CN118308407A - Application of gene GLR5 in regulation of rice grain shape - Google Patents
Application of gene GLR5 in regulation of rice grain shape Download PDFInfo
- Publication number
- CN118308407A CN118308407A CN202410510854.8A CN202410510854A CN118308407A CN 118308407 A CN118308407 A CN 118308407A CN 202410510854 A CN202410510854 A CN 202410510854A CN 118308407 A CN118308407 A CN 118308407A
- Authority
- CN
- China
- Prior art keywords
- glr5
- gene
- rice
- application
- use according
- 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
- 101100068845 Arabidopsis thaliana GLR3.7 gene Proteins 0.000 title claims abstract description 46
- 235000013339 cereals Nutrition 0.000 title claims abstract description 39
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 36
- 235000009566 rice Nutrition 0.000 title claims abstract description 36
- 230000033228 biological regulation Effects 0.000 title abstract description 9
- 240000007594 Oryza sativa Species 0.000 title 1
- 241000209094 Oryza Species 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 230000009261 transgenic effect Effects 0.000 claims abstract description 18
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims description 16
- 230000014509 gene expression Effects 0.000 claims description 14
- 230000002018 overexpression Effects 0.000 claims description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 108020004707 nucleic acids Proteins 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 13
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- 239000002299 complementary DNA Substances 0.000 claims description 11
- 230000000694 effects Effects 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 11
- 241000196324 Embryophyta Species 0.000 claims description 10
- 239000012620 biological material Substances 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 13
- 239000008187 granular material Substances 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 3
- 230000001488 breeding effect Effects 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 3
- 230000009456 molecular mechanism Effects 0.000 abstract description 3
- 238000010367 cloning Methods 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 230000004720 fertilization Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- 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 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 230000021759 endosperm development Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910009891 LiAc Inorganic materials 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 240000002582 Oryza sativa Indica Group Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 210000005069 ears Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 101710088566 Flagellar hook-associated protein 2 Proteins 0.000 description 1
- 101000577105 Homo sapiens Mannosyl-oligosaccharide glucosidase Proteins 0.000 description 1
- 102100025315 Mannosyl-oligosaccharide glucosidase Human genes 0.000 description 1
- 102100034408 Nuclear transcription factor Y subunit alpha Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002247 constant time method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000009630 liquid culture Methods 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
- 239000002245 particle Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000008117 seed development Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Abstract
The invention discloses an application of a gene GLR5 in regulating rice grain shape, belonging to the fields of botanic and molecular biology. The invention clones a new granule gene GLR5 by utilizing a reverse genetics method. Through transgenic verification, the gene is over-expressed to obviously increase the grain length of rice. The invention further deeply knows the function of the GLR5 gene, and the cloning and functional research of the gene are helpful for further knowing the molecular mechanism of rice grain shape regulation and control, thereby laying a material foundation for rice grain shape molecular design breeding.
Description
Technical Field
The invention belongs to the fields of botanic and molecular biology, and particularly relates to a rice grain-shaped gene GLR5 and application thereof.
Background
Grain shape is an important factor affecting rice yield and is also closely related to rice quality. The grain shape mainly comprises factors such as grain length, grain width, grain thickness, grain weight and the like, a plurality of grain shape genes, mainly including GS3, GW2, GW5, GL3.1, GLW7, GL5, GS9, GL10 and the like, are cloned by forward genetics and reverse genetics at present, and although a large number of grain shape genes are identified and cloned, the molecular mechanism for understanding the grain shape regulation is still limited, and the regulation relation among the grain shape genes is still little known. The granule genes participate in the regulation and control of granule shapes through various ways, and mainly comprise glume cells, seed grouting speed, plant hormones, transcription factors and the like.
Fertilization is an important process of rice seed development, but the relationship between genes or proteins involved in the fertilization process and grain size is not known at present, and there are few reports. HAP2/GCS1 is a gene involved in seed fertilization, which encodes a highly conserved protein, a homolog of which is present in each species. The current research on this gene has focused on the development of grain fertilization. The gene can be involved in fertilization development process and sucrose metabolism process.
The research of the grain shape genes is helpful for further understanding the genetic basis of grain shape regulation and deepening the understanding of the molecular network of grain shape regulation; meanwhile, more gene resources are provided for molecular design breeding of rice.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide application of a gene GLR5 related biological material.
Gene GLR5 has accession number in the RAP-DB (https:// rapdb. Dna. Affrc. Go. Jp /) database: os05g0269500, the full-length genome sequence is shown as SEQ ID NO.1, the coding region cDNA sequence is shown as SEQ ID NO.2, and the coding protein sequence is shown as SEQ ID NO. 3. The present invention finds that over-expression of GLR5 in the HJX74 genetic background results in a significant increase in rice grain length, and thus the present invention can be utilized to modify rice grain shape.
The aim of the invention is achieved by the following technical scheme:
Use of the gene GLR 5-related biomaterial, wherein:
the biological material is any one or more of the following substances A, B, C, D, E:
A. Full-length genomic DNA of gene GLR5 or its homologous nucleic acid molecule;
B. Gene GLR5 cDNA or a homologous nucleic acid molecule thereof;
C. GLR5 protein encoded by the gene GLR5 or a homologous amino acid sequence thereof;
D. A substance capable of increasing the expression level and/or activity of the substance A, B;
E. a substance capable of increasing the expression level and/or activity of substance C.
Further, the application is any one or more of the following applications 1), 2), 3):
1) Application in regulating rice grain shape;
2) Application in improving rice quality;
3) The application in cultivating transgenic rice.
Further, the nucleotide sequence of the full-length genome DNA described in A is shown as SEQ ID NO. 1.
Further, the homologous nucleic acid molecule described in A refers to a nucleic acid molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the full-length genomic DNA.
Further, the nucleotide sequence of the cDNA in the step B is shown as SEQ ID NO. 2.
Further, the homologous nucleic acid molecule in B is a nucleic acid molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to cDNA.
Further, the amino acid sequence of the GLR5 protein in the step C is shown as SEQ ID NO. 3.
Further, the homologous amino acid sequence described in C means an amino acid sequence having at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the amino acid sequence of the GLR5 protein.
Further, the application 1) includes: the application of increasing the grain length of the rice, and/or the application of reducing the grain width of the rice, and/or the application of increasing the aspect ratio of the rice.
Further, the substance D is selected from: recombinant vectors, expression cassettes, transgenic cell lines, transgenic plant tissues or recombinant bacteria containing the corresponding nucleic acid molecules.
Further, the recombinant vector is selected from the group consisting of overexpression vector pOX-eGFP.
Further, said substance E is selected from: recombinant vectors, expression cassettes, transgenic cell lines, transgenic plant tissues or recombinant bacteria containing the coding genes of the corresponding amino acid sequences.
Further, the rice is Hua japonica indica 74 (HJX 74).
Compared with the prior art, the invention has the following advantages and effects:
the invention clones a new granule gene GLR5 by utilizing a reverse genetics method. Through transgenic verification, the gene is over-expressed to obviously increase the grain length of rice.
The invention further deeply knows the function of the GLR5 gene, and the cloning and functional research of the gene are helpful for further knowing the molecular mechanism of rice grain shape regulation and control, thereby laying a material foundation for rice grain shape molecular design breeding.
Drawings
FIG. 1 is a histogram of GLR5 over-expressed plant expression levels; * Each represents a significance of the difference at 0.05,0.001 levels.
FIG. 2 is a graph of a phenotype of over-expressing GLR5 transgene granules; a. particle phenotype plot, scale 1cm; b. grain long histogram; c. grain width histogram; d. grain aspect ratio histogram; * Represents the significance of the difference at the 0.001 level.
FIG. 3 is a block diagram of the amino acid sequence of GLR 5; s represents a signal peptide sequence region; HAP2-GCS1 represents a functional domain; TM represents a transmembrane region; HR represents a histidine-rich region.
FIG. 4 is a histogram of expression of starch and sucrose related genes in a transgenic line;
FIG. 5 is a dynamic view of GLR5 endosperm development; a. development progress of endosperm, scale 5mm; b. fresh weight of endosperm; c. endosperm dry weight; d. endosperm length; e. endosperm width; * Each of these represents the significance of the difference at 0.05,0.01,0.001 levels.
FIG. 6 is a graph showing the self-activation activity of GLR5 protein in yeast.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The various raw materials and various devices used in the invention are conventional commercial products, and can be directly obtained through market purchase, and the primer sequences are synthesized by Shanghai JieRui bioengineering Co.
The overexpression vector pOX-eGFP used in the examples described below is disclosed in the literature "Zhan P, ma S, xiao Z, et al Natural variations IN GRAIN LENGTH (GL 10) regulate RICE GRAIN size [ J ]. Genetic newspaper: english edition, 2022,49 (5): 9.DOI: 10.1016/j.jgg.2022.01.008".
EXAMPLE 1 construction of GLR5 overexpression vector
The young spike cDNA of indica rice HJX74 is used as a template, the front primer is TYCZ-GLR5-F, the rear primer is TYCZ-GLR5-R, and KOD high-fidelity enzyme is used for amplifying the GLR5 coding region sequence, namely SEQ ID NO. 2.
PCR amplification reaction system: KOD Fx Neo Buffer 12.5.5. Mu.L of dNTPs 5. Mu.L, 0.75. Mu.L of each of the front and rear primers, KOD Fx Neo enzyme 0.5.5. Mu.L, sterilized ddH 2 O4. Mu.L, 1.5. Mu.L of cDNA template, and a total of 25. Mu.L system. The PCR reaction procedure was: pre-denaturation at 94℃for 4min; denaturation at 98℃for 10s, annealing at 55℃for 15s, elongation at 68℃for 3min,35 cycles; finally, the extension is carried out at 68 ℃ for 5min. The agarose gel recovery kit is used for recovering the target fragment, kpnI and HindIII are used as insertion sites, and the target fragment is connected to an over-expression vector pOX-eGFP by adopting a homologous recombination method, so that the over-expression vector pOX-Ubi: GLR5 is finally formed.
The primers and sequences used are (5 '-3'):
TYCZ-GLR5-F：
GTGTTACTTCTGCAGGGTACCATGCCTCGTCGTCGAGGAACC
TYCZ-GLR5-R：
CATAACGCGTACTAGTAAGCTTCACAGACGGGGAGTAGTGGCC
Example 2: construction of GLR5 overexpressing plants
Sequencing and comparing the constructed GLR5 over-expression vector, and after the GLR5 over-expression vector is completely correct, carrying out genetic transformation on the GLR5 over-expression vector by the Wohan long-distance biotechnology limited company, wherein the acceptor rice is HJX74, and identifying GLR5 over-expression positive transgenic seedlings by the obtained GLR5 over-expression plant:
(1) DNA extraction
Adding rice leaves into a corresponding centrifuge tube, and adding 800 mu L of TPS solution (special DNA extraction reagent); grinding with a grinder, 60Hz/sec,1min, repeating for 1 time; placing the centrifuge tube into a water bath kettle, and carrying out water bath at 75 ℃ for 40min; after the water bath is finished, putting the mixture into a centrifugal machine, and centrifuging the mixture for 10min at 13000 rpm; absorbing the supernatant in a new centrifuge tube, adding diploid pre-cooled absolute ethyl alcohol, slightly reversing for about 10 times, fully and uniformly mixing, then placing in a low-temperature refrigerator at-30 ℃, standing for 30min, and separating out DNA; after the completion of standing, the mixture was centrifuged at 13000rpm for 10 minutes, the supernatant was removed, and after air-drying, 150. Mu.L of sterilized ddH 2 O was added to dissolve DNA, and finally used for experiments or stored in a refrigerator at 4℃for use.
(2) Positive plant identification
The extracted transgenic seedling DNA is used as a template, and PCR amplification and identification are carried out by adopting a description method of a 2X EsTaqMasterMix kit. The reaction system is as follows: 2 XEsTaqMasterMix12.5. Mu.L, 1. Mu.L each of the front and rear primers, 1. Mu.L of DNA, and 25. Mu.L total of sterilized ddH 2 O9.5. Mu.L. The PCR amplification procedure was: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 45s,35 cycles; finally, the extension is carried out for 5min at 72 ℃. And obtaining 16 positive transgenic seedlings through PCR electrophoresis detection results.
FIG. 1 is a histogram of GLR5 over-expressed plant expression levels. As shown in fig. 1, the expression level of GLR5 in GLR5 overexpressing plants was significantly higher than HJX74.
FIG. 2 is a graph of the phenotype of the overexpression of GLR5 transgene in the form of a granule. As shown in fig. 2, the grain length of both over-expressed lines was significantly greater than control HJX74.
Example 3 expression of starch and sucrose-related genes in transgenic lines
(1) Extraction of young rice spike RNA and synthesis of reverse transcription cDNA
Young rice ears with the length of 3cm in the booting stage are taken, quickly frozen by liquid nitrogen and stored in a refrigerator with the temperature of-80 ℃. Fully washing the mortar, drying, and pre-cooling in liquid nitrogen; placing young ear 3cm into a precooled mortar, fully grinding into powder, placing into a 1.5ml centrifuge tube (1 mlTRIZOL solution is added in advance), fully vibrating and uniformly mixing, and standing at room temperature for 5min; adding 220 mu L chloroform into each tube, vigorously shaking for 10-15s, standing at room temperature for 2-3min, and allowing the chloroform to fully react; pre-cooling the centrifuge to 4 ℃ in advance at 12000rpm for 15min; after centrifugation, absorbing 500 mu L of colorless water layer, placing into a new centrifuge tube, adding equal volume of isopropanol, gently mixing, and standing for 10min at room temperature; centrifuging at 12000rpm at 4deg.C for 15min, and discarding supernatant; adding 1ml of 75% ethanol, washing, centrifuging again for 1-3min, discarding supernatant, air drying (note that complete drying is not required), adding 35 μl of DEPC water, and dissolving RNA; sucking 1 mu L of RNA solution for gel electrophoresis, and detecting the concentration and quality of RNA; placing in a refrigerator with ultralow temperature of-80deg.C for use. Reverse transcription first strand cDNA synthesis was performed according to the protocol described for the 5×all-In-One RT Master mix kit. 2. Mu.g of RNA sample is taken in a small centrifuge tube, and 2. Mu.L AccuRT Reaction Mix of water-supplementing Nuclear-freeH 2 O is added to 8. Mu.L; placing the sample into a PCR instrument, and incubating at 42 ℃ for 2min; after the completion, adding AccuRT Reaction Stopper in 2 mu L, and mixing uniformly; adding 4 μl of 5×all-One RT Mastermix and 6 μl of nucleic-free H 2 O, and mixing; the samples were placed in a PCR instrument with the following procedure: and cooling on ice after finishing at 25 ℃ for 10min, then at 42 ℃ for 50min and finally at 85 ℃ for 5min, and using the ice for experiments or preservation in a refrigerator at-30 ℃.
(2) Starch and sucrose related gene expression analysis
Real-time fluorescent quantitative PCR was performed using SYBR GREEN MASTER Mix kit protocol. The reaction system: cDNA template 0.5. Mu.L; SYBR GREEN MASTER Mix 10. Mu.L; 0.4. Mu.L each of the front primer and the rear primer; ddH 2 O8.7. Mu.L, 20. Mu.L total. The reaction procedure adopts a two-step method: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 10s, annealing/extension at 60℃for 30s,40 cycles; the relative quantitative calculation adopts a-delta CT method.
The primers and sequences used are (5 '-3'):
OsAGPL2-F：CGGACACAATCCTCCCGAAA
OsAGPL2-R：AGCCTGTAACATCCTCCAACA
OsAGPS2-F：CAACAATCGAAGCGCGAGAAA
OsAGPS2-R：GACAGGTGACGGTTCAGAGA
OsBEIIb-F：GAGCAAGAAAAACTAGTTGCCA
OsBEIIb-R：AAGCAAGCTGTCTAGTCTATCC
SPS4-F：GGGAGAATGCCTAGGATAGGTT
SPS4-R：CTAATGCCCGAGCAAGTTCTAC
FLO4-F：CTTGGTTTCCGTGGGTGC
FLO4-R：AGCCTGCTCTGCTATCTCAT
OsSSI-F：TCATGGATGTGAAGGAGCAA
OsSSI-R：TGGCAGTGAACCACAAACAT
SPK-F：ATGGAACCTATGGGAACAA
SPK-R：GAGTCTTGTATCCAAGGACAG
SSIIb-F：CGAGCACTACATCGATCATTTC
SSIIb-R：CATTGTCTTGATCTCCCACAGA
GluA2-F：GCAAGAGCAGGAACAAGGAC
GluA2-R：CCTCATGGTGCAAAAGGTCT
OSINV4-F：GCTTCGTCGACGTCGACATCG
OSINV4-R：CTAGTACTGCTACAACCACC
SPS1-F：CCACTGAGGGAACTTGCAAACC
SPS1-R：ACGAGTAGAGCTGGATGTTG
As shown in FIG. 4, most sucrose-related genes were expressed significantly more than HJX74 in OE-GLR5-2, except for the genes SSIIb and SPS 1.
EXAMPLE 4 study of dynamic development of endosperm of transgenic lines
The invention selects indica rice varieties HJX74 and GLR5 over-expression strain OE-GLR5-2 as materials, and determines dynamic change of endosperm development. During the flowering period of the rice, marking the small spikes of the flowers by using an oily pen every day in the period of 10-13 am, hanging a tag, and writing material information and time, wherein the marked small spikes are enough for subsequent statistics and analysis; sampling at 3 rd, 6 th, 9 th, 12 th, 15 th, 18 th, 21 th, 24 th, 27 th and 30 th days after flowering and pollination, picking up small ears at the same part as much as possible, and removing malformed kernels; in order to avoid water loss, the whole process is operated on ice, glume is carefully stripped, the squeezing is avoided, an analytical balance is used for weighing and recording in time, and the length and the width of endosperm in each period are counted and recorded; finally, the mixture was put into a constant temperature oven at 55℃and dried thoroughly, and the dry weight was measured (a in FIG. 5).
As shown in FIGS. 5 b and c, after flowering and fertilization, the fresh and dry weight increase rates of the OE-GLR5-2 endosperm were both greater than HJX74 until 6 days after endosperm development, whereas after 6 days there was no significant difference in endosperm fresh weights of HJX74 and OE-GLR 5-2; after 15 days of endosperm development, OE-GLR5-2 accumulates at a rate slightly greater than HJX74, either fresh or dry weight, and levels off for about 21 days. As shown by d and e in FIG. 5, the length of the OE-GLR5-2 endosperm is always greater than HJX74 after 3 days of grain fertilisation; the width of the endosperm of OE-GLR5-2 is larger than that of HJX74 in the early development period of endosperm, and the width of the endosperm of HJX74 is gradually larger than that of OE-GLR5-2 in the later development period, and the endosperm gradually becomes stable about 15 days.
EXAMPLE 5 analysis of the self-activating Activity of GLR5 protein in Yeast
The invention adopts DUALmembrane system to analyze the self-activation activity of the gene in yeast. NMY51 procedure for preparation of Yeast competence and Co-transformation: selecting NMY monoclonal with the size of about 3mm, inoculating the monoclonal into 3ml of YPDA liquid culture medium, and performing at 30 ℃ and 220rpm for 8 hours; mu.l of the seed solution was aspirated and added to a flask containing 50ml of YPDA medium at 30℃and 220rpm for 18-20h; transferring the culture solution into a 50ml centrifuge tube, centrifuging at room temperature and 2500g for 5min, removing supernatant, and collecting thalli; 2.5ml of sterilized ddH 2 O was added to the centrifuge tube, and the cells were resuspended, which is a yeast competent cell; PEG/LiAc mix was formulated: taking a 10ml centrifuge tube, adding 2.4ml of 50% PEG,360 μl of 1M LiAc and 250 μl of denaturation CARRIER DNA, and mixing; 1.5. Mu.g of plasmid was added to a 1.5ml centrifuge tube, 300. Mu.l of PEG/LiAc was added, and gently mixed; then 100 mu l of suspended yeast competence is added and mixed gently; water bath at 42 ℃ for 45min; after the water bath is finished, 700g is centrifuged for 5min, and the supernatant is removed; 150 μl of 0.9% NaCl solution was added to resuspend the cells, spread on the corresponding defect medium plates, and incubated at 30deg.C for 2-4d.
As shown in FIG. 6, the GLR5 protein has yeast self-activating activity, whereas the domain HAP2-GCS1 does not affect GLR5 self-activating activity, and the region affecting self-activating activity is located at the C-terminal end of the GLR5 protein.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The application of the gene GLR5 related biological material is characterized in that:
the biological material is any one or more of the following substances A, B, C, D, E:
A. Full-length genomic DNA of gene GLR5 or its homologous nucleic acid molecule;
B. Gene GLR5 cDNA or a homologous nucleic acid molecule thereof;
C. GLR5 protein encoded by the gene GLR5 or a homologous amino acid sequence thereof;
D. A substance capable of increasing the expression level and/or activity of the substance A, B;
E. A substance capable of increasing the expression level and/or activity of substance C;
the application is any one or more of the following applications 1), 2) and 3):
1) Application in regulating rice grain shape;
2) Application in improving rice quality;
3) The application in cultivating transgenic rice.
2. The use according to claim 1, characterized in that:
The nucleotide sequence of the full-length genome DNA in the A is shown as SEQ ID NO. 1.
3. The use according to claim 1, characterized in that:
the nucleotide sequence of the cDNA in the step B is shown as SEQ ID NO. 2.
4. The use according to claim 1, characterized in that:
the amino acid sequence of the GLR5 protein in the step C is shown as SEQ ID NO. 3.
5. The use according to claim 1, characterized in that:
The homologous nucleic acid molecule in A is a nucleic acid molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the full-length genomic DNA;
B means a nucleic acid molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to cDNA;
The homologous amino acid sequence in C is an amino acid sequence having at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the amino acid sequence of GLR5 protein.
6. Use according to any one of claims 1-5, characterized in that:
Application 1) comprises: the application of increasing the grain length of the rice, and/or the application of reducing the grain width of the rice, and/or the application of increasing the aspect ratio of the rice.
7. Use according to any one of claims 1-5, characterized in that:
The substance D is selected from the following substances: recombinant vectors, expression cassettes, transgenic cell lines, transgenic plant tissues or recombinant bacteria containing the corresponding nucleic acid molecules.
8. Use according to any one of claims 1-5, characterized in that:
the recombinant vector is used as a vector selected from the overexpression vector pOX-eGFP.
9. Use according to any one of claims 1-5, characterized in that:
the substance E is selected from the following substances: recombinant vectors, expression cassettes, transgenic cell lines, transgenic plant tissues or recombinant bacteria containing the coding genes of the corresponding amino acid sequences.
10. Use according to any one of claims 1-5, characterized in that:
The rice is Huajing-indica 74.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118308407A true CN118308407A (en) | 2024-07-09 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109161550B (en) | SlbHLH59 gene for regulating and controlling ascorbic acid content of tomato fruits and application method | |
| CN103451228B (en) | Method for regulating size and grain weight of rice seeds | |
| CN114752579B (en) | ZmMAPK protein and application of coding gene thereof in regulation and control of low-temperature stress tolerance of plants | |
| CN107266544B (en) | Application of protein SiNADP-ME3 and coding gene thereof in regulation and control of plant stress resistance | |
| CN109112227A (en) | Rape bloom key gene as the rape ecotype improvement and Mature breeding molecular labeling and application | |
| CN116445510A (en) | Application of OsAAP8 in regulation and control of rice tillering, grain size and grain weight | |
| CN114990136B (en) | Apricot PasLEA3-2 gene for kernel and application thereof in cold resistance and promotion of plant flowering or seed fruiting in advance | |
| CN114717241B (en) | Rice salt tolerance related gene OsMSRFP, encoding protein and application thereof | |
| CN102876687A (en) | SM gene for regulating and controlling cotton trichome, and application thereof | |
| CN114196678B (en) | Hedgehog interacting protein OsHIPL1 gene for controlling activity of rice seeds and engineering application thereof | |
| CN113584051B (en) | Application of GhGAI gene in regulation and control of plant flowering | |
| CN118308407A (en) | Application of gene GLR5 in regulation of rice grain shape | |
| CN108165557A (en) | Application of the wheat TaZCCT2 genes in the flowering of plant time is regulated and controled | |
| CN111662368B (en) | Rubber grass drought-enduring gene TkMYC2, protein, primer, vector, host bacterium and application thereof | |
| CN112226442B (en) | Wheat grain size character related gene TaSRK, encoding protein and application thereof | |
| CN114457094A (en) | Peony PoAGL15 gene and amino acid sequence and application thereof | |
| CN109082425B (en) | Rape boron high-efficiency gene BnA3NIP5; transposon insertion fragment TEQ of 1Q and primer and application thereof | |
| CN114164291B (en) | Application of rice grain length gene GL10 allele | |
| CN117363631B (en) | Application of Glyma.08g111100 gene in identification of salt-tolerant alkalinity of soybean | |
| CN111518184B (en) | Novel application of SiTGAL6 protein | |
| CN114457089B (en) | Gene for increasing length of tomato root hair and application thereof | |
| CN116121435B (en) | Method for regulating and controlling length of wheat root hair, molecular marker and application thereof | |
| CN110016479B (en) | Alfalfa MsGPF gene | |
| CN103667306A (en) | Sorghum wax synthesis regulation gene and application in Arabidopsis plant | |
| CN111875687B (en) | Application of sesame protein SiBRB in regulation and control of plant root system development |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication |