CN115725617A - Paddy rice adenosine triphosphate diphosphatase gene OsAPY2 and application thereof - Google Patents
Paddy rice adenosine triphosphate diphosphatase gene OsAPY2 and application thereof Download PDFInfo
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- CN115725617A CN115725617A CN202211067324.8A CN202211067324A CN115725617A CN 115725617 A CN115725617 A CN 115725617A CN 202211067324 A CN202211067324 A CN 202211067324A CN 115725617 A CN115725617 A CN 115725617A
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Abstract
The invention discloses a rice adenosine triphosphate diphosphatase geneOsAPY2And use thereof, the geneOsAPY2The nucleotide sequence of the protein is shown as SEQ ID NO.1, the nucleotide sequence of cDNA is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3. Will be provided withOsAPY2After the target gene is introduced into rice,OsAPY2the total phosphorus concentration of the over-expression material can be improved by nearly two times; at the same time, under the condition of low phosphorus stress,OsAPY2the length of the root hair and the phosphorus absorption of the knockout mutant material are obviously lower than those of wild rice. In the foregoing, the invention providesOsAPY2Gene in paddy phosphorusUse in regulation of uptake, overexpressionOsAPY2The invention can improve the low-phosphorus resistance of the rice and provides guarantee for cultivating new rice varieties suitable for phosphorus-poor soil.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice adenosine triphosphate diphosphatase geneOsAPY2And applications thereof.
Background
Rice (1)Oryza sativaL.) is the main food crop in China, and the planting area and the yield account for 30-40% of the total amount of the food crops. Phosphorus is one of three essential nutrient elements for plant growth and development, participates in enzymatic reaction and cell signal transduction processes in plants, and plays a vital role in plant growth and development. In order to improve the crop yield, the application amount of farmland phosphate fertilizers in China is continuously increased and accounts for about 27 percent of the total global phosphate fertilizer application amount. But the utilization rate of the phosphate fertilizer in season is only about 10 percent, so on one hand, the waste of phosphorus resources is caused, and on the other hand, a series of environmental pollution problems are caused. And the phosphorite is a non-renewable resource, and the global low-cost phosphate fertilizer resource is consumed by about 2050 years. Therefore, improving the utilization of phosphorus in crops is a major issue related to agricultural production and food safety in China.
The root system is the main organ for plants to absorb water and nutrients. The root hair is an important component of the plant root system, can greatly increase the contact area between the epidermal cells of the plant root and the soil, and is beneficial to the plant to absorb water and nutrients. Under the environment of nutrient deficiency, the rice root system can form a plurality of root hairs to promote the absorption of phosphorus of the rice. Paddy rice adenosine triphosphate diphosphatase geneOsAPY2It is involved in root hair development, but its effect on phosphorus uptake in rice is unclear. Therefore, the present invention proposes to utilizeOsAPY2To promote the phosphorus absorption of rice.
Disclosure of Invention
The invention aims to provide a rice adenosine triphosphate diphosphatase geneOsAPY2 Genes and uses thereof.
The purpose of the invention is realized by the following technical scheme:
paddy rice adenosine triphosphate diphosphatase geneOsAPY2The application of (1) to (4) is as follows:
1) Regulating the phosphorus content of the plant;
2) Regulating and controlling the phosphorus absorption of plants under the low-phosphorus condition;
3) Cultivating plants with increased phosphorus content;
4) Cultivating plants with low phosphorus tolerance;
the rice adenosine triphosphate diphosphatase geneOsAPY2The cDNA sequence of (A) is shown in SEQ ID NO. 2;
the plant is rice;
wherein the regulating the phosphorus content of the plant is increasing the phosphorus content of the plant; the regulation and control of the phosphorus absorption of the plant under the low-phosphorus condition is to promote the phosphorus absorption of the plant under the low-phosphorus condition;
wherein the phosphorus content is the total phosphorus content.
Paddy rice adenosine triphosphate diphosphatase geneOsAPY2The application of the encoded protein is at least one of the following 1) to 4):
1) Regulating the phosphorus content of the plant;
2) Regulating and controlling the phosphorus absorption of plants under the low-phosphorus condition;
3) Cultivating plants with increased phosphorus content;
4) Cultivating plants with low phosphorus tolerance;
the rice adenosine triphosphate diphosphatase geneOsAPY2The amino acid sequence of the encoded protein is shown as SEQ ID NO. 3;
the plant is rice;
wherein the regulating the phosphorus content of the plant is increasing the phosphorus content of the plant; the regulation and control of the phosphorus absorption of the plant under the low-phosphorus condition is to promote the phosphorus absorption of the plant under the low-phosphorus condition;
wherein the phosphorus content is the total phosphorus content.
