CN112094936B - 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 - Google Patents
6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 Download PDFInfo
- Publication number
- CN112094936B CN112094936B CN202011032023.2A CN202011032023A CN112094936B CN 112094936 B CN112094936 B CN 112094936B CN 202011032023 A CN202011032023 A CN 202011032023A CN 112094936 B CN112094936 B CN 112094936B
- Authority
- CN
- China
- Prior art keywords
- wheat
- tapfp
- fructose
- gene
- beta
- 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 54
- 241000209140 Triticum Species 0.000 title claims abstract description 40
- 235000021307 Triticum Nutrition 0.000 title claims abstract description 40
- 238000009395 breeding Methods 0.000 title abstract description 8
- 230000001488 breeding effect Effects 0.000 title abstract description 8
- 239000005715 Fructose Substances 0.000 claims abstract description 27
- 229930091371 Fructose Natural products 0.000 claims abstract description 22
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 21
- 230000035784 germination Effects 0.000 claims description 22
- 241000196324 Embryophyta Species 0.000 claims description 13
- 230000001105 regulatory effect Effects 0.000 claims description 9
- 238000011161 development Methods 0.000 claims description 6
- 230000018109 developmental process Effects 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 2
- 230000001276 controlling effect Effects 0.000 claims 5
- GSXOAOHZAIYLCY-UHFFFAOYSA-N D-F6P Natural products OCC(=O)C(O)C(O)C(O)COP(O)(O)=O GSXOAOHZAIYLCY-UHFFFAOYSA-N 0.000 abstract description 12
- BGWGXPAPYGQALX-ARQDHWQXSA-N beta-D-fructofuranose 6-phosphate Chemical compound OC[C@@]1(O)O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O BGWGXPAPYGQALX-ARQDHWQXSA-N 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 8
- 229910019142 PO4 Inorganic materials 0.000 abstract description 6
- 230000004060 metabolic process Effects 0.000 abstract description 6
- 239000010452 phosphate Substances 0.000 abstract description 6
- RNBGYGVWRKECFJ-ZXXMMSQZSA-N alpha-D-fructofuranose 1,6-bisphosphate Chemical compound O[C@H]1[C@H](O)[C@](O)(COP(O)(O)=O)O[C@@H]1COP(O)(O)=O RNBGYGVWRKECFJ-ZXXMMSQZSA-N 0.000 abstract description 5
- 230000023852 carbohydrate metabolic process Effects 0.000 abstract description 5
- 235000021256 carbohydrate metabolism Nutrition 0.000 abstract description 5
- 229940025237 fructose 1,6-diphosphate Drugs 0.000 abstract description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 5
