应用。
背景技术
随着白菜、甘蓝及甘蓝型油菜基因组数据的释放，芸薹属植物的研究取得了巨大的进步。CIPK基因家族作为丝氨酸/苏氨酸蛋白激酶家族中的成员，近年来在各个物种中的研究进展迅速，目前在拟南芥中鉴定出了26个CIPK蛋白家族成员，水稻中鉴定出33个，玉米中存在43个，陆地棉中有80个，它们在各自的物种中都发挥着重要的作用。CIPK家族中不同成员蛋白质可通过与CBL家族中的不同成员进行相互作用形成紧密的互作网络参与植株的各种非生物胁迫过程中。随着研究的深入，CIPK调控植株生长发育的功能也慢慢为人所知。已有研究表明，CIPK6能够调控生长素的运输影响根系的发育；CIPK6被CBL2募集到液泡膜中，并与液泡膜定位的糖转运体TST2相互作用，以促进棉花中糖的积累；CBL4/5-CIPK25形成的调控网络能够协调细胞分裂素和生长素之间的相互作用调控拟南芥根尖分生组织的大小；CIPK19参与花粉管极性生长和受精过程；CIPK14磷酸化核-质信号传递关键因子WHY1，从而控制其在细胞器内的分布，控制与衰老相关基因的表达及质体的发育；NtCIPK23不仅增强烟草种子发芽，而且还通过提高烟草幼苗的子叶绿化率，子叶扩展和下胚轴伸长来促进幼苗早期生长；种种证据表明CIPK能参与植株生长发育。但是未见CIPK有关调控角果长度的报道。
发明内容
有鉴于此，本发明的目的之一在于提供一种CIPK6蛋白激酶在调控植物角果长度中的应用；本发明的目的之二在于提供提高植物角果长度的方法；本发明的目的之三在于提供过表达CIPK6蛋白激酶在植物中上调丝原裂活蛋白激酶级联途径相关基因表达量中的应用。
为达到上述目的，本发明提供如下技术方案：
1、CIPK6蛋白激酶在调控植物角果长度中的应用。
优选的，所述植物为甘蓝型油菜或拟南芥。
优选的，抑制CIPK6蛋白激酶的编码基因表达或突变CIPK6蛋白激酶的编码基因至CIPK6蛋白激酶无活性或活性在降低使角果变长中的应用。
优选的，在植物中过表达CIPK6蛋白激酶编码基因在使角果变短中的应用。
优选的，所述CIPK6蛋白激酶为油菜CIPK6蛋白激酶，核苷酸序列如SEQ ID NO.3所示。
2、提高植物角果长度的方法，将植物中突变或干扰CIPK6蛋白激酶编码基因使CIPK6蛋白激酶无活性或活性降低。
优选的，所述植物为油菜或拟南芥。
优选的，所述CIPK6蛋白激酶为油菜CIPK6蛋白激酶，核苷酸序列如SEQ ID NO.3所示。
3、过表达CIPK6蛋白激酶在植物中上调丝原裂活蛋白激酶级联途径相关基因表达量中的应用。
本发明的有益效果在于：本发明公开了CIPK6蛋白激酶在调控植物角果长度中的应用，通过利用GUS组织化学染色发现Bna.CIPK6在各个组织中都有表达，但在角果及花中表达量最高，在种子中表达量最低；原生质体转化法探究了Bna.CIPK6蛋白定位于细胞质膜及细胞核；结合对转基因植株的农艺性状考察及转录组分析等结果，发现转基因Bna.CIPK6与野生型植株差异表达基因主要涉及MAPK及与激素相关的代谢途径，发现Bna.CIPK6能影响植株角果长度，因此可将CIPK6蛋白激酶用于调控植物角果长度，将植物中突变或干扰CIPK6蛋白激酶编码基因使CIPK6蛋白激酶无活性或活性降低。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为Bna.CIPK6启动子在拟南芥中的表达情况(b1-b4为拟南芥营养生长期整株及根和叶的表达情况，b5-b9为抽薹以后在茎、花、种子及角果皮中的表达情况)。
图2为Bna.CIPK6的亚细胞定位(ESID表示在明场，EGFP表示在绿色激发光状态下，ESID+EGFP表示叠加状态，标尺长度为5μm)。
图3为转基因农艺性状考察(a-c：拟南芥抽薹期长势比较(左边是野生型，右边是过表达)；d，e：45D拟南芥植株(从左到右依次是野生型，过表达，突变体)；f，h：成熟期角果长度比较g：成熟期种子大小比较(从左到右依次是野生型，过表达，突变体)；i：野生型和过表达植株株高比较)。
图4为差异表达基因KEGG富集Top 20柱状图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明使用材料如下试验材料：植株材料野生型拟南芥Col-0，过表达Bna.CIPK6拟南芥转基因植株、拟南芥Atcipk6突变体、实验所用菌株大肠杆菌DH5α、根癌农杆菌GV3101，载体pEarleyGate101、pCAMBIA1305.1。
转录组材料：将过表达拟南芥及野生型拟南芥消毒点播于1/2MS培养基中，4℃冰箱放置两天使种子处于相同的生长状态后置于组培间16小时黑暗22℃，8小时光照24℃培养至四叶期，移植至土培后，在植株抽薹前整个植株装入2mL无RNA酶的离心管(每个材料取三个重复)迅速置于液氮中，取样后立即冻存于-80℃备用。
实施例1、Bna.CIPK6基因克隆及表达情况
使用EZ-10DNA away RNA Mini-Preps Kit试剂盒按照说明书提取样品总RNA，用1.5％琼脂糖凝胶电泳检测样品RNA质量并检测RNA浓度，使用US EVERBRIGHT INC.的反转录试剂盒进行反转cDNA的合成。
