CN116004650B - 一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 - Google Patents
一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 Download PDFInfo
- Publication number
- CN116004650B CN116004650B CN202211053458.4A CN202211053458A CN116004650B CN 116004650 B CN116004650 B CN 116004650B CN 202211053458 A CN202211053458 A CN 202211053458A CN 116004650 B CN116004650 B CN 116004650B
- Authority
- CN
- China
- Prior art keywords
- gene
- ospk7
- rice
- male sterility
- pollen
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 33
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 26
- 235000009566 rice Nutrition 0.000 title claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 13
- 240000007594 Oryza sativa Species 0.000 title claims 2
- 101000913758 Oryza sativa subsp. japonica CBL-interacting protein kinase 12 Proteins 0.000 title abstract description 29
- 206010021929 Infertility male Diseases 0.000 title abstract description 19
- 208000007466 Male Infertility Diseases 0.000 title abstract description 19
- 230000001105 regulatory effect Effects 0.000 title abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 238000012216 screening Methods 0.000 claims description 2
- 241000209094 Oryza Species 0.000 abstract description 24
- 108091033409 CRISPR Proteins 0.000 abstract description 7
- 230000001276 controlling effect Effects 0.000 abstract description 4
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000010362 genome editing Methods 0.000 abstract description 3
- 230000008844 regulatory mechanism Effects 0.000 abstract description 2
- 239000013589 supplement Substances 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 241000196324 Embryophyta Species 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000000463 material Substances 0.000 description 9
- 230000007152 anther development Effects 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000003209 gene knockout Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 108020005004 Guide RNA Proteins 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 230000009261 transgenic effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 208000000509 infertility Diseases 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 208000021267 infertility disease Diseases 0.000 description 3