- 230000008635 plant growth Effects 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 230000035772 mutation Effects 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 102000027487 Fructose-Bisphosphatase Human genes 0.000 description 7
- 108010017464 Fructose-Bisphosphatase Proteins 0.000 description 7
- 244000098338 Triticum aestivum Species 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 5
- 229920000410 poly[9,9-bis((6'-N,N,N-trimethylammonium)hexyl)fluorenylene phenylene dibromide] polymer Polymers 0.000 description 5
- 238000000692 Student's t-test Methods 0.000 description 4
- 108090000992 Transferases Proteins 0.000 description 4
- 102000004357 Transferases Human genes 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 235000011180 diphosphates Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000743774 Brachypodium Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 239000001177 diphosphate Substances 0.000 description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 3
- XPPKVPWEQAFLFU-UHFFFAOYSA-N diphosphoric acid Chemical compound OP(O)(=O)OP(O)(O)=O XPPKVPWEQAFLFU-UHFFFAOYSA-N 0.000 description 3
- 210000005069 ears Anatomy 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000034659 glycolysis Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 2
- 206010053759 Growth retardation Diseases 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 231100000001 growth retardation Toxicity 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 229940005657 pyrophosphoric acid Drugs 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 230000007226 seed germination Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 241000209758 Aegilops Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101001126433 Arabidopsis thaliana Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha 2 Proteins 0.000 description 1
- 101000999895 Arabidopsis thaliana Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit beta 2 Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000003793 Fructokinases Human genes 0.000 description 1
- 108090000156 Fructokinases Proteins 0.000 description 1
- 108010042889 Inulosucrase Proteins 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 102000012435 Phosphofructokinase-1 Human genes 0.000 description 1
- 108010022684 Phosphofructokinase-1 Proteins 0.000 description 1
- 102000001105 Phosphofructokinases Human genes 0.000 description 1
