CN106868022B - Nitrogen transport gene OsNPF2.4b for promoting increase of effective spike number of rice and application thereof - Google Patents
Nitrogen transport gene OsNPF2.4b for promoting increase of effective spike number of rice and application thereof Download PDFInfo
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Abstract
The invention discloses a nitrogen transport gene for promoting the improvement of effective spike number of riceOsNPF2.4bAnd the application thereof, belonging to the field of plant genetic engineering.OsNPF2.4bThe amino acid and cDNA sequence of the gene coding protein are shown in SEQ ID NO.1 and 2. The invention constructs riceOsNPF2.4bOver-expression of the gene, found to be improvedOsNPF2.4bThe expression of the gene can promote the growth of rice, increase the tiller number and effective spike and increase the yield. By constructing interfering plants, a reduction is foundOsNPF2.4bThe gene expression can reduce the biomass of rice, the tiller number and effective spike, and the yield is reduced. Thus, it is possible to provideOsNPF2.4bThe gene can be used for promoting the growth of rice to improve the biomass and the yield of the rice.OsNPF2.4bThe gene has important application in improving the plant type and increasing the yield of rice.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a nitrogen transport gene OsNPF2.4b capable of promoting improvement of effective ears of rice.
Background
Plants obtain nitrogen by absorbing ammonia, nitrate, amino acids, soluble peptides, etc. in soil; nitrogen uptake and transport is mainly achieved by transporters such as ammonium transport protein (AMT), nitrate transport protein (NRT), amino acid transport protein (AAT), and peptide transport Protein (PTR) (Williams L, Miller A. transporters response for the uptake and purification of nitrogenes solubles. Annu Rev Plant Biol and Plant Mol Biol,2001,52: 659. sup. 688.). Ammonium is taken up by the plant AMT and then glutamine and glutamate are synthesized by Glutamine Synthetase (GS) and glutamate synthase (GOGAT), which in turn further form other amino acids (Sonoda Y, Ikeda A, Saiki S, et al. feedback regulation of the ammonium transporter gene amino acid AMT1by glutamine in rice. plant Cell Physiol,2003,44: 1396-. Plants can absorb environmental nitrates via NRT2 of the High Affinity Transport System (HATS) and NRT1 of the Low Affinity Transport System (LATS), form ammonium upon reduction by Nitrate Reductase (NR) and nitrite reductase (NiR), and further form amino acids (Paungfoo-Lonhienne C, Lonhienne T G, Rentsch D, et al. plants can use protein as and source from among other organisms. PNAS,2008,105: 4524-.
The NRT1/PTR family (NRT1/PTR family, NPF) refers to proteins capable of mediating transmembrane transport of substances such as nitrate and small peptides of 2-3 amino acid residues (Rentsch D, Schmidt S, Tegger M. Transporter for uptake and allocation of organic nitro compounds in plants FEBS Let,2007,581: 2281-. Members of the NRT1/PTR family are involved in plant growth and development, as well as in the accumulation of proteins during seed formation and in the transport of small molecule polypeptides after protein degradation during germination (Martre P, Porter J R, Jamieson P D, et al.
Partial members of the NPF family of rice can promote plant growth and increase biomass and yield. Overexpression of the OsNPF8.20 gene in rice increases ammonium uptake and tillering number (Fang ZM, Xia KF, Yang X, et al. Alteredexpression of the PTR/NRT1homologue OsPTR9 afflictions nigrens diagonalizing efficiency, growth and grain yield in rice plant Biotech J,2013,11: 446) 458. The OsNPF6.5 gene encodes a nitrate transport protein, can influence each link of absorption, transport, assimilation and the like of nitrate, and can influence the tillering number of rice (Hu B, Wang W, Ou S, et al.variation in NRT1.1B controls. today genes. Nature Genetics,2015,47: 834. 838). OsNPF7.2 has low affinity transport for nitrate and is able to affect plant root growth at high nitrate concentrations (Hu R, QiuD, Chen Y, et al. knock-down of a toplast localized low-affinity nitrogen nitrate transporter OsNPF7.2affects force growth under high nitrate supply, 2016, 7). Recent studies have shown that overexpression of the high affinity nitrate transport gene OsNRT2.3b promotes rice growth, improves nitrogen utilization efficiency, and increases yield (Fan X, Tang Z, Tan Y, et al. overexpression of a pH-sensitive nitrate transporters in rice channels PNAS,2016,113:7118 + 7123).