利用油菜基因组网站Brassica napus database(http://www.genoscope.cns.fr/brassicanapus/)获取CIPK家族成员染色体位置信息，并根据Bna.CIPK6基因的ORF序列设计引物，具体引物如下：
上游引物：5’-caccatggtcggagcaaaacctatagag-3’(SEQ ID NO.1)；
下游引物：5’-ggctggtgtagtagaagtcc-3’(SEQ ID NO.2)；
然后以提取的cDNA为模板，进行PCR扩增，扩增产物经测序得到1251bp的cDNA序列，具体序列如SEQ ID NO.3所示。
利用pENTR/D-TOPO Cloning Kit，将扩增的Bna.CIPK6基因cDNA重组到pENTR/D-TOPO入门载体，转化大肠感受态DH5α。利用引物SEQ ID NO.1和SEQ ID NO.2鉴定阳性克隆后，使用EasyPure Plasmid MiniPrep Kit提取质粒，将其与pEarleyGate101表达载体进行LR重组反应，形成用于过量表达和亚细胞定位的pEarleyGate101-Bna.CIPK6载体。
根据Bna.CIPK6基因启动子序列，设计特异性引物(SEQ ID NO.4和SEQ ID NO.5)扩增启动子上游1473bp序列，具体序列如SEQ ID NO.6所示，通过重组法将其重组在pCAMBIA1305.1载体上得到重组载体pCAMBIA1305.1-Bna.CIPK6，获得重组载体转化农杆菌GV3101并侵染野生型拟南芥获得T3代转基因植株，利用GUS组织化学染色法对其进行染色，结果如图1所示。结果显示，在拟南芥生长的各个时期各个组织部位都有一定的表达，且表达情况和BrassicaEDB网站(https://brassica.biodb.org/)预测结果一致：根据GUS组织化学染色结果可以看出Bna.CIPK6具有组成型表达的特征，在各个时期各个部位都存在表达，但是表达量高低存在差异；在营养生长期中，叶片及根部存在少量表达；在生殖生长期间，叶片中表达量较高，茎和花中表达量最高，角果中表达量次之，种子中表达量较低。
为了确定Bna.CIPK6蛋白在细胞内的表达位置，利用载体上具有融合的黄色蛋白标记基因YFP的表达载体pEarleyGate101-Bna.CIPK6，利用荧光蛋白的定位确定Bna.CIPK6的表达定位，结果如图2所示。结果表明，Bna.CIPK6蛋白同时定位于细胞核和细胞膜。
实施例2、Bna.CIPK6对拟南芥角果长度的影响
为了探究转基因拟南芥发生的变化，将pEarleyGate101-Bna.CIPK6通过农杆菌介导转化拟南芥，将过表达Bna.CIPK6拟南芥与野生型拟南芥在相同情况下培养45天后，对其株高、角果长度等性状进行考察。为了进一步研究CIPK6的功能，从ABRC拟南芥突变体中心购买了CIPK6沉默的Atcipk6突变体(编号：WiscDsLox345-348H12)，与野生型拟南芥在相同情况下培养45天后，对其株高、角果长度、种子大小等性状进行考察。
在拟南芥从1/2MS培养基移栽在土里及后续的营养生长阶段，植株生长情况基本一致，不存在明显差异；但是在它们进入生殖生长阶段，过表达转基因植株抽薹速度明显低于野生型，如图3中a-c所示；在拟南芥生长45天后，突变体拟南芥株高显著高于野生型植株，野生型拟南芥株高显著高于过表达拟南芥株高如图3中d所示；从突变体、野生型和过表达群体中各随机挑选了植株，从植株基部分叉处第三个角果往上数，共取十个角果，依次测量其长度，测量结果如图3中e-f，根据测量结果显示，拟南芥突变体角果长度显著长于野生型角果长度，野生型角果长度显著长于过表达植株的角果长度；随后利用体式镜拍照记录并测量了相应的拟南芥种子的大小，结果如图3中g-h，结果表明，突变体种子大小大于野生型种子大小，野生型植株的种子大小显著大于过表达植株种子大小。
实施例3、转录组结果分析
为了分析过表达Bna.CIPK6转基因植株的分子机制，揭示其角果长度变短的原因，对抽薹前期的过表达Bna.CIPK6转基因植株行转录组测序，以同一时期野生型的转录组数据作为对照。研究利用相应的分析流程对转录组的测序结果进行分析；结果显示，在同一时期野生型拟南芥和过表达Bna.CIPK6转基因拟南芥植株中共鉴定出342个差异表达基因，对差异基因进行KEGG富集分析，总共富集到了42个KEGG条目共100个差异基因，如图4所示。结果显示，富集的前20个主要可能调控的KEGG通路，其中包括丝裂原活蛋白激酶级联途径(MAPK signaling pathway)、植物激素信号转导通路(Plant hormone signaltransduction)、半乳糖代谢途径(Galactose metabolism)及植物与病原体的相互作用途径(Plant-pathogen interaction)等。测序结果显示，过表达植株中，丝原裂活蛋白激酶级联途径相关基因表达量显著上调，说明在拟南芥中过量表达Bna.CIPK6能够激活MAPK级联途径从而提高植株抗性，这个也从印证了前期有关Bna.CIPK6调控拟南芥及油菜抗性的研究结果。
