CN106967745B - Application of nitrogen nutrition transport gene OsNPF7.1 in improving tillering and spike number of rice - Google Patents
Application of nitrogen nutrition transport gene OsNPF7.1 in improving tillering and spike number of rice Download PDFInfo
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Abstract
The invention discloses a nitrogen nutrition transport geneOsNPF7.1Application in improving tillering and spike number of rice, belonging to the field of plant genetic engineering.OsNPF7.1The amino acid sequence of the gene coding protein is shown as SEQ ID NO.1, and the cDNA sequence is shown as SEQ ID NO. 2. The invention constructs riceOsNPF7.1Gene over-expression plant,OsNPF7.1Gene interfering plants, found by increasingOsNPF7.1The gene expression can increase the tillering number and the spike number of normal rice, therebyOsNPF7.1The gene can be used in rice breeding to improve the rice yield.OsNPF7.1The gene has important application value in the aspects of explaining the influence of nitrogen on the growth and development process of plants and the improvement of rice plant types.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a nitrogen nutrition transport geneOsNPF7.1Application in improving tillering and spike number of rice.
Background
Nitrogen nutrition is closely related to rice tillering development because of the need to provide more nutrients in the early stage of tillering bud growth. It has been reported that when the nitrogen content of the stem sheath is less than 1.3% and the nitrogen content of the leaf blade is less than 2%, the differentiation and development of tillering buds are stopped, and when the nitrogen content of the stem sheath is 2.7% -3.3% and the nitrogen content of the leaf blade is 5%, tillering of rice can normally occur (Jianpenyan, Hongxiang, Von. et al. influence of nitrogen concentration on the absorption of nitrogen and tillering of rice under hydroponic conditions. report on crops, 1997, 23: 191-. Rice glutamineThe synthetase GS1 is capable of assimilating ammonium to glutamine (Sukanya R, LiM, Snastd P. Root-and shot-specific responses of inorganic glutamine synthesis genes of mail to nitrate and ammonium Plant Molecular Biology,1994, 26(6): 1935-OsGS1;2Plays a major role in rice ammonium assimilation (Funayama K, Kojima S, Tabuchi-Kobayashi M, et al. cytologic glutamine synthesis 1;2 isocyanate for the primary analysis of ammonium in rice roots, Plant and cell Physiology, 2013: pct 046.). At the same time, the riceOsGS1;2Is very important for the extension of lateral buds of tillers,OsGS1;2the deletion(s) results in a severe reduction in rice tillering shoots and in lignin accumulation (Ohashi M, Ishiyama K, Kusano M, et al, rock of cytolytic glutamine synthesis 1;2 in vacuum of aqueous soil customers reduction in The outer growth and division of The metabolic substrate in rice seed Journal 2015, 81: 347) in The same manner.
Members of the NPF family of nitrogen transport play an important role in growth and development (Rentsch D, Schmidt S, TegederM. Transporters for uptake and allocation of organic nitrile compounds. Febs Letters, 2007, 581: 2281-. Members of the NPF family of riceSP1Plays an important role in controlling The growth and seed set of rice (Li S, Qian Q, Fu Z, et al. Short panicle 1 encodes adaptive PTR family transporter and standards rice size. The plant journal, 2009, 58(4): 592) 605.). OsNPF2.2 mediates the unloading of xylem nitrate, affecting rice growth and seed filling and setting (Li Y, Ouyang J, Wang Y, et al, precipitation of the rice transporter OsNPF2.2 roots-to-shoot nitrate and regulator concentrations. Scientific reports 2015, 5: 9635.).OsNPF7.2Has low affinity transport for nitrate and can affect plant growth (Hu R, Qiu D, Chen Y, et al, Knock-down of aeroplast localized low-affinity nitrate transportOsNPF7.2Affects, procegrowth under high nitrate supply, Frontiers in plant science, 2016, 7.). Overexpression of high-affinity nitrate transport genesOsNRT2.3bCan promote the growth of rice and improve the utilization efficiency of nitrogenRate, increase in yield (Fan X, Tang Z, Tan Y, et al. Overexpression of a pH-sensitive nitrate transporter in production channels, Proceedings of the National Academy of Sciences,2016, 113(26): 7118 + 7123.).
More than 80 members of the NPF family of rice are excavated to obtain the nitrogen efficient transport gene, particularly the key gene for controlling the plant type of the rice, which is beneficial to the cultivation of high-yield rice varieties. Although it is known that nitrogen fertilizer can affect plant growth, how nitrogen fertilizer affects plant growth is not known at present. In particular, how nitrogen is transported by some key genes in the NPF family and how nitrogen causes plant growth and development changes is not reported at present. The invention discovers that the NPF family has the characteristics of high purity and high purityOsNPF7.1The gene has important control effect on rice tillering, effective spike and the like, and can be applied to plant type improvement so as to increase the yield of rice.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provide a rice NPF gene family memberOsNPF7.1The application of the gene in improving tillering and spike number of rice.
The purpose of the invention is realized by the following technical scheme:
the invention uses the NPF gene family members of riceOsNPF7.1The gene is used as an object and is cloned from rice flower 11OsNPF7.1The cDNA sequence of (1). By constructionOsNPF7.1The gene over-expression vector adopts agrobacteriumEHA105The mediated genetic transformation method is to introduce the over-expression vector into the normal japonica rice variety medium flower 11 to obtainOsNPF7.1Compared with the control wild type middle flower 11, the tillering number and the spike number of the gene over-expression plant are obviously improved. Construction by RNAi techniqueOsNPF7.1Gene interference expression vector, introducing the interference expression vector into middle flower 11 to obtainOsNPF7.1The tillering number and the spike number of the interference plant with the reduced gene expression amount are obviously reduced compared with those of the middle flower 11. These results show that by increasingOsNPF7.1The expression of the gene can increase the tillering number and the spike number of normal rice, thereby improving the rice yield.
Based on the discovery of the present inventionOsNPF7.1The function of the gene(s) is,OsNPF7.1the gene can be used for rice breeding. The rice breeding is to improve the tillering number and the spike number of the rice, thereby improving the rice yield. In particular by increasingOsNPF7.1The expression of the gene increases the tillering number and the ear number of each plant of the rice, thereby achieving the purpose of improving the yield of the rice.
OsNPF7.1The genes may also be used to increase yield in other plants, e.g.by transgenesisOsNPF7.1The gene is (over) expressed in the plant to increase the number of branches of the plant, thereby increasing the yield of the plant. The plant is monocotyledon or dicotyledon; such as: wheat, tomato, turf grass or alfalfa and the like.
SaidOsNPF7.1The amino acid sequence of the gene-coded OsNPF7.1 protein is shown in SEQ ID NO. 1; saidOsNPF7.1The cDNA sequence of the gene is preferably shown in SEQ ID NO. 2.
It is understood that the amino acid sequence shown in SEQ ID NO.1 can be variously substituted, added and/or deleted by one or several amino acids by those skilled in the art to obtain an amino acid sequence having equivalent functions without affecting the activity of the OsNPF7.1 protein (i.e., without being in the active center of the protein). Therefore, the OsNPF7.1 protein also comprises a protein with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1. Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.
The invention has the advantages and effects that:
(1) cloned according to the inventionOsNPF7.1The tillering capacity of rice is enhanced after the gene is over-expressed, which shows thatOsNPF7.1The gene has obvious influence on the improvement of the rice yield, so the improvement is realized by the gene engineering technologyOsNPF7.1Expression of the gene can improve plant yield. This not only facilitates the cultivation of high-yielding rice under normal nitrogen application conditions, but also allows the variety improvement of plants through molecular breeding.
（2）OsNPF7.1The successful cloning of the gene further proves the important function of the NPF family in the nitrogen absorption process, has important significance for clarifying the biological function of the NPF family, and has great promotion effect on further understanding the plant nitrogen metabolic pathway and improving the nitrogen absorption efficiency.
(3) Although some genes have been cloned to improve plant yield, the molecular mechanisms for plant yield increase are still unclear. And cloned according to the inventionOsNPF7.1The gene can improve the yield of rice and has great promotion effect on determining the key factor of plant yield increase.
Drawings
FIG. 1 shows a control flower 11,OsNPF7.12 strains of gene over-expression plants andOsNPF7.1the phenotype of the whole plant of 2 lines of the gene interference plant.
FIG. 2 shows the flower 11 in the control,OsNPF7.12 strains of gene over-expression plants andOsNPF7.1statistical histogram of tillering number of 2 lines of gene-interfered plants, data were analyzed for variables (ANOVA) using SPSS software, significance of differences was analyzed at 0.05 level using Duncan's, and asterisks (#) of different groups indicated significant differences from the control.
FIG. 3 shows the flower 11 in the control,OsNPF7.1The effective ear phenotype of single plant of 2 lines of gene over-expression plant.
FIG. 4 shows the flower 11 in the control,OsNPF7.12 strains of gene over-expression plants andOsNPF7.1statistical histogram of effective panicles of 2 lines of gene-interfered plants, data were analyzed by variable analysis (ANOVA) using SPSS software, significance of differences was analyzed at 0.05 level using Duncan's, and asterisks (asterisks) of different groups indicated significant differences from the control.
FIG. 5 shows the flower 11 in the control,OsNPF7.12 strains of gene over-expression plants andOsNPF7.12 strains of gene interference plantsOsNPF7.1Statistical histograms of relative gene expression were obtained by variable analysis (ANOVA) using SPSS software and significance analysis of differences at 0.05 level using Duncan's, with different group asterisks indicating significant differences from the control.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. 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 1OsNPF7.1Construction of Gene-overexpressing plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F1：5'-GGTACCATGGCGTTTCTTTTTTTTTTTTGTGGCACAGTTCCT-3'（kpnI），
R1：5'-GGATCCGACTTGGATGACAGCCTTCTTCAA-3'（BamHI）；
amplification by PCROsNPF7.1After cDNA of the gene, bykpnI、BamHI was digested and ligated into pCAMBIA-1306 vector (pCAMBIA-1306 vector from Cambia Co.) to constructOsNPF7.1Overexpression vector of geneOsNPF7.1-p 1306. By using AgrobacteriumEHA105The mediated 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, planting the seedlings in a field when the seedlings grow to be about 10cm in height, extracting genome DNA (deoxyribonucleic acid) and detecting transgenic plants through PCR (polymerase chain reaction), wherein a detection primer pair is as follows:
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
if 517bp fragments are amplified, the transgenic plants are positive plants. The positive plants are harvested and planted individually until homozygous transgenic plants are identified at T2 generation, and the transgenic plants are obtainedOsNPF7.1And (3) gene overexpression plants.OsNPF7.1The tillering number and the effective spike number of the gene over-expression plant are far more than those of the flower 11 plants in the controlStrains, the differences were significant as shown in figures 1, 2, 3, 4.
GetOsNPF7.1Extracting RNA from the leaf of the plant with gene over-expression, reverse transcribing the RNA into cDNA, and detecting the cDNA by real-time fluorescent quantitative PCROsNPF7.1The results of the expression level of the gene show (FIG. 5) that the plant is over-expressedOsNPF7.1The expression level of the gene was significantly increased compared to the control flower 11. The primer pair used for the real-time fluorescent quantitative PCR is as follows:
F3：5'-TTGTGGCACAGTTCCTCAGCAAGA-3'，
R3：5'-GTTGCGCGACTATGGGGATGCCTC-3'。
example 2OsNPF7.1Construction of Gene-interfering plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F4：5'-GGTACCCACAGTTCCTCAGCAAGACAAGCG-3'（KpnI），
R4：5'-GGATCCGGTCTCCCCTCCCCTGTGGCGGCT-3'（BamHI）；
F5：5'-ACTAGTCACAGTTCCTCAGCAAGACAAGCG-3'（SpeI），
R5：5'-GAGCTCGGTCTCCCCTCCCCTGTGGCGGCT-3'（SacI）；
respective PCR amplificationOsNPF7.1After the cDNA fragment of the gene is cut by corresponding restriction enzyme, the cDNA fragment is connected with pTCK303 vector to constructOsNPF7.1Interfering expression vector of geneOsNPF7.1-pTCK 303. By using AgrobacteriumEHA105The mediated genetic transformation method is to introduce the interference expression vector into the normal japonica rice variety flower 11.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, planting the seedlings in a field when the seedlings grow to be about 10cm in height, extracting genome DNA (deoxyribonucleic acid) and detecting transgenic plants through PCR (polymerase chain reaction), wherein a detection primer pair is as follows:
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
if 517bp fragments are amplified, the transgenic plants are positive plants. Harvesting seeds of single positive plantAnd planting until homozygous transgenic plants are identified at T2 generation, namely obtainingOsNPF7.1The gene interferes with the plant.OsNPF7.1The tillering number and the effective spike number of the gene interference plant are far less than those of the flower 11 plant in the control, and the difference is obvious, as shown in figures 1, 2 and 3.
GetOsNPF7.1Gene interference of plant leaf, extracting RNA and reverse transcription to cDNA, real-time fluorescent quantitative PCR detectionOsNPF7.1The results of the expression of the genes showed (FIG. 5) that the plants interferedOsNPF7.1The expression level of the gene was significantly reduced compared to the control flower 11. The primers used in real-time fluorescent quantitative PCR were the same as in example 1.
The above results show that by increasingOsNPF7.1The expression of the gene can increase the tillering number and the spike number of the rice, thereby improving the rice yield.
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
<110> Wuhan bioengineering college
Application of <120> nitrogen nutrition transport gene OsNPF7.1 in improving tillering and spike number of rice
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<170>PatentIn version 3.3
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Claims (3)
- The application of the OsNPF7.1 gene in improving the tillering number of rice is characterized in that: the tillering number of the rice is increased by improving the expression of the OsNPF7.1 gene; the amino acid sequence of the OsNPF7.1 protein coded by the OsNPF7.1 gene is shown as SEQ ID NO. 1.
- The application of the OsNPF7.1 gene in the improvement of the rice panicle number is characterized in that: the rice spike number is increased by improving the expression of the OsNPF7.1 gene; the amino acid sequence of the OsNPF7.1 protein coded by the OsNPF7.1 gene is shown as SEQ ID NO. 1.
- 3. Use according to claim 1 or 2, characterized in that: the cDNA sequence of the OsNPF7.1 gene is shown as SEQ ID NO. 2.
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| CN105112504A (en) * | 2015-07-23 | 2015-12-02 | 毕节市农业科学研究所 | Molecular marker identification method of paddy high nitrogen use efficiency gene |
| 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 |
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| CN105112504A (en) * | 2015-07-23 | 2015-12-02 | 毕节市农业科学研究所 | Molecular marker identification method of paddy high nitrogen use efficiency gene |
| 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 |
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| Title |
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| REDICTED: Oryza sativa Japonica Group protein NRT1/ PTR FAMILY 8.3 (LOC4343841), transcript variant X1, mRNA;NCBI;《GenBank Database》;20160301;Accession Number: XM_015792364.1 * |
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