The OsNPF2.4b related by the invention is a short transcript form formed by the rice NPF gene family gene OsNPF2.4 through post-transcriptional processing and splicing. The invention discovers that the OsNPF2.4b gene has important effect on the influence of the effective ears of the rice, and can be applied to the improvement of the nitrogen utilization efficiency of the plant rice, the improvement of the plant type of the rice and the improvement of the yield.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nitrogen transport gene OsNPF2.4b for promoting the improvement of the tillering number and the effective spike number of rice and application thereof.
In the first aspect of the invention, a nitrogen transport gene OsNPF2.4b for promoting the improvement of tillering number and effective spike number of rice is provided, and the amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 1.
It is understood that amino acid sequences with equivalent functions can be obtained by various substitutions, additions and/or deletions of one or more amino acids of the amino acid sequence shown in SEQ ID NO. 1by those skilled in the art without affecting the activity of the OsNPF2.4b protein (i.e., without being in the active center of the protein).
Therefore, the protein coded by the rice OsNPF2.4b gene also comprises the protein coded by the rice OsNPF2.4b gene with the same activity, which is obtained by replacing, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1.
Further, the invention provides a nucleotide sequence SEQ ID NO.2 of the cDNA of the gene OsNPF2.4b.
It is understood that, considering the degeneracy of codons and the preference of codons for different species, one skilled in the art can obtain the nucleotide sequence of cDNA of the gene OsNPF2.4b using codons suitable for the expression of a particular species, as necessary.
Further, the present invention provides a cloning vector or an expression vector comprising a sense sequence or an antisense sequence of the above cDNA polynucleotide or a fragment thereof, a host cell containing the vector, a transformed plant cell and a transgenic plant containing the nucleotide sequence or a fragment thereof.
In the second aspect of the invention, the application of the gene OsNPF2.4b in rice breeding is provided, and the rice breeding aims to improve the tillering number and the effective spike number of rice, thereby improving the yield.
The third aspect of the invention provides a detection kit for nitrogen transport gene OsNPF2.4b transgenic plants for promoting the increase of the effective spike number of rice, which detects the expression level of the OsNPF2.4b gene by amplifying the primer pair by using the cDNA of the transgenic rice to be detected as a template through real-time fluorescent quantitative PCR, wherein the primer pair is as follows:
F2：5'-GTTTGGGTGTGCAGGGAACG-3'(SEQ ID NO.5)，
R2：5'-TAGCGGCCGAGGTAGGCGTC-3'(SEQ ID NO.6)。
if the expression level of the OsNPF2.4b gene of the transgenic plant is obviously increased compared with that of the flower 11 in the control, the transgenic plant is indicated as a transgenic positive plant.
The technology for realizing the invention is as follows:
the invention takes the rice nitrogen transport gene OsNPF2.4b as an object, and clones the cDNA sequence of OsNPF2.4b from rice middle flower 11. The overexpression vector is introduced into the normal japonica rice variety middle flower 11 by constructing the overexpression vector of the OsNPF2.4b and adopting an agrobacterium EHA105 mediated genetic transformation method, so that an overexpression plant of the OsNPF2.4b gene is obtained, and the tillering number and the effective spike number of the overexpression plant are remarkably improved compared with those of a control wild type middle flower 11 in a rice field. Meanwhile, an interference vector of the OsNPF2.4b gene is constructed, the interference vector is introduced into the middle flower 11 to obtain an interference plant of the OsNPF2.4b gene, the interference plant is planted in a paddy field, and the tiller number and the effective spike number of the interference plant are obviously reduced compared with the middle flower 11. These results indicate that by improving the OsNPF2.4b gene expression, the rice growth can be promoted, and the tiller number and the effective spike number can be increased; the OsNPF2.4b gene has application value in the aspect of improving the rice yield, and can be applied to the improvement of rice varieties through molecular breeding.
The invention has the advantages and effects that:
(1) after the expression of the cloned OsNPF2.4b gene is improved, the tillering number and the effective ears of rice can be increased under low nitrogen, which shows that the OsNPF2.4b gene has obvious effect on improving the biomass and the yield of the rice, therefore, the plant biomass and the yield can be improved by improving the expression of the OsNPF2.4b gene through a gene engineering technology. This not only contributes to the cultivation of high-yielding rice by reducing the use of nitrogen fertilizers, but also contributes to the variety improvement of plants by molecular breeding.
(2) The successful cloning of the OsNPF2.4b gene further proves the important function of different splicing forms of the NPF family gene in the nitrogen transportation process, has important significance for explaining the biological function of the NPF transport family, and has great promotion effect on further understanding the plant nitrogen metabolic pathway and improving the nitrogen absorption efficiency.
(3) Although several genes affecting plant growth in the nitrogen nutrient pathway have been cloned, the molecular mechanisms underlying plant growth and development need to be further studied. The OsNPF2.4b gene cloned by the invention can improve the yield of rice and has great promotion effect on determining key factors of plant yield increase.
Drawings
FIG. 1 is a table diagram of the entire plant of flower 11, 2 lines of OsNPF2.4b gene overexpression plants and 2 lines of OsNPF2.4b gene interference plants in the control under field planting;
FIG. 2 is the analysis of relative gene expression levels in control flower 11, OsNPF2.4b gene over-expressed plant 2 lines and OsNPF2.4b gene interfering plant 2 lines, the data were analyzed by SPSS software for variance analysis (ANOVA), and Duncan's were used to analyze the significance of the differences at 0.05 level, all the values of the material were compared with the control, and the difference was expressed as;
FIG. 3 is a statistical analysis of the number of effective ears in control flowers 11, 2 lines of OsNPF2.4b gene overexpressing plants and 2 lines of OsNPF2.4b gene interfering plants, the data were analyzed by variance analysis (ANOVA) using SPSS software, and the significance of differences was analyzed at 0.05 level using Duncan's, all material values were compared to control and are expressed as significant differences.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to recombinant techniques already described (see molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.); the materials, reagents and the like used are all commercially available.
Example 1 construction of OsNPF2.4b Gene overexpression plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F1：5'-GGTACCATGGAGGTGTGGGGGGTTTGGGTG-3'(Kpn I)，(SEQ ID NO.3)
R1：5'-TCTAGACTTGCATCTCAGTTGGCGGCT-3' (Xba I); (SEQ ID NO.4) the cDNA of the OsNPF2.4b gene is amplified by PCR, and then is cut by Kpn I and Xba I and then is connected into a pCAMBIA-1306 vector to construct the overexpression vector OsNPF2.4b-p1306 of the OsNPF2.4b gene. Adopting agrobacterium EHA105 mediumThe introduced genetic transformation method is to introduce the overexpression vector into the flower 11 of the normal rice variety.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, soaking 50 rice transgenic seedlings into a hygromycin solution with the concentration of 50mg/L prepared by 500mL of distilled water for 48 hours when the seedlings are about 10cm long, and then taking plants with green leaves and good growth states as positive transgenic plants; while plants with withered and yellow leaves and curly leaves were negative plants and died immediately. And planting and harvesting a single positive plant until a homozygous transgenic plant without any withered and yellow leaf and curling in the hygromycin solution is identified by the T2 generation, and obtaining the OsNPF2.4b gene overexpression plant. Soaking the over-expression plant and the seeds of the middle flower 11 contrast on a culture dish by using distilled water for 3 days, culturing for 7 days, transferring into a rice nutrient solution for culturing for 15 days, and transferring into a field for planting after the formula of the nutrient solution is referred to the formula of the international rice institute. As can be seen from figure 1, under field planting, the biomass of the OsNPF2.4b gene overexpression plant is improved compared with that of a control medium flower 11 plant, which is specifically represented by the increase of tillering number and effective spike and achieves obvious difference.
The leaf of the OsNPF2.4b gene overexpression plant is taken, RNA is extracted and is reversely transcribed into cDNA, the expression level of the OsNPF2.4b gene is detected by real-time fluorescent quantitative PCR, and the result shows that (figure 2) the expression level of the OsNPF2.4b gene of the overexpression plant is obviously increased compared with that of the control medium flower 11. The primer pair used for the real-time fluorescent quantitative PCR is as follows:
F2：5'-GTTTGGGTGTGCAGGGAACG-3'(SEQ ID NO.5)，
R2：5'-TAGCGGCCGAGGTAGGCGTC-3'(SEQ ID NO.6)。
the number of effective spikes of the OsNPF2.4b overexpression plants and the control are counted in the reproductive period, and the number of the effective spikes of the overexpression plants is found to be remarkably higher than that of the control of the medium flower 11, as shown in figure 3.
Example 2 construction of OsNPF2.4b Gene-interfering plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F3：5'-GGTACCGTTTGGGTGTGCAGGGAACG-3'(Kpn I)(SEQ ID NO.7)，
R3：5'-GGATCCACATGAACAGCACCGCGAACTG-3'(BamH I)(SEQ ID NO.8)，
F4：5'-ACTAGTGTTTGGGTGTGCAGGGAACG-3'(Spe I)(SEQ ID NO.9)，
R4：5'-GAGCTCACATGAACAGCACCGCGAACTG-3'(Sac I)(SEQ ID NO.10)；
the cDNA fragments of the OsNPF2.4b gene are amplified by PCR respectively, and are cut by the corresponding restriction enzyme and then are connected into a pTCK303 vector to construct an interference expression vector OsNPF2.4b-pTCK303 of the OsNPF2.4b gene. The interference expression vector is introduced into the normal japonica rice variety middle flower 11 by adopting an agrobacterium EHA105 mediated genetic transformation method.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, soaking 50 rice transgenic seedlings into a hygromycin solution with the concentration of 50mg/L prepared by 500mL of distilled water for 48 hours when the seedlings are about 10cm long, and then taking plants with green leaves and good growth states as positive transgenic plants; while plants with withered and yellow leaves and curly leaves were negative plants and died immediately. And planting and harvesting a single positive plant until a homozygous transgenic plant without any withered and yellow leaf and curling in the hygromycin solution is identified by the T2 generation, and obtaining an OsNPF2.4b gene interference plant. And soaking the interfering plant and the control seeds in distilled water for 3 days on a culture dish, culturing for 7 days, transferring to a rice nutrient solution for culturing and culturing for 15 days, wherein the formula of the nutrient solution refers to the formula of the international rice institute. Then the seeds are transferred to a field for planting. As can be seen in FIG. 1, the OsNPF2.4b gene interference plants have reduced biomass and effective panicle compared to the control, flower 11 plants.
The leaf of the OsNPF2.4b gene interference plant is taken, RNA is extracted and is reversely transcribed into cDNA, the expression level of the OsNPF2.4b gene is detected by real-time fluorescent quantitative PCR, and the result shows that (figure 2) the expression level of the OsNPF2.4b gene of the interference plant is obviously reduced compared with that of the flower 11 in the contrast. The primer pair used for the real-time fluorescent quantitative PCR is as follows:
F2：5'-GTTTGGGTGTGCAGGGAACG-3'，
R2：5'-TAGCGGCCGAGGTAGGCGTC-3'。
the number of effective ears of the OsNPF2.4b interfering plants and the control are counted in the reproductive period, and the number of the effective ears of the interfering plants is found to be obviously lower than that of the middle flowering 11 control, as shown in figure 3.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
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<212>DNA
<213> Artificial sequence
<400>4
tctagacttg catctcagtt ggcggct 27
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<400>5
<210>6
<211>20
<212>DNA
<213> Artificial sequence
<400>6
<210>7
<211>26
<212>DNA
<213> Artificial sequence
<400>7
ggtaccgttt gggtgtgcag ggaacg 26
<210>8
<211>28
<212>DNA
<213> Artificial sequence
<400>8
ggatccacat gaacagcacc gcgaactg 28
<210>9
<211>26
<212>DNA
<213> Artificial sequence
<400>9
actagtgttt gggtgtgcag ggaacg 26
<210>10
<211>28
<212>DNA
<213> Artificial sequence
<400>10
gagctcacat gaacagcacc gcgaactg 28
Claims (1)
1. Of riceOsNPF2.4bApplication of gene in rice breedingOsNPF2.4bThe amino acid sequence of the gene coding protein is shown as SEQ ID NO.1, and is characterized in that the protein is obtained by increasingOsNPF2.4bThe expression of the gene realizes the application, and the rice breeding aims to improve the tillering number and the effective spike number of the rice, thereby improving the yield.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710241339.4A CN106868022B (en) | 2017-04-13 | 2017-04-13 | Nitrogen transport gene OsNPF2.4b for promoting increase of effective spike number of rice and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710241339.4A CN106868022B (en) | 2017-04-13 | 2017-04-13 | Nitrogen transport gene OsNPF2.4b for promoting increase of effective spike number of rice and application thereof |
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| CN106868022A CN106868022A (en) | 2017-06-20 |
| CN106868022B true CN106868022B (en) | 2020-05-29 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982828B (en) * | 2020-01-02 | 2022-08-30 | 南京农业大学 | Nitrate transport protein gene specifically induced by rice arbuscular mycorrhiza and application thereof |
| CN111808867B (en) * | 2020-07-27 | 2022-07-19 | 南京农业大学 | Application of OsNRT2.3b in reduction of emission of methane and nitrous oxide |
| CN111793633B (en) * | 2020-07-27 | 2022-08-02 | 南京农业大学 | Application of OsNRT2.3b in improving yield and rice quality |
Citations (3)
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|---|---|---|---|---|
| CN101421295A (en) * | 2006-02-09 | 2009-04-29 | 先锋高级育种国际公司 | Genes for enhancing nitrogen utilization efficiency in crop plants |
| CN106119262A (en) * | 2016-07-28 | 2016-11-16 | 武汉生物工程学院 | Improve Oryza sativa L. nitrogen use efficiency and the gene OsPTR10 of yield and purposes |
| CN106222180A (en) * | 2016-07-28 | 2016-12-14 | 武汉生物工程学院 | Improve rice yield and the gene OsNPF7.3 of grain of rice protein content and purposes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005185101A (en) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | VEGETABLE FULL-LENGTH cDNA AND UTILIZATION THEREOF |
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2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101421295A (en) * | 2006-02-09 | 2009-04-29 | 先锋高级育种国际公司 | Genes for enhancing nitrogen utilization efficiency in crop plants |
| CN106119262A (en) * | 2016-07-28 | 2016-11-16 | 武汉生物工程学院 | Improve Oryza sativa L. nitrogen use efficiency and the gene OsPTR10 of yield and purposes |
| CN106222180A (en) * | 2016-07-28 | 2016-12-14 | 武汉生物工程学院 | Improve rice yield and the gene OsNPF7.3 of grain of rice protein content and purposes |
Non-Patent Citations (5)
| Title |
|---|
| A putative 6-transmembrane nitrate transporter OsNRT1.1b plays a key role in rice under low nitrogen;Xiaorong Fan et al;《J Integr Plant Biol》;20150918;第58卷(第6期);第590-599页 * |
| Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields;Xiaorong Fan et al;《Proc Natl Acad Sci U S A》;20160606;第113卷(第16期);第7118-7123页 * |
| 植物功能基因选择性剪接研究进展;周新成 等;《热带农业科学》;20120215;第32卷(第2期);第36-41页 * |
| 水稻低亲和硝酸盐转运蛋白基因OsNPF2.4生物学功能分析;夏秀东;《中国博士学位论文全文数据库农业科技辑》;20160815(第08期);第17页-89页 第三章-全文结论部分 * |
| 水稻高亲和硝酸盐转运蛋白基因OsNRT2.3a/b生物学功能分析;唐仲;《中国博士学位论文全文数据库农业科技辑》;20131215(第12期);D047-10 * |
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| CN106868022A (en) | 2017-06-20 |
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