在差异基因的KEGG中，还富集到了植物激素信号转导通路(Plant hormonesignal transduction)，这个通路中相关的激素主要包括生长素、细胞分裂素、赤霉素、脱落酸等，富集的差异基因主要参与生长素代谢通路，也有涉及赤霉素、脱落酸、茉莉酸和水杨酸部分基因。这些基因主要调控细胞分裂、细胞增大、果实成熟等植株发育过程。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
<120> CIPK6蛋白激酶在调控植物角果长度中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccatggtc ggagcaaaac ctatagag 28
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggctggtgta gtagaagtcc 20
<210> 3
<211> 1251
<212> DNA
<213> 甘蓝型油菜(Brassica napus L.)
<400> 3
atggtcggag caaaacctat agagaatgaa tctgacggtg gagcaagcac gggtcttctc 60
cacggacgtt acgagctagg ccgtcttcta ggccacggaa cattcgctaa agtgtaccac 120
gcacgtaacg taacaactgg caaaagcgtg gcgatgaaag tcgtcggaaa agagaaggtg 180
gtgaaggtcg gcatggtgga ccagatcaaa cgagagatct cagtgatgag gatggtgaag 240
catccgaaca tcgtcgagct ccacgaggtg atggcgagca aaaccaagat ctacttcgct 300
atggagctcg tgcgaggcgg ggagctgttc gccaaagtcg ccaaaggaag gctgcgcgag 360
gacgtggcgc gcgtgtactt ccagcagctg atctccgccg tcgatttctg ccacagccgg 420
ggggtttacc accgagatct caagccggag aatctcttgc ttgacgaaga aggcaacctc 480
aaggtcaccg actttggtct ctccgctttc accgagcatt tgaagcaaga cgggcttctc 540
cacacgacct gtggaactcc ggcgtacgtc gcgccggagg ttatactgaa gaaaggatac 600
gacggagcga aggcggatct ttggtcttgc ggtgttatcc tcttcgtgct cctcgcgggt 660
tacttgccgt ttcaggatga taatcttgtc aacatgtata ggaagatcta cagaggagac 720
ttcaagtgcc ctggatggct ctcctccgat gcgaggaggc tcgtgacgaa gcttctggat 780
ccgaatccga atacacggat cactatcgat aaggtgatgg attcacactg gttcaagaag 840
tcctcaacga gatcaagaaa cgagccagtg gccgcaacac cagaggctga ggaggatgtt 900
gatgtgtccg tgcacaagtc caaggaagag acggagacgc taaacgcgtt tcacatcatt 960
gcgttatctg aagggttcga tctctcgccg ttgttcgagg agaagaagaa agaggagaag 1020
gtggagatga ggttcgcgac gtctcggccg gcgatgagga tggaagggaa gctgagtggt 1080
cggaaaggga agttggcggt ggaggcggag atattcgcgg tggctccgtc gtttgttgtc 1140
gtggaggtga agaaagatca tggggacacg cttgagtaca ataacttttg tagtacggct 1200
cttagaccag ccctcaagga catcttctgg acttctacta caccagcctg a 1251
<210> 4
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgggatcctt gtcgatctat tgctgcatca ccactc 36
<210> 5
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ccatggggtt cagacagccg aaagaggagt gttta 35
<210> 6
<211> 1473
<212> DNA
<213> 甘蓝型油菜(Brassica napus L.)
<400> 6
ttgtcgatct attgctgcat caccactcaa atcagatgca tataaaagat acgcatgtta 60
taagaaaaaa tatattttgt aaatgaaagt aacttgatta tagcatttgt tgtttgtaat 120
agttgccatt taataaaaaa aaagcaatag ttgccattta tcaactaata atcagtttaa 180
gactatcata ataaaacatg tgaaatataa gaaaatagta taacagatgt agaatatatt 240
atcttaatca tagtttatgc cgtatggcgt atggtgttga tatgtgtata catattttca 300
tttaattgcg tataaagatt tgtatatacg tatgtgttta atttgtacat ttctgtaact 360
atacaaaagt tacacgcacg tgacttccca attgcttctt gtgtgctaac caatccaaat 420
tgtttccttc acaagttgcg actggcatat gattttagat tattcccagc tatgcttact 480
cctcgggcac tggttaaaac cgcgaaaact gtcgagtggg tgtataagag atactttgat 540
gcatgatgta tgctcattca gtatattgac ttattgagaa ttatttgtta ttagtataca 600
tgatggttac taatatgatc cctataattc cttggtaaaa gattttattg tccattatct 660
cataatgcgt gttatgctta taaactgtgc aattagcttt tcactggttt tgtttttatt 720
attcctaacc agcgtatctt atacgtatac gtgatacttt gctacaaatt tccacagctc 780
aatatatatt ttgttatgtt aaatttcaaa gattaattac agaaagtatt aagattaaga 840
ttagtctcgg cataactaga aacgaacttc ccattttgta tctactgtct ccgtgtacaa 900
aaaaacaaag agatagaagt agaaaaccct agaatcaaca cattattggt ccacaaggac 960
cacaacgaat gaaaacgatt aaacaaaaat aaaagaaaaa aatggtagga atctatagaa 1020
aatgagcatg tcctacatta tatggcacca atatctttta aaatgaattc ttcatctttg 1080
tagatgcaga aactaaatca aattaatata attaatgata ttttccctca attagcatat 1140
gcattgtata gatgcaatgc atgcaatgca aattggtcag caagccggtc aacgagagag 1200
tgattaactt tccaagaaat gtctacctta tactagtaac aaagaccaga ggggtaggat 1260
agtaaaaact tatctcaacc ggtgaactaa ggacacgtgc cttcatcgct atcaaataat 1320
tgacaatccc gaagccgcaa cgagtacatc tctcgacacc tgtcagtcag tgaacggaga 1380
ttaaacggtg tagttatcac tactaataag ggcagttacg taattacgca ttcattaaga 1440
cctatataaa cactcctctt tcggctgtct gaa 1473
Claims (1)
1.提高植物角果长度的方法，其特征在于：将植物中CIPK6基因突变使CIPK6蛋白激酶无活性；所述植物为拟南芥；所述CIPK6蛋白激酶为油菜CIPK6蛋白激酶核苷酸序列如SEQID NO.3所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111062538.1A CN113604451B (zh) | 2021-09-10 | 2021-09-10 | Cipk6蛋白激酶在调控植物角果长度中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111062538.1A CN113604451B (zh) | 2021-09-10 | 2021-09-10 | Cipk6蛋白激酶在调控植物角果长度中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604451A CN113604451A (zh) | 2021-11-05 |
| CN113604451B true CN113604451B (zh) | 2024-02-02 |
Family
ID=78310338
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111062538.1A Active CN113604451B (zh) | 2021-09-10 | 2021-09-10 | Cipk6蛋白激酶在调控植物角果长度中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604451B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115807016B (zh) * | 2022-11-22 | 2024-05-10 | 西南大学 | 甘蓝型油菜Bna.Arf基因在提高植物生物量中的应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102458098A (zh) * | 2009-04-10 | 2012-05-16 | 陶氏益农公司 | 植物snf1相关蛋白激酶基因 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9029638B2 (en) * | 2008-01-31 | 2015-05-12 | National Institute For Biological Sciences | Plants having altered growth and/or development resulted from modulated expression of ubiquitin-specific proteases and a method for making the same |