- 230000021121 meiosis Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000037039 plant physiology Effects 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- JXCKZXHCJOVIAV-UHFFFAOYSA-N 6-[(5-bromo-4-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine Chemical compound [NH3+]C1CCCCC1.O1C(C([O-])=O)C(O)C(O)C(O)C1OC1=CNC2=CC=C(Br)C(Cl)=C12 JXCKZXHCJOVIAV-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 108091060290 Chromatid Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229930002875 chlorophyll Natural products 0.000 description 2
- 235000019804 chlorophyll Nutrition 0.000 description 2
- 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 2
- 210000004756 chromatid Anatomy 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 230000036571 hydration Effects 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 239000004848 polyfunctional curative Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 2
- 101100313923 Arabidopsis thaliana TIP1-2 gene Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010027344 Basic Helix-Loop-Helix Transcription Factors Proteins 0.000 description 1
- 102000018720 Basic Helix-Loop-Helix Transcription Factors Human genes 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 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
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102000016285 Guanine Nucleotide Exchange Factors Human genes 0.000 description 1
- 108010067218 Guanine Nucleotide Exchange Factors Proteins 0.000 description 1
- 206010021928 Infertility female Diseases 0.000 description 1
- 108010059597 Lanosterol synthase Proteins 0.000 description 1
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 1
- 101000898937 Oryza sativa subsp. japonica Potassium transporter 1 Proteins 0.000 description 1
- 101100099674 Oryza sativa subsp. japonica TIP2 gene Proteins 0.000 description 1
- 101100313935 Oryza sativa subsp. japonica TIP2-1 gene Proteins 0.000 description 1
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 229920002334 Spandex Polymers 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 102000021115 calcium ion binding proteins Human genes 0.000 description 1
- 108091011132 calcium ion binding proteins Proteins 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- VWTINHYPRWEBQY-UHFFFAOYSA-N denatonium Chemical compound [O-]C(=O)C1=CC=CC=C1.C=1C=CC=CC=1C[N+](CC)(CC)CC(=O)NC1=C(C)C=CC=C1C VWTINHYPRWEBQY-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000008119 pollen development Effects 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- -1 small molecule compound Chemical class 0.000 description 1
- 239000004759 spandex Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Abstract
本发明公开了一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用。该基因OsPK7的核苷酸序列如SEQ ID NO.1所示，其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。本发明通过CRISPR/Cas9基因编辑技术对OsPK7基因进行敲除后，敲除株系表现为雄性不育性，表明该基因在花粉中具有至关重要的功能，能够为水稻雄性不育系的培育提供基因资源，并且也对雄性不育调控机理进行补充。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用。
背景技术
水稻是全世界大部分人口赖以生存的主要口粮，也是我国最重要的主食作物。随着经济发展对劳动力的需求，全世界人口不断增加，我国人口数量也增加到全世界人口的四分之一，然而农村年轻劳动力进城务工，导致农村从事农业生产人员大量减少，耕地面积急剧减少，水稻产量的提升急需解决。利用杂种优势的水稻三系杂交育种是提高水稻产量的重要途径，三系杂交育种包含恢复系、保持系和不育系，其中不育系是雄性不育水稻，雄性不育种质资源的发掘和利用对于杂交育种至关重要，因此，水稻雄性不育基因的研究有利于拓展不育系种质资源。具有重要的理论意义和生产应用潜力。
目前，关于影响水稻雄性不育的基因被克隆了很多，相关的基因根据其作用的时期分为二类，一类是影响花粉发育时期的减数分裂、花药绒毡层降解和花粉壁形成的相关基因，如OsRAD1编码核酸外切酶同源蛋白，参与减数分裂时期抑制非同源末端连接，影响花粉粒育性，导致不育(Hu et al,Plant Physiology,2016)。OsRAD21-3编码姐妹染色单体粘连和分离关键因子，参与减数分裂中姐妹染色单体的粘连和分离(Tao et al,The PlantJournal,2007)。TIP2编码bHLH转录因子，影响花药绒毡层细胞程序化死亡的中断，导致花粉无活性，完全雄性不育(Fu et al,The Plant Cell,2014)。OsDEX1编码钙离子结合蛋白，基因功能丧失导致花药中绒毡层退化延迟，不能形成成熟花粉粒，完全雄性不育(Yu etal,Plant Physiology,2016)。OsABCG15编码ABC转运蛋白，参与花药中绒毡层细胞脂质分子向花药药室运输，进而影响花粉外壁发育(Zhao et al,Plant Physiology,2015)。OsOSC12编码氧化鲨烯环化酶，影响花粉外壁中花粉包被物质积累，导致花粉粒快速失水，表现出不育(Xue et al,Nature Communications,2018)。第二类影响花粉粒水合萌发和花粉管伸长，例如OsmLO12编码七跨膜结构域蛋白，可能通过与钙调素相互作用调控花粉水合作用(Yi et al,Plant Reproduction,2014)。OsRopGEF2/3/6/8编码鸟嘌呤核苷酸交换因子，在水稻花粉粒萌发中起着至关重要的作用(Xu et al,Development,Growth&Differentiation,2021)。RUPO编码类受体激酶，通过磷酸化OsHAK1/19/20三个钾离子转运蛋白，调控花粉管K+稳态影响花粉管伸长(Liu et al,PLoS Genetics,2016)。GORI编码WD-40基序蛋白，介导水稻花粉管生长的内吞和外排复合体中起着关键作用(Kim et al ThePlant Journal,2020)。虽然雄性不育相关基因报道不少，但是花粉中糖酵解相关酶导致水稻完全雄性不育很少报道。
发明内容
针对现有技术中的上述不足，本发明提供一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种调控水稻雄性不育的基因OsPK7，该基因OsPK7的核苷酸序列如SEQ ID NO.1所示。具体如下：
ATGGCGGTGATGGAGGAGCAGCAGGAGGGAGCCGCGGGGGTGATGCGGCGGAGGCCCAAGACGAAGATCGTGTGCACGCTGGGGCCGGCGTCGAGGTCGGTGGAGATGATCGGGCGGCTGCTCCGCGCCGGGATGTGCGTCGCGCGCTTCAACTTCTCCCACGGATCCCACGAGTACCACCAGGAGACGCTCGACAACCTCCGCGCCGCCATGGAGAGCACCGGCATCCTCTGCGCCGTCATGCTCGACACCAAGGGGCCAGAGATCCGAACTGGATTTCTGAAAGATGGAAAACCCGTACAGTTGAAGAAGGGTCAAGAAATCACAGTTTCAACTGATTATAGCATAAAGGGTGATGACAACATGATATCAATGAGCTACAAGAAGCTAGCGGTGGATCTGAAGCCAGGCAGTGTAATATTATGTGCTGATGGCACCATCACTCTTACTGTCCTTCACTGTGATAAAGAGCAAGGCTTGGTTCGCTGCCGTTGTGAGAACACTGCTATGCTTGGTGAGAGGAAGAATGTTAACCTTCCAGGAGTTATTGTTGATCTCCCGACACTTACTGAGAAGGACAAGGAGGATATTCTTAAATGGGGTGTCCCAAACAAGATTGACATGATTGCTCTGTCTTTTGTTCGTAAAGGCTCAGACCTTGTAGAGGTCAGGAAGGTGCTTGGGAAGCATGCAAAGTCAATAATGCTGATGTCAAAGGTTGAGAATCAAGAGGGAGTGGCTAACTTTGATGATATCCTGGCACAATCTGATGCTTTTATGGTTGCAAGGGGTGATTTGGGAATGGAAATTCCGATAGAGAAGATCTTTTATGCACAGAAGGTGATGATTTTCAAGTGCAATATTCAGGGCAAGCCAGTTGTGACTGCAACCCAGATGTTGGAATCTATGATCAAGTCTCCCCGCCCTACTAGAGCAGAAGCGACTGATGTTGCCAATGCAGTTCTTGATGGCACTGACTGTGTCATGCTCAGTGGTGAGACAGCTGCTGGGGCTTACCCGGAACTGGCTGTACGGACCATGGCCAAGATCTGCCTGCAAGCGGAGTCATGCGTCGACCATGCCGCTGTTTTCAAGTCCATTACCGCATCAGCTCCAATTCCCATGAGCCCATTGGAGAGCCTTGCATCATCAGCTGTGCGTACAGCGAACTCTGCCAAGGCGGCGCTCATCTTGGTCCTGACTAGGGGAGGAACAACTGCTAGGCTGGTGGCCAAGTACAGGCCATCCATGCCGATTCTGTCCGTCGTGGTTCCTGAGCTGAAACAGACCGACTCCTTCGACTGGACCTGCAGCGACGAAGCTCCTGCGCGGCACAGCCTCATCGTCAGGGGAGTGATCCCGATGCTGAGTGCAGCCACTGCCAAGGCCTTCGACAACGAAGCCACTGAAGAAGCTCTCGGGTTCGCCATCAGCAACGCCAAGGCGATGGGGCTCTGCAACTCCGGTGAGTCCGTCGTCGCCCTCCACCGGATCGGAACTGCATCTGTCATCAAGCTCCTGACAGCGAACTAG。
进一步地，基因OsPK7的序列为与SEQ ID NO.1所示核苷酸序列具有80％以上同源性，且编码相同功能蛋白的序列。
上述基因OsPK7编码的蛋白，该蛋白的氨基酸序列如SEQ ID NO.2所示，具体如下：
MAVMEEQQEGAAGVMRRRPKTKIVCTLGPASRSVEMIGRLLRAGMCVARFNFSHGSHEYHQETLDNLRAAMESTGILCAVMLDTKGPEIRTGFLKDGKPVQLKKGQEITVSTDYSIKGDDNMISMSYKKLAVDLKPGSVILCADGTITLTVLHCDKEQGLVRCRCENTAMLGERKNVNLPGVIVDLPTLTEKDKEDILKWGVPNKIDMIALSFVRKGSDLVEVRKVLGKHAKSIMLMSKVENQEGVANFDDILAQSDAFMVARGDLGMEIPIEKIFYAQKVMIFKCNIQGKPVVTATQMLESMIKSPRPTRAEATDVANAVLDGTDCVMLSGETAAGAYPELAVRTMAKICLQAESCVDHAAVFKSITASAPIPMSPLESLASSAVRTANSAKAALILVLTRGGTTARLVAKYRPSMPILSVVVPELKQTDSFDWTCSDEAPARHSLIVRGVIPMLSAATAKAFDNEATEEALGFAISNAKAMGLCNSGESVVALHRIGTASVIKLLTAN*。
一种表达盒、表达载体或克隆载体，其包括上述核苷酸序列。
含有上述基因OsPK7、或者表达盒、表达载体或克隆载体的工程菌。
上述基因OsPK7在水稻雄性不育品种筛选中的应用。
上述基因OsPK7在制备转基因植物中的应用。
一种调控水稻雄性不育的制剂，该制剂包括抑制上述基因表达，或敲除上述基因的活性成分。
进一步地，活性成分为小分子化合物、shRNA、gRNA或短肽。
本发明的有益效果：
本发明通过CRISPR/Cas9基因编辑技术对OsPK7基因进行敲除后，敲除株系表现为雄性不育性，表明该基因在花粉中具有至关重要的功能，能够为水稻雄性不育系的培育提供基因资源，并且也对雄性不育调控机理进行补充。
附图说明
图1为OsPK7基因CRISPR/Cas9靶位点信息；
图2为野生型ZH11和敲除突变体植株及穗部表型；
图3为野生型ZH11和敲除突变体植株花粉碘染；
图4为OsPK7基因在水稻全生育期时空表达谱分析；
图5为报告基因GUS载体图谱；
图6为OsPK7 GUS转基因材料染色观察及半薄切片；
图7为野生型ZH11和敲除突变体植株正反交结果。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1OsPK7基因敲除体的构建
1、OsPK7基因敲除载体构建及转化
利用华南农业大学刘耀光老师建立的网站(http://skl.scau.edu.cn/)设计向导RNA(guide RNA,gRNA)靶位点序列，并构建到植物抗性为潮霉素的pYLCRISPR/Cas9 Pubi-H载体上，具体方法参照(Yao-Guang Liu,Current Protocols in Molecular Biology,2016)，基因编辑载体转化材料受体为野生型ZH11，实验转化由百格基因公司完成(见图1)。
2、花粉碘染
采集野生型ZH11和敲除突变体植株将要开花的成熟颖花，取出花药置于载玻片上，加1-2滴I2-KI溶液，用镊子充分捣碎，盖上盖玻片，用显微镜观察花粉染色情况。结果显示敲除突变体花粉碘染成功，说明敲除突变体花粉有淀粉积累。(见图2和图3，其中，KO1和KO2为敲除突变体)