- 108010069341 Phosphofructokinases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- BJHIKXHVCXFQLS-UYFOZJQFSA-N fructose group Chemical group OCC(=O)[C@@H](O)[C@H](O)[C@H](O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000011392 neighbor-joining method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000004108 pentose phosphate pathway Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000007425 progressive decline Effects 0.000 description 1
- 229940048084 pyrophosphate Drugs 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- C12Q2600/158—Expression markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Botany (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了6‑磷酸果糖转移酶TaPFP基因在小麦育种中的应用,属于农作物育种技术领域。本发明首次系统分析了小麦中磷酸果糖代谢的相关基因TaPFP、TaPFK、TaFBP对植物生长的影响,研究结果证实TaPFP基因的突变影响小麦的产量。本发明有益于理解与果糖‑6‑磷酸(F‑1‑P)和果糖‑1,6‑二磷酸(F‑1,6‑P2)平衡有关的基因,为评价相关基因在小麦的碳水化合物的代谢方面作用奠定了基础。
Description
技术领域
本发明属于农作物育种技术领域,具体涉及6-磷酸果糖转移酶(TaPFP)基因在小麦育种中的应用。
背景技术
小麦是世界上主要粮食作物之一,随着全球人口剧增,耕地面积递减,再加上全球气候异常变化的增多和加剧,提高小麦单位面积上的产量已成为迫在眉睫的问题。小麦产量构成因素包括单位面积收获穗数、每穗粒数和粒重,产量及其构成因素是由多基因控制的,易受环境影响。对小麦遗传杂交育种来说,解析影响小麦产量的遗传因素显得特别重要。
随着分子标记的迅速发展,用分子标记来评价作物遗传多样性,相对于形态标记、系谱资料、杂种优势和生物化学等方法,具有一定的优越性。因为分子标记具有不受外界环境因素等的影响,速度快、效率高、灵敏度高等优势。
6-磷酸果糖转移酶(TaPFP)可逆催化6-磷酸果糖和1,6-二磷酸果糖之间的转化,涉及糖酵解和糖异生等碳水化合物代谢,同时还涉及代谢产物焦磷酸的利用。目前,还未有关于TaPFP调控小麦发育的报道。
发明内容
本发明的目的在于提供了6-磷酸果糖转移酶(TaPFP)基因在小麦育种中的应用。
发明人在前期采用多个与产量相关的分子标记在小麦8个亲本的遗传群体和花培3号的诱变群体中进行分析。在分析的基础上,发现花培3号的诱变群体与碳水化合物代谢相关的基因焦磷酸:6-磷酸果糖转移酶(TaPFP)基因的突变,降低了小麦的产量。
TaPFP可逆催化6-磷酸果糖和1,6-二磷酸果糖之间的转化。植物体内还有果糖激酶(PFK)单向催化6-磷酸果糖形成1,6-二磷酸果糖,同时1,6-二磷酸果糖磷酸酶(FBP)催化1,6-二磷酸果糖脱磷酸形成6-磷酸果糖。因此本发明同时研究了三个基因的分子特点以及TaPFP突变对植物生长、其他两个基因的表达的影响进行了研究。
本发明首次系统分析了小麦中磷酸果糖代谢的相关基因,并说明了其中一个基因的突变影响小麦的产量。
1、虽然βtapfps可以正常生长,但田间试验发现下调的βTaFPFs导致生长迟缓(株高降低,千粒重,种子发芽率降低,花粉数量减少)。
2、在发芽过程中,果糖在突变株系中的积累高于野生型花培3号,果糖含量与发芽率呈负相关。
3、对αTaPFPs和TaPFKs的转录水平也有影响。
本发明有益于理解与果糖-6-磷酸(F-1-P)和果糖-1,6-二磷酸(F-1,6-P2)平衡有关的基因,为评价相关基因在小麦的碳水化合物的代谢方面作用奠定了基础。
附图说明
图1为小麦PFK、PFP和FBP的进化树、保守基序和基因结构。A为PFK和PFP,B为FBP。利用氨基酸序列,在MEGA7.0软件中,通过邻接法构建了进化树。PFK/PFP和FBP的氨基酸序列分别提交给MEME(http://meme-suite.org/tools/meme)获得它们间的保守基序。。基因的外显子用矩形表示,其他序列用直线表示。
图2为普通小麦中国春(CS)不同发育阶段的TaPFKs、TaPFPs和TaFBPs的转录谱。A为TaPFKs、B为TaPFPs、C为TaFBPs。R:根;St:茎;L:叶子;Sp:小穗;G:籽粒。基因的相对水平通过每百万转录本(TPM)取log2对数进行显示。。
图3为快中子诱导的HP3突变种群体中βTaPFPs基因突变鉴定。A为单株的基因型,保守引物F1和R1的PCR产物通过6%PAGE分离。1-3泳道686株系;4-6泳道557株系;7-9泳道785株系;第10-12条带,分别是中国春缺体-四体系N7DT7B,N7BT7A和N7DT7B(N:缺失;T:添加);419株系的电泳结果未显示。B为1.5%琼脂糖凝胶检测突变株系的RNA转录本。以cDNA为模板的βPFP-A(上),-B(中)和-D(下)特异性引物的PCR产物。泳道1花培3号,泳道2 419突变株系;泳道3 686突变株系;泳道4 557突变株系;泳道5 785突变株系。
图4为βtapfp突变体表型。A为WT和四个突变株系的株高。平均值±SD,n≥50。B为千粒重(01-2019年两年数据)。n≥3;(C-H)雄蕊和雌蕊的形态。C和F为花培3号(WT);D和G为419株系;E和H为557株系。F-G花粉用1%I–KI染色。图中bar=1mm或2mm。“*”表示在P<0.05时有显着差异(student-t检验)
图5为βtapfp突变株的发芽表型和可溶性糖含量。A为WT和四个突变株吸水萌发发40小时(H)后的状态,bar=2cm。B为0-112H期间的发芽率。C-D为胚芽鞘和主根的长度。平均值±SD,n≥3。“*”表示P<0.05时有显着差异(student-t检验)。每次至少利用100粒种子,3次重复。E为果糖含量。F为葡萄糖含量。G为蔗糖含量。E-G均为平均值±SD。n≥3。“*”表示P<0.05时有显着差异(student-t检验)。
图6为在WT和四个突变株发芽期间,TaPFKs,TaPFPs和TaFBPs的表达。在发芽0、24、48、72和96小时(h)后检测基因的转录水平。将所有表达水平标准化为26S rRNA表达。平均值±SD,n=3。“*”表示P<0.05时有显着差异(student-t检验)。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用的试剂可以商业购买得到。实施例中未注明具体条件的实验方法,通常为本领域常规方法。
实施例1
1.禾本科植物的PFKs、PFPs和FBPs家族的基因数目发生了复制和丢失现象
在拟南芥、水稻、短柄草、大麦、乌拉尔图小麦、粗山羊草(Ae.tauchilii)和普通小麦的基因组中寻找含有PFK(PF00365)结构域的基因。进一步将TaPFK、TaPFP和TaFBP的52个成员定位在小麦染色体上,发现这些成员都分布在普通小麦的21条染色体上。
用邻接法构建了PFK/FPF的进化树,结果表明:PFKs聚集在一组中,而PFPs聚集在另一组中。发明人在PFK_A和PFP_ALPHA亚组中发现基因成员丢失。五个PFK_A成员存在于水稻和短柄草中基因组,但在大麦和小麦的相关物种中只有4个,这表明在短柄草属/小麦族物种分化后发生了基因丢失的事件。
总之,在禾本科植物基因组进化过程中,基因丢失和重复发生在不同的时间,它们决定了禾本科植物三个基因家族中基因成员的变异性。这表明禾本科植物的基因组在这些位点受到了进化的选择。
2.TaPFKs、TaPFPs和TaFBPs蛋白的的特征
从表1和图1A中,可以看出来自小麦部分同源染色体的同源蛋白通常具有相似的肽长度,理论pI值、分子量,细胞亚定位和外显子-内含子结构。发现TaPFKA4,TaPFKC2和TacpFBP1的三个成员的pI值的差异大于0.5pH单位。另外,某些亚家族成员之间的pI存在着巨大的差异(例如TaPFKA1、2、3和4;TaPFKC1、2和3;TacpFBP1和2)。这意味着它们的同工酶可以在较广的pH环境下工作(例如,从酸性到碱性环境)。可能增加小麦应对不同环境的能力,进而增加小麦的抗逆活力。
TaPFK/PFP成员间具有五个保守基序1-4和9,它们的排列顺序相同(图1A)。进一步将TaPFK/PFP与PDB1PFK(大肠杆菌PFK)比对,发现底物F-6-P结合位点位于基序1-4和9中。TaPFK的特异底物ATP的结合位点位于基序8中(图1A)。
由于在cpFBPs的N-末端有信号肽,所以它们多肽长度比cyFBPs长(如表1所示)。除了cpFBP1含有一个额外的基序9,所有TaFBPs有9个保守基序(图1B)。进一步比较TaFBPs于AtcyFBP结构,发现底物或镁离子结合位点在FBP家族中普遍保守;F-1,6-P 2结合位点RYxG(对应于AtcyFBP 246-249)和KLRL(对应于AtcyFBP 277-280)在TacpFBP2中,分别被RYxC和HLRL取代;此外,在KLRL保守基序之前发现了六个氨基酸残基的缺失。但这些突变在植物的植物cpFBP2蛋白中很常见。
表1 TaPFK,TaPFP和TaFBP的特性
注:普通小麦(中国春);NE:外显子数目。PFK:依赖ATP磷酸果糖激酶;PFP:焦磷酸依赖果糖-6-磷酸1-磷酸转移酶;FBP:果糖-1,6-二磷酸酶。
3.TaPFKs,TaPFPs和TaFBPs在小麦不同发育阶段的表达模式
在中国春(CS)小麦参考序列(IWGSC Ref V1.0)搜索了三个基因家族的所有成员,在小麦不同发育阶段的中表达。图2显示,同一亚基因家族的A、B和D亚基因组中的三个同源基因通常具有相似的表达趋势,例如,TaPFK1-A、B和D在根中表达较高,在茎中表达较低。在8个TaPFKs(总共24个成员)中,只有A1和A3是相对表达较高的成员,而其他的则在某些组织中表达较低或在特定的器官中表达较高,例如TaPFK-A2在根中高度表达(图2A)。
在三种TaαPFPs中,αPFP2在根,茎和穗中高表达,叶和发育中的籽粒相对较低;βPFP与αPFP2具有非常相似的表达方式式。由于TaαPFP编码PFP蛋白的调节亚基的,而TaβPFP编码催化亚基的,由此可知,TaαPFP2s和TaβPFPs是小麦发育过程中的发挥主要功能的PFP基因(图2B)。
cpFBP1s在绿色组织中特别表达较高(80-150TPM),在根、发育中的籽粒中几乎不表达(不超过1TPM)。cyFBP1s具有与cpFBP1s相似的表达模式。D基因组编码的两个cpFBP2基因中,cpFBP2-D2的表达水平低于cpFBP2-D1的表达水平(图2C),且与cpFBP2-B的表达模式不相近(图1B)。
4.普通小麦βTaPFP基因的鉴定突变
为了揭示TaPFP的功能,发明人在普通小麦品种花培3(HP3)的快速中子诱导突变群体中鉴定了其突变株。使用一对保守引物同时检测所有三个βTaPFP的突变。从6%非变性PAGE电泳结果中,发现CS的第7同源群中NT缺失系中分别缺失对应于βTaPFP-A,B和D的条带(图3A)。该结果证实βTaPFP-A,B和D分别位于7A,7B和7D上。根据基因分型结果,发现突变群体中少数单株植物缺乏对应于βTaPFPA的条带,而大多数单株植物在419/686株系具有βTaPFP的所有三个拷贝。使用相同的方法,得到了βtapfp-b的突变体。βtapfp-d在M2群体中为杂合突变,筛选M3单株获得纯合的βtapfp-d突变体。
检测突变体的引物如下:
βPFP conserve F1:5′-CACAATGTGATCTGCGGCATC-3′(SEQ ID NO.1);
βaPFP conserve R1:5′-CTTGAATCCATACATGGTGCTG-3′(SEQ ID NO.2)。
以上引物用于基因组水平的突变体的鉴定。针对该引物的扩增特异条件为:退火温度为56℃,延伸时间为30秒。
通过基因特异性引物在RNA水平上进一步鉴定了所有四个突变体系的表达。结果表明在419(686),557和785株系中分别缺少来自βTaPFP-A,B和D的转录本(图3B)。这些结果表明,获得了全部三个同源的βTaPFP敲除突变体:βtapfp-a(419和686株系),βtapfp-b(557株系)和βtapfp-d(785株系)。
用于检测βPFP三个成员特异的表达的引物如下:
βPFP-A F:5′-GATGGATGTTGAGAGGAGGCAAG-3′(SEQ ID NO.3)
βPFP-A R:5′-GGGCTGATGT ATCGGTTCTT G-3′(SEQ ID NO.4)
检测TaβPFP-A的表达。
βPFP-B(D)F:5′-TTAATAAGAGGTTGAGTTGGAGTGG-3′(SEQ ID NO.5)
βPFP-B R:5′-ATAGCCTATGGTTCAGACAGTTATAGG-3′(SEQ ID NO.6)
检测TaβPFP-B的表达。
βPFP-B(D)F与βPFP-D R:5′-AATAGCCTATGGTTCAGACAGTTGG-3′(SEQ ID NO.7)检测TaβPFP-D的表达。
5.βtapfp突变体的表型
收集两年的田间试验数据,发现所有突变株系的株高和千粒重(TKW)均下降,668(βtapfp-a)和557(βtapfp-b)突变系表现更为明显,但所有株系均具有正常的籽粒。由于我们的杂交实验,未能获得βtapfp-a/b,βtapfp-a/d和βtapfp-b/d双突变体,进一步观察了花的形态。图4C-E显示野生型和四个突变株的雌蕊的子房和柱头是正常的,但所有突变株中的花粉量都减少了(图4的C-E)。可见大量花粉散布在HP3的柱头上(图4F),但少量花粉分散在分布在419株系的柱头上(图4G)。557(βtapfp-b)株系的花粉凝聚在一起(图4H)。通过植物的株高、千粒重、以及花粉数量等的观察,发现缺乏βTaPFPs其中一个基因会降低植物的活力。根据四个突变系的表型,可推论出三个同源的βTaPFP拷贝调节普通小麦的植物生长方面具有加性效应。
6.βtapfp突变体萌发时期可溶性糖含量变化
进一步观察了WT(HP3)和4个βtapfp突变株的发芽过程。发现两个株系(419和557系)的发芽率,主根长度和胚芽鞘与HP3相似,但在686系(βtapfp-a,P<0.05)显着降低,βtapfp-d(株系785系),平均值与野生型无统计学差异(图5A-D)。离子交换色谱法检测果糖、葡萄糖和蔗糖等可溶性糖,发现果糖和葡萄糖在发芽过程中都有增加的趋势(图5E-F)。蔗糖含量在24-96H发芽过程中表现出先升后降的动态趋势(图5G)。萌发24h后,所有突变株中果糖的含量均高于HP3,但是24-72h内,突变株中果糖浓度的增加量相对较小。这些表明,萌发过程中,敲除的βTaPFP了基因,对萌发过程中果糖的动态变化有影响。此外,在686株系中,果糖含量在发芽后,比其他株系高2-3倍。果糖水平高可能意味着与果糖代谢相关的下游代谢:如糖酵解、磷酸戊糖途经受到阻碍,将导致ATP、DNA、RNA、蛋白质生物合成所需的原料的短缺,这可能是686株系发芽率降低或幼苗生长慢的原因(图5A,B和E)。
7.TaPFP在糖酵解途径中发挥作用
为了揭示导致突变株和野生型果糖含量不同的机理,进一步检测了TaPFK,TaPFP和TaFBP的表达。从表达数据中看出:1)在发芽过程中仅有少数TaPFKs,TaPFPs和TaFBPs成员表达。例如,在发芽过程中,TacyFBP2s的是唯一表达的TaFBP(图6);2)TaPFKA2和αTaPFP2是HP3中高表达的基因,而其他基因是低表达的基因;3)在突变株中,TaPFKA2,αTaPFP2和βTaPFP的表达水平低于HP3。果糖含量与总TaPFPs的表达水平呈负相关(R2=0.6077-0.7787),而与TaPFK的表达水平无关(图5E,图6);显然,敲除了TaβPFPs对F-6-P和F-1,6-P2代谢相关的基因有影响,这可能是果糖在四个突变体和野生型之间发生差异的原因。此外,可能还存在协调TaPFPs两个亚基(α和β)基因表达的机制。从TaPFPs的表达水平与果糖含量之间的负相关关系,可推断果糖主要通过TaPFP转化为ATP,核糖以及合成氨基酸需要的丙酮酸等下游物质,进而影响了植物的生长。
本发明首次系统分析了小麦中磷酸果糖代谢的相关基因,并说明了其中一个基因的突变影响小麦的产量。
1、虽然βtapfps可以正常生长,但田间试验发现下调的βTaFPFs导致生长迟缓(株高降低,千粒重,种子发芽率降低,花粉数量减少)。
2、在发芽过程中,果糖在突变株系中的积累高于花培3号(WT),果糖含量与发芽率呈负相关。
3、对αTaPFPs和TaPFKs的转录水平也有影响。
本发明有益于理解与果糖-6-磷酸(F-1-P)和果糖-1,6-二磷酸(F-1,6-P2)平衡有关的基因,为评价相关基因在小麦的碳水化合物的代谢方面作用奠定了基础。
序列表
<110> 南通大学
<120> 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用
<130> 20200927
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cacaatgtga tctgcggcat c 21
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cttgaatcca tacatggtgc tg 22
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gatggatgtt gagaggaggc aag 23
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gggctgatgt atcggttctt g 21
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttaataagag gttgagttgg agtgg 25
<210> 6
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atagcctatg gttcagacag ttatagg 27
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aatagcctat ggttcagaca gttgg 25
Claims (1)
1.βTaPFP基因在调控小麦发育中的应用,其特征在于,βTaPFP基因用于调控小麦株高和千粒重,βTaPFP基因用于调控小麦种子发芽率,βTaPFP基因用于调控小麦花粉数量,β TaPFP基因用于调控小麦发芽过程中果糖的含量。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011032023.2A CN112094936B (zh) | 2020-09-27 | 2020-09-27 | 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011032023.2A CN112094936B (zh) | 2020-09-27 | 2020-09-27 | 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112094936A CN112094936A (zh) | 2020-12-18 |
CN112094936B true CN112094936B (zh) | 2023-12-26 |
Family
ID=73756317
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011032023.2A Active CN112094936B (zh) | 2020-09-27 | 2020-09-27 | 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112094936B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100833476B1 (ko) * | 2007-03-09 | 2008-05-29 | 경희대학교 산학협력단 | 성장이 향상된 AtPFP 형질전환 식물체 |
CN101457234A (zh) * | 2007-12-14 | 2009-06-17 | 中国科学院遗传与发育生物学研究所 | 一种提高植物产量的方法及其表达盒 |
CN105907862A (zh) * | 2016-05-05 | 2016-08-31 | 中国科学院遗传与发育生物学研究所 | 一种检测与千粒重相关的TaAGPS基因等位变异的方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050177901A1 (en) * | 2001-06-22 | 2005-08-11 | Syngenta Participations Ag | Identification and characterization of plant genes |
-
2020
- 2020-09-27 CN CN202011032023.2A patent/CN112094936B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100833476B1 (ko) * | 2007-03-09 | 2008-05-29 | 경희대학교 산학협력단 | 성장이 향상된 AtPFP 형질전환 식물체 |
CN101457234A (zh) * | 2007-12-14 | 2009-06-17 | 中国科学院遗传与发育生物学研究所 | 一种提高植物产量的方法及其表达盒 |
CN105907862A (zh) * | 2016-05-05 | 2016-08-31 | 中国科学院遗传与发育生物学研究所 | 一种检测与千粒重相关的TaAGPS基因等位变异的方法 |
Non-Patent Citations (2)
Title |
---|
Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase;Hwei-Fang Cheng等;《Journal of Biological Chemistry》;第265卷(第4期);第2173-2177页 * |
高等植物中焦磷酸:果糖-6-磷酸1-磷酸转移酶;胡志元等;《植物生理学通讯》;第29卷(第3期);第161-168页 * |
Also Published As
Publication number | Publication date |
---|---|
CN112094936A (zh) | 2020-12-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Su et al. | A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat | |
Chourey et al. | Sugar–hormone cross-talk in seed development: two redundant pathways of IAA biosynthesis are regulated differentially in the invertase-deficient miniature1 (mn1) seed mutant in maize | |
Zhang et al. | Characterization of the temporal and spatial expression of wheat (Triticum aestivum L.) plant height at the QTL level and their influence on yield-related traits | |
Li et al. | Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses | |
CN107384937B (zh) | 控制水稻粒长、粒重、产量和籽粒外观品质的基因及其应用 | |
Zalewski et al. | Expression patterns of HvCKX genes indicate their role in growth and reproductive development of barley | |
CN101652480A (zh) | 具有提高的胁迫耐受性和产量的转基因植物 | |
Sun et al. | Haplotype analysis of Viviparous-1 gene in CIMMYT elite bread wheat germplasm | |
Liu et al. | TaTPP‐7A positively feedback regulates grain filling and wheat grain yield through T6P‐SnRK1 signalling pathway and sugar–ABA interaction | |
Kim et al. | Inheritance of (1–3)(1–4)-beta-D-glucan content in barley (Hordeum vulgare L.) | |
Liu et al. | Co-silencing E1 and its homologs in an extremely late-maturing soybean cultivar confers super-early maturity and adaptation to high-latitude short-season regions | |
Ma et al. | Structure and expression of barley starch phosphorylase genes | |
Zhang et al. | Genetic dissection of stalk lodging-related traits using an IBM Syn10 DH population in maize across three environments (Zea mays L.) | |
Jia et al. | Comparative gene retention analysis in barley, wild emmer, and bread wheat pangenome lines reveals factors affecting gene retention following gene duplication | |
KR101226485B1 (ko) | 벼의 분질배유 유전자 FLO(a)와 분자마커 및 유전자 부위 정밀유전자지도 | |
Dong et al. | Cloning of TaSST genes associated with water soluble carbohydrate content in bread wheat stems and development of a functional marker | |
CN112094936B (zh) | 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用 | |
Zuo et al. | Genome‐wide association study reveals loci associated with seed longevity in common wheat (Triticum aestivum L.) | |
Zhang et al. | Functional markers developed from TaGS3, a negative regulator of grain weight and size, for marker-assisted selection in wheat | |
KR20220141844A (ko) | 수박에서 종자 크기를 선택하고 토마토-종자 크기 유전자에서 변형을 생성하는 방법 | |
Gallagher et al. | Fructan in temperate forage grasses; Agronomy, physiology, and molecular biolo | |
Rose et al. | Molecular characterization and sequence diversity of genes encoding the large subunit of the ADP-glucose pyrophosphorylase in wheat (Triticum aestivum L.) | |
CN101374952A (zh) | 海藻糖-6-磷酸合酶调节植物生长的用途 | |
Agreda-Laguna et al. | TREHALOSE ACCUMULATION PROVIDES DROUGHT TOLERANCE TO GENETICALLY-MODIFIED MAIZE IN OPEN FIELD TRIALS. | |
US9120864B2 (en) | Flowering inhibition |
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 |