A method for producing transgenic rice, comprising the step of increasing the expression level and/or activity of the protein according to claim 4 in recipient rice to thereby obtain transgenic rice;
the transgenic rice satisfies at least one phenotype of the following 1) to 2):
1) The phosphorus content is higher than that of the receptor rice;
2) Low phosphorus tolerance is higher than that of the recipient rice;
the recipient rice is Nipponbare rice;
the method for increasing the expression level and/or activity of the protein of claim 4 in recipient rice by introducing the rice apyrase gene of claim 1 into recipient riceOsAPY2The implementation is carried out;
wherein the phosphorus content is the total phosphorus content.
The invention has the beneficial effects that:
1. the invention provides for the first time through systematic researchOsAPY2The gene has the function of improving the phosphorus absorption of rice.
2、OsAPY2The gene over-expression rice can obviously improve the length of rice root hair and promote the absorption of phosphorus.
In conclusion, the invention is obtained by transgenic meansOsAPY2The length of the root hair of the gene over-expression material is obviously increased, and the gene over-expression material plays an important role in improving the absorption of the phosphorus in the rice.OsAPY2When the gene is introduced into rice as a target gene, the over-expression plant can improve the phosphorus concentration of the rice by nearly two times, improve the low phosphorus resistance of the rice and provide guarantee for cultivating a new rice variety suitable for phosphorus-poor soil.
Drawings
FIG. 1:OsAPY2in the gene overexpression materialOsAPY2Relative expression amount of (3).
FIG. 2: created by CRISPR-Cas9 technologyOsAPY2And (4) identifying the mutant.
FIG. 3: under low phosphorus treatmentOsAPY2The gene over-expression material root hair length.
FIG. 4: under low phosphorus treatmentOsAPY2The phosphorus concentration of the overground part of the gene over-expression material.
FIG. 5: under low phosphorus treatmentOsAPY2The phosphorus concentration of the root system of the gene over-expression material.
FIG. 6: under low phosphorus treatmentOsAPY2The length of the root hair of the gene knockout mutant material.
FIG. 7: low phosphorus spotTake the following reasonOsAPY2The aboveground phosphorus concentration of the knockout mutant material.
FIG. 8: under low phosphorus treatmentOsAPY2The phosphorus concentration of the root system of the mutant material is knocked out.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Paddy rice adenosine triphosphate diphosphatase geneOsAPY2The nucleotide sequence of (A) is shown as SEQ ID NO.1, the cDNA sequence is shown as SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3.
Total RNA of the root system of a seedling of Nipponbare of a rice variety is extracted, and the purity and concentration of the extracted RNA are measured by using a ultramicro spectrophotometer NanoDrop-2000 (Thermo Scientific). The extracted RNA is reversely transcribed into cDNA serving as template DNA by using a HiScript IIQRT SuperMix kit of Vazyme company, and the rice adenosine triphosphate diphosphatase gene is carried out by adopting a primer pair consisting of P1 and P2OsAPY2The PCR amplification of (1).
P1：5’-cagtGGTCTCacaacatgcgccgctactccgcgct-3’，
P2：5’-cagtGGTCTCatacatcaagatgcaacctcaatgg-3’。
The PCR reaction system is as follows:
the PCR reaction program is: 5min at 94 ℃; 30s at 94 ℃, 45s at 50 ℃, 84s at 72 ℃ and 30 cycles; 10min at 72 ℃; 30min at 16 ℃.
Recovering 1404bp PCR product, cloning the PCR product to pBWA (V) HU vector (purchased from holotype gold Biotechnology Co., ltd.) according to the instruction of TA cloning kit (purchased from holotype gold Biotechnology Co., ltd.), transferring the ligation product to Escherichia coli DH5 alpha, selectively culturing on LB medium containing 50. Mu.g/mL kanamycin, selecting positive single colony, sending positive single colony after PCR detectionSequencing the sex clone. Extracting plasmid from the positive clone-containing bacterial colony, carrying out BsaI/Eco 31I double enzyme digestion identification, and naming the obtained over-expression vector asOsAPY2-OE。
The constructed super expression vectorOsAPY2the-OE is transferred into agrobacterium tumefaciens EHA105 by an electric transformation method and is used for transforming rice.
The genetic transformation of rice is carried out by a method referred to [ Xu Feiyun. Aquaporin OsPIP1 ], 2 functional study on CO2 transport in rice leaves [ D ]. Nanjing university of agriculture ]. Mature wild Nipponbare seeds are used for inducing 2~3 weeks and subculturing for 4-6 d, the embryonic callus is infected by an agrobacterium-mediated method, the culture is carried out for 60 days, and a T1 generation transgenic plant is obtained through selective culture, differentiation, rooting and seedling hardening. If the obtained T2 generation plants are all transgenic lines, the T1 generation transgenic lines are homozygous transgenic materials. Three homozygous transgenic lines OE # 1, OE # 2 and OE # 3 were selected for subsequent analysis.
Taking the root system of the 4-leaf transgenic line seedling, extracting total RNA, carrying out reverse transcription to obtain cDNA serving as template DNA, and carrying out fluorescent quantitative PCR detection by adopting a primer pair consisting of P3 and P4.
P3：5’-TACGCAGACACAGTTGGAGT-3’；
P4：5’-AGCCTTCACTGCATCCTTCT-3’。
The fluorescent quantitative PCR reaction system is as follows: SYBR PremixExTaq 10 muL, upstream and downstream primers 0.4 muL respectively, cDNA template 2 muL, rox Reference Dye 0.4 muL, ddH 2 O 6.8µL。
The fluorescent quantitative PCR reaction conditions are as follows: 40 cycles, 95 ℃ 1min,95 ℃ 5s,60 ℃ 30s,95 ℃ 15s,60 ℃ 1min,95 ℃ 5s.
Quantitative results according to 2 -△Ct And (4) calculating and analyzing the method. As shown in FIG. 1, NIP indicates Nipponbare, and OE # 1, OE # 2 and OE # 3 indicate Nipponbare, rice cultivarsOsAPY2Gene overexpression transgenic strains;OsAPY2in the gene over-expression transgenic strain line,OsAPY2 the relative expression quantity of the gene is obviously higher than that of the Nipponbare rice.
Example 2
Rice adenosine triphosphate diphosphoric acid by using CRISPR/Cas9 gene editing technologyEnzyme genesOsAPY2Designing and constructing the target siteOsAPY2Knocking out the carrier, and carrying out genetic transformation on the rice variety to obtain a transgenic rice plant.
Designing a target site:
Target1：5’-GTACGCCAATAACCCGCAGG-3’，
Target2：5’-GTGGTTCCCGTGGAATTGCG-3’。
selecting an expression vector Pos-sgRNA, digesting the expression vector with Bsa I to linearize the expression vector, recovering a digestion product, connecting a target site to the expression vector, transforming Escherichia coli DH5 alpha competent cells by the connection product, and obtaining a positive clone through PCR verification. The cloning is proved to be successful after sequencing and comparison.
The constructed knockout vector is used for transforming agrobacterium-induced EHA105, and genetic transformation is carried out on a rice variety Nipponbare by utilizing an agrobacterium-mediated rice callus infection method, so as to obtain a T0 generation positive transformation seedling with kanamycin resistance. In the positive transformation of seedlings by T0 generationOsAPY2Sequencing gene and analyzing its mutation site, selecting three different mutation sitesOsAPY2Mutant lines were knocked out and their relative gene expression was determined. The genetic transformation method and the fluorescent quantitative PCR detection method of the rice variety are the same as those in example 1.
The results are shown in FIG. 2, and three gene knockout mutant strains are obtained through identification; at the same time, the mutant strain root systemOsAPY2The gene expression is obviously lower than that of wild rice.
Example 3
To the obtainedOsAPY2After detecting the gene over-expression transgenic strain, the trilobate seedlings of different strains are cultured and grown for 10 days under the condition of normal phosphorus (300 mu M KH2PO 4) or low phosphorus (2 mu M KH2PO 4). Using an SMZ microscope and DS-U3
Shooting by a camera, wherein in order to ensure the accuracy of the experimental result, the root hair shooting part of each seedling is controlled at the same position. Root hair length measurements were then made using Image J software. The results are shown in FIG. 3, where the root hair length of the over-expression lines is significantly higher than that of the wild type, both under normal and low phosphorus treatment.
To the obtainedOsAPY2After the gene overexpression transgenic strains are detected, different strains of the three-leaf stage seedlings are respectively cultured and grown for three weeks under the conditions of normal phosphorus (300 mu M KH2PO 4) or low phosphorus (2 mu M KH2PO 4). And respectively taking overground part and root system samples, and determining the total phosphorus concentration. As shown in FIGS. 4 and 5, the phosphorus concentrations in the overground part and the root system of the rice of the over-expression line were significantly higher than those of the wild-type rice under the normal phosphorus and low phosphorus treatment.
Example 4
To the obtainedOsAPY2After detecting the transgenic strains of the gene knockout mutants, the trilobate seedlings of different strains are cultured and grown for 10 days under the conditions of normal phosphorus (300 mu M KH2PO 4) or low phosphorus (2 mu M KH2PO 4). Shooting by using an SMZ type microscope and a DS-U3 camera, and controlling the root hair shooting part of each seedling at the same position in order to ensure the accuracy of the experimental result. Root hair length measurements were then made using Image J software. The results are shown in fig. 6, where the root hair length of the mutant lines is significantly lower than that of the wild type rice.
To the obtainedOsAPY2After the detection of the gene knockout mutant transgenic line, the trilobate seedlings of different lines are cultured and grown for three weeks under the conditions of normal phosphorus (300 mu M KH2PO 4) or low phosphorus (2 mu M KH2PO 4). And respectively taking overground part and root system samples, and determining the total phosphorus concentration. As shown in FIGS. 7 and 8, the phosphorus concentrations in the overground part and the root system of the mutant line rice were significantly lower than those of the wild type rice under the normal phosphorus and low phosphorus treatments.
Claims (8)
1. Paddy rice adenosine triphosphate diphosphatase geneOsAPY2The application of (2), which is characterized in that: at least one of the following 1) to 4):
1) Regulating the phosphorus content of the plant;
2) Regulating and controlling the phosphorus absorption of plants under the low-phosphorus condition;
3) Cultivating plants with increased phosphorus content;
4) Cultivating plants with low phosphorus tolerance;
the rice adenosine triphosphate diphosphatase geneOsAPY2The cDNA sequence of (A) is shown in SEQ ID NO. 2;
the plant is rice.
2. Use according to claim 1, characterized in that: regulating the phosphorus content of the plant to increase the phosphorus content of the plant; the regulation and control of phosphorus uptake of plants under low phosphorus conditions is to promote phosphorus uptake of plants under low phosphorus conditions.
3. Use according to claim 2, characterized in that: the phosphorus content is the total phosphorus content.
4. The rice apyrase gene as set forth in claim 1OsAPY2Use of the encoded protein, characterized in that: is at least one of the following 1) to 4):
1) Regulating the phosphorus content of the plant;
2) Regulating and controlling the phosphorus absorption of plants under the low-phosphorus condition;
3) Cultivating plants with increased phosphorus content;
4) Cultivating plants with low phosphorus tolerance;
the rice adenosine triphosphate diphosphatase geneOsAPY2The amino acid sequence of the encoded protein is shown as SEQ ID NO. 3;
the plant is rice.
5. Use according to claim 4, characterized in that: regulating the phosphorus content of the plant to increase the phosphorus content of the plant; the regulation and control of phosphorus uptake of plants under low phosphorus conditions is to promote phosphorus uptake of plants under low phosphorus conditions.
6. Use according to claim 5, characterized in that: the phosphorus content is the total phosphorus content.
7. A method for breeding transgenic rice, which is characterized in that: comprising the step of increasing the expression level and/or activity of the protein of claim 4 in a recipient rice plant to thereby obtain a transgenic rice plant;
the transgenic rice satisfies at least one phenotype of the following 1) to 2):
1) The phosphorus content is higher than that of the receptor rice;
2) Low phosphorus tolerance is higher than that of the recipient rice;
the recipient rice is Nipponbare rice;
the method for increasing the expression level and/or activity of the protein of claim 4 in recipient rice by introducing the rice apyrase gene of claim 1 into recipient riceOsAPY2And (5) realizing.
8. The method of claim 7, wherein: the phosphorus content is the total phosphorus content.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851264A (en) * | 2011-06-30 | 2013-01-02 | 复旦大学 | An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof |
| CN104946674A (en) * | 2015-06-17 | 2015-09-30 | 山东大学 | Adenosine triphosphate/dual phosphatase and coding gene and application thereof |
| CN109068642A (en) * | 2015-11-12 | 2018-12-21 | 得克萨斯州大学系统董事会 | Improvement plant containing the apyrase assortment of genes and the method for being used to prepare the improvement plant with apyrase combination |
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- 2022-09-01 CN CN202211067324.8A patent/CN115725617A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102851264A (en) * | 2011-06-30 | 2013-01-02 | 复旦大学 | An apyrase encoding gene of Aedes albopictus, and preparation method and application of protein thereof |
| CN104946674A (en) * | 2015-06-17 | 2015-09-30 | 山东大学 | Adenosine triphosphate/dual phosphatase and coding gene and application thereof |
| CN109068642A (en) * | 2015-11-12 | 2018-12-21 | 得克萨斯州大学系统董事会 | Improvement plant containing the apyrase assortment of genes and the method for being used to prepare the improvement plant with apyrase combination |
Non-Patent Citations (2)
| Title |
|---|
| TANAKA, T.，等: "RecName: Full=Probable apyrase 2; Short=OsAPY2; AltName: Full=ATP-diphosphatase; AltName:Full=ATP-diphosphohydrolase; AltName: Full=Adenosine diphosphatase;Short=ADPase", UNIPROTKB/SWISS-PROT登录号Q6Z4P2.1, pages 1 - 25 * |
| TANAKA，T.，等: "RecName: Full=Probable apyrase 2; Short=OsAPY2; AltName: Full=ATP-diphosphatase; AltName:Full=ATP-diphosphohydrolase; AltName: Full=Adenosine diphosphatase;Short=ADPase", UNIPROTKB/SWISS-PROT登录号：Q6Z4P2.1 * |
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