实施例2OsPK7基因在水稻全生育期时空表达谱分析
荧光实时定量PCR分析具体过程：采用Vazyme公司生产的ChamQ SYBR qPCRMaster Mix进行实验，以OsActin基因为内参，前引物为ACCATTGGTGCTGAGCGTTT，后引物为CGCAGCTTCCATTCCTATGAA，OsPK7基因为检测基因，前引物为GGCACCATCACTCTTACTGT，后引物为GGTCTGAGCCTTTACGAAC，使用10μL反应体系，其中包含1μL反转录产物，5μLChamQ SYBRqPCR Master Mix和4μL 1μM基因引物。荧光实时定量PCR检测目的基因设置三个技术重复，实验所需仪器为Bio-Rad CFX96，试验过程枪头，离心管均为无DNase和RNase型。
提取R498背景的水稻根、叶、节、节间、幼穗、开花后5d、10d、15d、20d、25d颖果中总RNA，以及ZH11背景花药发育时期为6-7、7-8、8-9、9-10、10-11、12-13颖花总RNA。使用Nanodrop测定各个样品RNA浓度，取500ng总RNA用FORE GENE公司生产的RT EasyTMII(WithgDNase)反转录成cDNA，利用荧光实时定量PCR进行表达量分析，见图4。
图4中，(A)为从R498背景下提取的R(根)；N(孕穗期倒一节)；IN(孕穗期倒一节节间)；L1(苗期叶片)；L2(灌浆期叶片)；P1(孕穗期2cm长幼穗)；P2(孕穗期12cm长幼穗)；5DAF-25DAF(受精后5-25天胚乳)颖果中RNA的表达量分析检测。
(B)为ZH11野生型植株花药发育不同时期的颖花，St6-7(花药发育第6-7期颖花)；St7-8(花药发育第7-8期颖花)；St8-9(花药发育第8-9期颖花)；St9-10(花药发育第9-10期颖花)；St10-11(花药发育第10-11期颖花)；St12-13(花药发育第12-13期颖花)中RNA的表达量分析检测。
如图4所示，由于OsPK7基因敲除突变体为雄性不育，因此提取了ZH11背景花药发育时期为6-7、7-8、8-9、9-10、10-11、12-13颖花RNA，反转录后定量分析，结果显示OsPK7基因表达量在颖花中逐渐增加，花药后期表达量更高。
实施例3OsPK7基因在水稻各时期组织中的表达
1、GUS载体构建及转化
以NIP的DNA为模板，通过PCR扩增ATG起始密码子上游的1kb(OsPK7)基因组片段，并将PCR产物胶回收用单片段重组酶连接到以线性化(HindIII,BamhI)的DX2181载体上。并送百格基因转粳稻ZH11材料(如图6所示)。
2、GUS材料染色
取阳性GUS转基因植株的各个组织器官进行GUS染色分析。
(1)按1:50的比例将X-Gluc Solution(50x)和GUS Buffer充分混匀，配成X-Gluc染色液。
(2)将各个组织放入5mL离心管中，加入适量染色液，使组织完全浸没于染液中。
(3)37℃避光孵育1-24h，直至组织上显现蓝色。
(4)用70％乙醇反复脱去组织中的叶绿素，直至叶绿素被彻底清除。
(5)样品保存于70％乙醇中，并用体式显微镜(LEICA S60，Germany)进行观察和拍照。
3、GUS材料半薄切片
(1)取材及固定：将GUS染色成功的叶片和颖花放入FAA固定液中固定24h以上。
(2)冲洗和脱水：将固定好的材料，先用蒸馏水清洗30min，再依次用30％、50％、70％、80％、85％、90％、95％、100％的乙醇进行梯度脱水，每级处理30min，除100％酒精重复三次外，其余浓度均处理一次。
(3)透明：将脱水后的材料放入二甲苯与无水乙醇体积比为1:1的透明剂中，处理45min。然后转入纯二甲苯中，处理45min。
(4)预渗透：各取等体积的无水乙醇与7100基液(base liquid)混合，制成混合树脂，然后将透明后的样品放入适量混合液中60℃孵育1-2h。
(5)渗透：将1g hardener I加入到100mL基液(base liquid)中，充分溶解配成制备液。然后将样品放入足量的制备液中室温浸泡12h。
(6)聚合反应：按照hardener II与制备液体积比为1:15的比例将两种试剂混匀。吸取500μl混合液到胶囊模具中，接着放入渗透过的样品，调整好位置，室温2h即可凝固。
(7)切片与烘片：将修整好的树脂块用半薄切片机(Leica RM2255,Germany)切成厚度2μm的薄片，用镊子移至盛有水滴的载玻片上展片，然后将载玻片置于42℃烤片机上过夜烤片。
(8)染色：对烤片完成后的切片依次做以下处理：高碘酸溶液处理5-10min；流水冲洗5min，擦干切片上多余的水分；滴加Schiff试剂，染色10-15min；流水冲洗5min，擦干切片上多余的水分；滴加0.1％的考马斯亮蓝R250溶液，染色1min；流水冲洗5min，擦干切片上多余的水分，置于烤片机上烘干。
(9)拍照：用莱卡光学显微镜(Leica,DM2700P,Germany)进行观察及拍照(见图7)。
由图5和图6的GUS染色显示，OsPK7基因在叶片，节，节间，花药中的花粉中有明显的GUS信号，其在水稻生长发育的各个时期均有表达。
实施例2敲除OsPK7基因对雄性的影响
采用正反交技术验证敲除OsPK7基因后雄性植株的可育性，具体如下：
田间早上6点半时，剪去颖花二分之一左右，喷水使花粉吸水失活，然后去点雄蕊的花药。并用杂交袋套袋，避免雌蕊授粉，待田间少量ZH11开花时，取作父本的穗子喷水，使穗子湿润，放入黑色塑料袋中，10分钟左右取出，将花粉抖落到相应的母本穗子上，最后使用回形针封口。正反交为母本野生型ZH11和父本敲除突变体、母本敲除突变体和父本野生型ZH11。通过正反交分析OsPK7敲除突变体不育表型是雄性不育还是雌性不育，见图7。
如图7所示，母本野生型ZH11和父本敲除突变体不结实，母本敲除突变体和父本野生型ZH11正常结实，说明OsPK7敲除突变体花粉障碍，表现为雄性不育表型。
以上所述仅为本发明的若干个具体实施方式，应当指出，对于本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (2)
1.基因OsPK7在水稻雄性不育品种筛选中的应用，其特征在于，所述基因OsPK7编码的蛋白的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的应用，其特征在于，所述基因OsPK7的核苷酸序列如SEQ IDNO.1所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211053458.4A CN116004650B (zh) | 2022-08-31 | 2022-08-31 | 一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211053458.4A CN116004650B (zh) | 2022-08-31 | 2022-08-31 | 一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116004650A CN116004650A (zh) | 2023-04-25 |
| CN116004650B true CN116004650B (zh) | 2024-02-23 |
Family
ID=86017993
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211053458.4A Active CN116004650B (zh) | 2022-08-31 | 2022-08-31 | 一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116004650B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952810A (zh) * | 2012-09-04 | 2013-03-06 | 新疆农业科学院核技术生物技术研究所 | 小麦一个抗旱基因TaPK及其编码蛋白与应用 |
| CN106497936A (zh) * | 2016-09-29 | 2017-03-15 | 北京大学 | 控制水稻雄性育性的蛋白及其编码基因和应用 |
| CN108004218A (zh) * | 2018-01-19 | 2018-05-08 | 四川农业大学 | 一种控制水稻千粒重的基因OsPK3及应用 |
| CN109337884A (zh) * | 2018-12-21 | 2019-02-15 | 中国农业科学院北京畜牧兽医研究所 | 一种丙酮酸激酶基因及其应用 |
| CN114262699A (zh) * | 2021-12-28 | 2022-04-01 | 中国水稻研究所 | 水稻雄性不育基因OsLDDT1及其分子标记和应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005185101A (ja) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | 植物の全長ｃＤＮＡおよびその利用 |
| EP2478760A1 (en) * | 2005-05-10 | 2012-07-25 | Monsanto Technology LLC | Genes and uses for plant improvement |
| CA2738105A1 (en) * | 2008-10-23 | 2010-04-29 | Basf Plant Science Gmbh | A method for producing a transgenic cell with increased gamma-aminobutyric acid (gaba) content |
| CA2768331A1 (en) * | 2009-07-23 | 2011-01-27 | Basf Plant Science Company Gmbh | Plants with increased yield |
-
2022
- 2022-08-31 CN CN202211053458.4A patent/CN116004650B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952810A (zh) * | 2012-09-04 | 2013-03-06 | 新疆农业科学院核技术生物技术研究所 | 小麦一个抗旱基因TaPK及其编码蛋白与应用 |
| CN106497936A (zh) * | 2016-09-29 | 2017-03-15 | 北京大学 | 控制水稻雄性育性的蛋白及其编码基因和应用 |
| CN108004218A (zh) * | 2018-01-19 | 2018-05-08 | 四川农业大学 | 一种控制水稻千粒重的基因OsPK3及应用 |
| CN109337884A (zh) * | 2018-12-21 | 2019-02-15 | 中国农业科学院北京畜牧兽医研究所 | 一种丙酮酸激酶基因及其应用 |
| CN114262699A (zh) * | 2021-12-28 | 2022-04-01 | 中国水稻研究所 | 水稻雄性不育基因OsLDDT1及其分子标记和应用 |
Non-Patent Citations (10)
Also Published As
| Publication number | Publication date |
|---|---|
| CN116004650A (zh) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016355682B2 (en) | Haploid induction compositions and methods for use therefor | |
| Yoshida et al. | Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis | |
| Yan et al. | Disruption of OPR7 and OPR8 reveals the versatile functions of jasmonic acid in maize development and defense | |
| Xing et al. | ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2 | |
| Alva et al. | Pollen morphology and boron concentration in floral tissues as factors triggering natural and GA-induced parthenocarpic fruit development in grapevine | |
| Ge et al. | Transcriptional profiling of genes involved in embryogenic, non-embryogenic calluses and somatic embryogenesis of Valencia sweet orange by SSH-based microarray | |
| WO2019062895A1 (zh) | 玉米基因ZmABCG20在调控作物雄性育性中的应用以及与玉米雄性生育力相关的DNA分子标记及其应用 | |
| Wang et al. | Transcriptomic analysis of grapevine Dof transcription factor gene family in response to cold stress and functional analyses of the VaDof17d gene | |
| CN109295246B (zh) | 与玉米雄性生育力相关的dna分子标记及其应用 | |
| Li et al. | Gynoecy instability in cucumber (Cucumis sativus L.) is due to unequal crossover at the copy number variation-dependent Femaleness (F) locus | |
| CN109207513A (zh) | Dcm1蛋白及其编码基因在调控植物雄性育性中的应用 | |
| KR20210023827A (ko) | 고추의 바카툼 세포질 웅성 불임 시스템에 대한 복원 유전자좌 | |
| CN110241126A (zh) | OsDGD2β基因在培育雄性不育水稻品种中的应用 | |
| CN108103074B (zh) | 二穗短柄草抗旱基因和表达载体及其编码蛋白质与应用 | |
| Wang et al. | Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.) | |
| CN116004650B (zh) | 一种调控水稻雄性不育的基因OsPK7及其编码蛋白和应用 | |
| KR101432278B1 (ko) | 신규 유채 품종 | |
| Gao et al. | A kelch‐repeat superfamily gene, ZmNL4, controls leaf width in maize (Zea mays L.) | |
| CN112028979B (zh) | 一种提高黄瓜植株抗低温能力的方法 | |
| Kovacik et al. | Isolation of high purity tissues from developing barley seeds | |
| CN113583990A (zh) | 水稻全育期半矮化表型调控基因sd38及其应用 | |
| Shamina et al. | Two novel meiotic restitution mechanisms in haploid maize (Zea mays L.) | |
| Pagalla et al. | Induction of microspore embryogenesis of eggplant (Solanum melongena L.)‘Gelatik’ | |
| Li et al. | A novel maize gene, glossy6 involved in epicuticular wax deposition and drought tolerance | |
| Szécsi et al. | Genetic and phenotypic analyses of petal development in Arabidopsis |
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 |