| CN102958347B (zh) * | 2009-09-28 | 2015-03-11 | Fb科技控股公司 | 减少植物应激的方法 |
| WO2018144831A1 (en) * | 2017-02-02 | 2018-08-09 | Duke University | Compositions and methods for controlling gene expression |
-
2021
- 2021-09-10 CN CN202111062538.1A patent/CN113604451B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102458098A (zh) * | 2009-04-10 | 2012-05-16 | 陶氏益农公司 | 植物snf1相关蛋白激酶基因 |
Non-Patent Citations (5)
| Title |
|---|
| Arabidopsis CBL-interacting protein kinase (CIPK6) is involved in plant response to salt/osmotic stress and ABA;Liang Chen等;Mol Biol Rep;第4759–4767页 * |
| CBL-CIPKs信号系统的研究进展;汤湖斌等;中国细胞生物学学报;第100–105页 * |
| Identification of miRNAs that regulate silique development in Brassica napus;Li Chen等;Plant Science;第106–117页 * |
| Relationship between Endogenous Hormones and Pod-length Development in Brassica napus L ．;SHUI Hong-xia等;Agronomy and Forestry;第710 -714页 * |
| The Brassica napus Calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signalling;Liang Chen等;ournal of Experimental Botany;第6211-6222页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604451A (zh) | 2021-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Leng et al. | A subclass of HSP70s regulate development and abiotic stress responses in Arabidopsis thaliana | |
| US9546377B2 (en) | Methods for the identification of genes involved in abiotic stress tolerance in plants | |
| US11535858B2 (en) | Polynucleotide construct for improving agricultural characteristics in crop plants | |
| KR100647767B1 (ko) | 신규 녹색지속유전자 및 녹색지속성 변이식물체의 제조방법 | |
| CN114369599B (zh) | 一种增加水稻产量的长链非编码rna基因及其应用 | |
| Šimášková et al. | KIL1 terminates fertility in maize by controlling silk senescence | |
| CN116218876A (zh) | 一种调控水稻垩白的基因OsB12D3及其编码蛋白和应用 | |
| Li et al. | Effects of two apple tonoplast sugar transporters, MdTST1 and MdTST2, on the accumulation of sugar | |
| CN113604451B (zh) | Cipk6蛋白激酶在调控植物角果长度中的应用 | |
| CN114540357A (zh) | 玉米长链非编码RNA lncRNA25659及其用途 | |
| CN112592393B (zh) | 水稻早衰控制基因es1-3及其应用 | |
| EP3095863B1 (en) | Parthenocarpy regulation gene and use thereof | |
| Wang et al. | Expression and functional analysis of VviABCG14 from Vitis vinifera suggest the role in cytokinin transport and the interaction with VviABCG7 | |
| CN112301046A (zh) | 调控植物茎和侧枝发育的基因GhD14及其应用 | |
| CN112522283A (zh) | 一种花粉发育相关基因及其应用 | |
| CN112322645A (zh) | OsHDA710表观调控因子基因在水稻发育和抗逆中的应用 | |
| CN115960189B (zh) | 一种文冠果蛋白及其编码基因在提高植物花瓣中花青素的含量中的应用 | |
| CN113583990B (zh) | 水稻全育期半矮化表型调控基因sd38及其应用 | |
| CN116004708A (zh) | 甘蓝型油菜BnaA01.SnRK1.1基因的应用 | |
| US20230279419A1 (en) | Enhancement of productivity in c3 plants | |
| Nguyen et al. | Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system | |
| CN115807016B (zh) | 甘蓝型油菜Bna.Arf基因在提高植物生物量中的应用 | |
| CN110272907B (zh) | 一种调控番茄茎杆发育的基因sd1及其应用 | |
| US20240052002A1 (en) | Tomato-derived sijul gene regulating phloem development and use thereof | |
| CN112831509B (zh) | 番茄SlOST1基因及应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |