CN117821503B - Application of OsWD gene 40-115 gene in regulation and control of rice quality - Google Patents
Application of OsWD gene 40-115 gene in regulation and control of rice quality Download PDFInfo
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- CN117821503B CN117821503B CN202310712227.8A CN202310712227A CN117821503B CN 117821503 B CN117821503 B CN 117821503B CN 202310712227 A CN202310712227 A CN 202310712227A CN 117821503 B CN117821503 B CN 117821503B
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 44
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- 241000209094 Oryza Species 0.000 claims abstract description 43
- 230000001105 regulatory effect Effects 0.000 claims abstract description 9
- 238000009395 breeding Methods 0.000 claims description 4
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Abstract
The invention discloses an application of OsWD gene 40-115 gene in regulating rice quality. After OsWD-115 gene knockout, the chalkiness and chalkiness of rice are increased, and endosperm starch grains are mostly irregularly round and loosely arranged. The gene of the rice OsWD-115 is involved in regulating the formation of chalkiness of the rice, and can be used for genetic improvement of rice quality.
Description
Technical field:
The invention belongs to the field of rice breeding, and relates to application of OsWD-115 genes in regulating and controlling rice quality.
The background technology is as follows:
rice quality includes processing quality, appearance quality, cooking taste quality, and nutritional quality. The appearance quality mainly comprises three indexes of particle type, chalky (chalky rate and chalky whiteness) and transparency.
Studies have shown that chalkiness are mainly affected by several factors: chalkiness vary greatly between varieties and between years; chalkiness of late rice are generally lower than that of early rice; the influence of climate on chalkiness is large, grouting is influenced by high-temperature induced maturity or low-temperature overcast and rainy, and the grouting period is an important stage directly influencing the yield and quality of rice. In addition, environmental conditions such as illumination, moisture management, geographical location, cultivation mode, etc. have a great influence on chalkiness. Generally, chalkiness are a complex quantitative trait, primarily determined by genetic genes, and are susceptible to environmental factors. Therefore, research on the mechanism of chalkiness formation has important scientific value and production guidance significance for genetic improvement of rice chalkiness. Genes associated with chalkiness have been identified to focus on genes involved in starch and storage protein synthesis, plastids and mitochondrial development. There are also few transcriptional regulators involved in chalkiness but concentrated on several genes containing specific families of domains. Therefore, the transcription regulating factors influencing chalkiness are further identified, and the method has important scientific value and production guidance significance for genetic improvement of chalkiness.
In view of this, the present invention has been made.
The invention comprises the following steps:
The invention aims to provide the function of OsWD genes in regulating the chalkiness of rice, and provides technical support for improving the quality of rice by utilizing OsWD genes to 115 genes.
The first object of the invention is to provide an application of OsWD gene 40-115 in regulating rice quality, wherein the nucleotide sequence of OsWD gene 40-115 is shown as SEQ ID NO. 1.
Preferably, the OsWD gene in the knocked-out rice is applied to the regulation of rice quality.
Preferably, the rice quality is an increase in the chalky grain rate of rice.
Preferably, the rice quality is an increase in rice chalkiness.
A second object of the present invention is to provide the use of OsWD-115 gene in rice breeding.
Preferably, it is used for improving the chalkiness and/or chalkiness of rice.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides OsWD genes which have functions in regulating the chalkiness of rice. OsWD115 gene contains NB-ARC domain, and literature search shows that research on regulation of rice chalkiness by the gene is not reported yet. Therefore, the OsWD gene has novel function and application of regulating the chalkiness of rice.
Description of the drawings:
Fig. 1: osWD40 construction of a transgenic line with a gene knockout of 40-115. Nip represents the wild type material Nipponbare, cr-1, cr-2 and Cr-3 represent 3 OsWD, respectively 40-115, gene knockout transgenic lines.
Fig. 2: osWD40 influence of gene knockout on rice chalkiness 40-115. Panel A shows the chalky phenotype of wild type Nip and OsWD-115 gene knockout lines (Cr-1, cr-2 and Cr-3), and panels B and C show the statistics of chalky rate (PGWC) and chalky Degree (DEC), respectively. * Represents a significant difference in p < 0.05 in t-test, and represents a significant difference in p < 0.01 in t-test.
Fig. 3: osWD40 influence of gene knockout on rice endosperm starch grains 40-115. Scale bar=200 μm (up), 20 μm (middle) and 4 μm (down), and the line frame indicates the site of magnification observation by scanning electron microscope.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof. The specific experimental conditions and methods are not noted in the following examples, and the techniques employed are conventional and well known to those skilled in the art.
Example 1: construction of OsWD Gene knockout transgenic lines 40-115
The sequence of OsWD-115 gene (Locus No. LOC_Os05g15040) whose nucleotide sequence is shown in SEQ ID NO.1 is obtained by using RGAP (http:// price. Plant biology. Msu. Edu /) database.
OsWD40 target sequence for gene knockout of 40-115:
T1：GGATCAGGTTAGAGATCTTA CGG
T2：CGTTGTCCTCAAGTCTCTAG CGG
primers for amplifying the sgRNA expression cassette are:
OsWD40-115-gRT1:GATCAGGTTAGAGATCTTAgttttagagctagaaat
OsWD40-115-OsU6aT1:TAAGATCTCTAACCTGATCCggcagccaagccagca
OsWD40-115-gRT2:CGTTGTCCTCAAGTCTCTAGgttttagagctagaaat
OsWD40-115-OsU6bT2:CTAGAGACTTGAGGACAACGCaacacaagcggcagc
The OsU a is used as a template, and primers OsWD40-115-gRT1 and OsWD40-115-OsU aT1 are used for amplification to obtain a PCR product of the target 1. Using OsU b as a template, PCR products of target 2 were amplified using primers OsWD40-115-gRT2 and OsWD40-115-OsU6bT 2. The PCR procedure was: 95 ℃ for 2min;94℃for 20s,60℃for 20s,68℃for 25s,25 cycles, 68℃for 10min. And purifying and recycling the obtained PCR product, and then carrying out connection transformation. The connection system is as follows: 10 XCut buffer 1.5. Mu.L, 10 XT 4 buffer 1. Mu.L, bsaI-HF (NEB Co.) 0.5. Mu.L, T4 ligase (NEB Co.) 0.25. Mu.L, cas9 cassette 60-80ng, end product of target 1 10-15ng, end product of target 2 10-15ng, and water to 15. Mu.L. The procedure for enzyme digestion and ligation is as follows: 5min at 37 ℃;10 ℃ for 5min,20 ℃ for 5min,10-15 circulation and 37 ℃ for 5min. The ligation product was transformed into E.coli DH 5. Alpha. And plated on LB plates containing 50mg/L kanamycin, after overnight at 37℃5 single clones were selected for sequencing, and the correct Crispr vector was finally obtained. After the constructed knockout vector is transformed into agrobacterium EHA105, infection transformation is carried out on callus of Japanese sunny by using the agrobacterium, and the obtained transgenic line is subjected to seed reproduction and separation and is subjected to primer F:5'-GGGAGGCATCCTACGAAA-3', R:5'-CCGACCCACTGGAACAAA-3' the isolated transgenic individuals were subjected to amplification sequencing, and compared with the Japanese sunny sequences to obtain OsWD-115 homozygous knocked-out transgenic lines (FIG. 1, cr-2 and Cr-3).
OsU6A and OsU B are described in (reference ：A robust CRISPR/Cas9 system for convenient,high efficiency multiplex genome editing in monocot and dicot plants;Ma XL,Zhang QY,Zhu QL,et al.,Molecular Plant,2015,8:1274–1284.).
Example 2: effect of OsWD gene knockout on chalkiness in rice
Wild control material Nip and OsWD-115 gene transgene lines (Cr-1, cr-2 and Cr-3) are planted in a transgene greenhouse, and seeds are harvested and naturally dried. Husking with a rice huller (JLG-III), grinding the brown rice into polished rice with a rice mill (JNM-III), selecting whole polished rice, and measuring chalky rice with a rice appearance quality determiner (SC-E). The chalkiness and chalkiness of OsWD-115 gene knockout lines were significantly increased compared to control Nip (fig. 2).
Example 3: influence of OsWD Gene knockout on Rice endosperm starch grains
The formation of rice chalkiness is often closely related to the physicochemical properties of the endosperm, in particular to the filling of the starch grains in the endosperm. We analyzed the morphology of the starch grains in the endosperm of the control Nip and Cr strains, the endosperm starch grains of the control Nip being polyhedral and densely arranged, while the endosperm starch grains of the Cr strains were mostly irregularly rounded and loosely arranged (fig. 3).
OsWD the gene sequence of OsWD-115 is as follows (SEQ ID NO. 1):
atggaggacatcgtcgacacattcttggtgcacgacgatggccctgaccccaccgatccacacatgctcctgcgcctccggaagaagttagg
caagctgttcaagaagagcaaggctcgccgtaagatctctaacctgatccaagacattaacaagaagttcgagcaggtgaaagcaaggcat
gtcaagtacacagtcgacagcgttgtcctcaagtctctagcggcaacgaccatcgatcctcgtctccagaatctgtacaaaagggcaacaga
gcttgttggcattgacgggccaagggacgatctcataaacaagctatccctggggggtgatgttgatgtgtccgacaagaagatgaaagtcat
ctctgtcgttggatttggaggactcggcaagaccactcttgccaaagctgtctatgaccacttcaaaccgcatttcaagtgcagggcttctgttcc
agtcgaccagaatcctgacatgaataaagcattcgatgaccaggttgaaccgcatttcgaatgcggagcttttgttccagtgggtcggaatcct
gacatgaagaaagtctttagagacattctcattgatcttgacaaggaaaagtacacgaatccgaatatgatggtgttggatgaaaagcagctca
tggacgaactcaaggaattcgtcaaggagaagaggtgtttcattgttattgatgatatatgggacaaggagtcatggaaattaatcagatgtgct
ctgcaagaaagtgattgtggaagtagacttgtcataactactcgtatttctgaagttgccacacatgctggtgaagcttacaaaattcagccacttt
ctcgtgataactctgaaaaattattgtatgcaagaatagttgatggtgaatggaagtatttcgataatccatcagctaaggcatgtgaaaaaatttt
gaagaaatgtggtggtgtaccattagctatcattacaatagctagtttgttggccagtaaaccaggggaggactggtctgaggtctacaactctg
tcggttttgggcatgaagacaatgatgatgtaggtaatactagaaggatattgtcttttagctactatgatctgtcttcacatttaaaagattgcttatt
gtatctatgtgtatttcccgaagatcatgtgatagagaaaaatagtttgatatggaagtggatagctgaaggtttcatccatgaggaactagcagc
agggttaggattatttgagctcggggagaggtgcttcaatgagttgataaatagaagcatgatccagccagtggagacaaaacatgaagggt
atgtagatggttgctgcgttcatgatatggttcttgatatgatccactcattatcgagtgaagaaaattttgtcactgtattgaatggtgatgagcga
cagaaacttccggggagcattgctcgcaggttagccctgcagcgtgttctagtgcacaaggatggtcagctagctaatatcactctgaagaat
gtgagatcccttattgccagtcagtgcttcggttcattatgcctacacactccggttttacgtgtattagatgtgataaactgtgcagaaagtgaga
cgcctaatgttttggaagatagggacaaaactatgccaagccatcttgggagtttacttcacctgaggtacctcgggttagtagatacatatatca
ctaagctgcccagggaagtaaggtatctcaagtttctgcagacactggatttaaggggaagtcgcataaaggagctgccagaggaggtggg
actgctgacacaacttgtttgcctacgagctgattgggaaataatggtgaagattggtttgatcggtaagttgacctcattgcaagagctttggat
agagcctgctgctgctgtttatgatgacgatgctgcttctgttgatgatgatgatgatgctgctgctgttgatgatactacgaagtctacgatgaag
tttgtgaatgagctgggcctactgagggaactaagggtgctccggacttattttgaggatttgaatgagagcatggagagagctttgttggaatc
cctaagcaagctgcacaatatccgggacctgcacattgagggcccacccctagtgacaaggagagtgacaagcagtgcaagatctgtctcc
tgcctgcatctccgatacctgtgtctgaattgctttgtgttctctggactgccagcatggattaaatcatcgcttgctccgaacctctcctgtctgga
tgtggttgtgctaattgtgaaagagcaggacatggaaacccttgcgaggttgccagagctccgctgccttgtgctgcgtttgcatgagaccga
attggttagcataaagattcatactgaagatgttggctactttcggaagttgagaatcttaaggatacttggtacatctatctggtttgatctgcacg
gcagcaagtgcaacatcacagacaatagaagcttagcatctattatcatgccaagtcttgaattcctcgagtttgctgtccatatgcggttcctga
aagatgcaacccagcttggttttgacaagctgcttagcttcgagaagcttggaagaagttcgctccagaaagtcaccatagtactgaaatgtgg
agatgcccgtctgtcagaggtggaggaagtggctgctgcattgatgaactcagctgcggtccatcccaaacacccaactgttcaaaccaaat
ggcaagacgaacaagctgtgctctcaccctaccaagaggcatatatgcatgtgagcagaaccccggagttggtaaatgaggcctggaagg
cgaacattgttagctctggccgtattcgagcactgcgtatgccagttacagaagcatcttcaagcaaagttatctgtctgctttatagaaaaagtg
ggaagggacttttagctctcagctcaaatgctgttcataagctgtggaaatgggagagcaatgacaagaatcctgctggcatgtccaccacat
ctgtcccacctcaagtgtggcaaccggaaagtgacattctaatgaatgacactgctaatggcaaccctgaagaagcagctgcctgcagtctac
tatccaaaaatgactgctatctaatttctgcatctggtggcaaagtttcgttgttcaacatgttaaatttcaagaccatgactactttcatagcacctc
caccctctgcaactttcctcgcattccacccgcatgacaataacatcatagctattggaacggatgactcttcgattctactctacaatatccgtgt
tgacgaggttaaatgggtgctcaagggccatcagaaaaagataactggactggtattctcgctatcaatgaatgtgcttgtatcttcaggtgctg
attctcagctatgtgtttggagtatggaagattgggcgaagaagaaatccagatatatccaacctccatctaatcattctggagctttagttggtga
tataatggtgcagtttcactatgaccaaaagcgccttttagtagttcatgagagccaattggcgatctacgatgaggagctagaatgcttgtgctc
atggttcccaagtgatccactctctgctccagtttcgagtgcagtatactcatcggatggcttattgatctacgctggattctgtgatggtgcaattg
gaatatttcaagtggaatctctcatgttacagtgcaggattgcaccttctgcctatataccatcttcagtttctagcggtggtgaaattgtgtatccc
atggttgtcgctgcgcatccctggaagcctaaccagtttgcagtcggcatgagcgatggtgcagttcatgtgctggagccgttggacacaccc
gatgatatcatcagcaattcaattcagcaggggcgttttggtgacagcggatga。
Claims (2)
1. The application of OsWD-115 genes in knocking out rice in regulating and controlling the increase of the chalkiness rate and/or the chalkiness degree of rice is characterized in that the nucleotide sequence of OsWD-115 genes is shown as SEQ ID NO. 1.
The application of the OsWD40-115 gene in rice breeding is characterized in that the nucleotide sequence of the OsWD-115 gene is shown as SEQ ID NO.1, and the rice breeding is to improve the chalkiness rate and/or chalkiness degree of rice.
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| CN105063206A (en) * | 2015-08-03 | 2015-11-18 | 南京大学 | Molecular breeding method using plant anti-disease gene enriched clusters, and applications thereof |
| CN107142276A (en) * | 2017-05-23 | 2017-09-08 | 华南农业大学 | A kind of transgenic breeding method that anthocyanidin is synthesized in Crop Seed Endosperm |
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| WO2016000238A1 (en) * | 2014-07-03 | 2016-01-07 | Pioneer Overseas Corporation | Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving low nitrogen tolerancegenes |
| CN115786362B (en) * | 2022-09-05 | 2024-02-13 | 四川农业大学 | Heat shock protein family gene HSP110-3 for controlling rice quality and application thereof |
| CN116218876A (en) * | 2023-04-12 | 2023-06-06 | 扬州大学 | Gene OsB12D3 for regulating rice chalkiness, encoding protein and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105063206A (en) * | 2015-08-03 | 2015-11-18 | 南京大学 | Molecular breeding method using plant anti-disease gene enriched clusters, and applications thereof |
| CN107142276A (en) * | 2017-05-23 | 2017-09-08 | 华南农业大学 | A kind of transgenic breeding method that anthocyanidin is synthesized in Crop Seed Endosperm |
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