CN116143892A - Application of OsGN11 gene in improving rice grain number per ear character - Google Patents

Application of OsGN11 gene in improving rice grain number per ear character Download PDF

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CN116143892A
CN116143892A CN202310328272.3A CN202310328272A CN116143892A CN 116143892 A CN116143892 A CN 116143892A CN 202310328272 A CN202310328272 A CN 202310328272A CN 116143892 A CN116143892 A CN 116143892A
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徐铨
李丰成
崔鑫
陈昊
于之雯
王潇澈
马丽
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Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of an OsGN11 gene in improving the grain number per ear character of rice. The invention adopts the over-expression technology to the rice OsGN11 gene, so that the gene expression level is obviously increased, and the over-expression rice of the OsGN11 gene is constructed. The research of the invention shows that the overexpression of the OsGN11 gene can obviously increase the grain number per ear, thereby obviously increasing the rice yield. The result provides a brand new and simple and effective method for cultivating the rice with excellent properties, and can be put into large-scale popularization and use.

Description

Application of OsGN11 gene in improving rice grain number per ear character
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of an OsGN11 gene in improving the grain number per ear character of rice.
Background
Rice is an important food crop that provides staple food to more than 50% of the population worldwide. The grain number per ear is one of three factors of yield, and directly affects the yield of rice. The number of grains per ear of rice is mainly determined by the number of primary and secondary branches. The regulation of the number of the rice branches is significant in agricultural production and developmental biology. The number and the positions of the primary branches are established in the process of the transformation of the rice plant from vegetative growth to reproductive growth, so that the excavation of new genes and dominant alleles affecting the primary branches and the secondary branches of the rice is beneficial to expanding and controlling the gene network of the grain number per ear, and the cognition of the NPF family genes of the rice is enriched and deepened.
The plant NPF (NRT 1/PTR FAMILY) family is homologous to the animal peptide transporter SLC15/PepT/PTR/POT family, with a total of 11 members in rice, where the function of most NPF family genes and their methods of use in breeding remain unknown.
Disclosure of Invention
Based on the technical problems, the invention provides the following technical scheme:
the invention provides application of an OsGN11 gene in improving the grain number per ear character of rice, and the nucleotide sequence of the OsGN11 gene is shown as SEQ ID NO. 1.
Furthermore, the OsGN11 gene positively regulates the grain number per ear of rice.
Furthermore, the number of grains per ear of rice is increased by over-expressing the OsGN11 gene.
Further, the over-expression of the OsGN11 gene is achieved by transferring the OsGN11 gene or related biological materials into rice or exogenously applying substances capable of improving the expression of the OsGN11 gene.
Still further, the OsGN11 gene-related biological material includes an expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line that contains the OsGN11 gene.
Based on the same inventive concept, the invention also provides application of the OsGN11 gene or the expression product thereof in directional selection or identification of the grain number per ear character of rice.
Further, the application includes: and (3) identifying the level of the OsGN11 gene or the expression product thereof in the test rice, and if the level of the OsGN11 gene or the expression product thereof in the test rice is obviously higher than the average value of the rice, obtaining the rice with excellent grain number per spike.
Further, the method for identifying and testing the level of the OsGN11 gene in rice comprises fluorescent quantitative PCR, and the primers used are shown as SEQ ID NO. 5-6.
Furthermore, the OsGN11 gene expression product is an OsGN11 protein, and the amino acid sequence is shown as SEQ ID NO. 2.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an application of an OsGN11 gene in improving the grain number per ear character of rice. The over-expression technology is adopted to increase the expression level of the OsGN11 gene, so that the over-expression rice of the OsGN11 gene is constructed. Researches show that the over-expression of the OsGN11 gene can obviously increase the grain number per spike and further obviously increase the rice yield, and the result provides a brand new, simple and effective method for cultivating the rice with excellent properties, and can be put into large-scale popularization and use.
Drawings
FIG. 1 shows the OsGN11 gene expression levels of wild-type and OsGN11 gene overexpressing plants.
FIG. 2 shows the spike type (A) and grain number per spike comparison (B) of wild type and OsGN11 gene over-expressed plants.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The invention adopts the over-expression technology to increase the expression level of the OsGN11 gene, and obtains a germplasm with increased grain number per ear. The nucleotide sequence of the OsGN11 gene is shown in a sequence table SEQ ID NO.1, and the encoded amino acid sequence is shown in a sequence table SEQ ID NO. 2. The high-yield germplasm prepared by the invention has increased grain number per ear, can obviously increase the yield of rice when being applied to rice breeding, and has good application prospect.
Example 1
Construction of Rice over-expressing OsGN11 Gene
1. The complete CDS sequence of the rice OsGN11 gene is obtained from the TIGR retrieval of a rice genome annotation database, and is shown as SEQ ID NO.1, and is delivered to Jiangsu Bo biotechnology Co., ltd for gene synthesis;
2. enzyme digestion reaction: the pCAMBIA1300S vector is linearized by double enzyme digestion and then the vector fragment is obtained by gel recovery. The enzyme digestion reaction system is as follows:
TABLE 1 cleavage reaction System
Figure BDA0004154001280000031
3. Recombination reaction: the target DNA fragment and vector were reacted at 37℃for 1 hour using 2 XHiFi Clone PG-1 recombinase to transform Top10 competent cells and plating. The reaction system is as follows:
TABLE 2 recombination reaction System
Figure BDA0004154001280000041
4. Gene editing vector transformation agrobacterium: transforming the correctly constructed gene editing vector into agrobacterium (EHA 105) competent, extracting the transformed agrobacterium plasmid, transforming the agrobacterium plasmid into escherichia coli (DH 5 alpha) competent again, extracting the escherichia coli plasmid for sequencing verification, and determining that the constructed gene editing vector is correctly transferred into agrobacterium.
5. Tissue culture
1) Callus induction: selecting full mature Sasanishiki rice seeds without mildew spots, removing hulls, washing with sterile water for 3 times, treating with 70% ethanol for 1min, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 12min each time for 30s, washing with sterile water for 7 times, each time for 30s, placing the seeds on sterilized filter paper to absorb water, and then clamping onto an induction culture medium with sterile forceps at 28 ℃ for more than 4 weeks.
2) Subculture of callus: embryogenic callus with bright, compact and dry color was selected and placed on a secondary medium for dark culture for 10 days at 28 ℃.
3) Infection with agrobacterium: the constructed agrobacterium is streaked and cultured for one time, and then precultured for 2 days. Transferring into a centrifuge tube containing suspension medium, vortex shaking to re-suspend Agrobacterium, and adjusting suspension of Agrobacterium to OD 600 0.9; selecting embryo callus which is bright, compact and dry in color, mixing with agrobacterium suspension, and soaking for 30 minutes; transferring the calluses to sterilized filter paper, and drying on an ultra-clean workbench; the calli were placed on co-culture medium with a layer of filter paper laid down and incubated at 28℃for 3 days.
4) Selection and culture: transferring the co-cultured calli into a sterilized triangular flask, and fully washing the calli with sterilized distilled water; soaking the callus in sterilized water containing 400mg/L carbenicillin for 30min, and shaking from time to time; transferring the callus to sterilized filter paper to absorb water, and airing in an ultra-clean workbench; the calli were transferred to selection medium containing 50mg/L hygromycin for 2 weeks (first selection concentration of carbenicillin 400mg/L and second selection concentration of 250 mg/L).
5) And (3) differentiation culture: the resistant calli were transferred to differentiation medium and incubated under light at 28 ℃.
6) Rooting culture: the roots produced during differentiation were cut off and then transferred to rooting medium, which was light-cultured for more than 2 weeks at 28 ℃.
7) When the seedling is about 10cm in height, the root system is better, the bottle cap is opened, sterile water is injected into the bottle, after seedling hardening is carried out for 2 days, residual culture medium on the root is washed off, and the seedling is transplanted. In the first few days, a preservative film is covered, strong light irradiation is avoided, and moisture is kept moist.
Example 2: identification of OsGN11 overexpressing plants
1. Identification of Positive transgenic lines
The rice seedlings obtained in example 1 are subjected to hygromycin marking PCR identification by extracting leaf DNA, and a transgenic positive strain is obtained, wherein PCR amplification primers are as follows:
OsGN11-F(SEQ ID NO.3)：5′-TTCGAGCTCGGTACCCGGGGATCCATGGATGTTGAGTCAAGGCAA-3′；
OsGN11-R(SEQ ID NO.4)：5′-CTTGCATGCCTGCAGGTCGACTCAGTGACCCATGTTGACCT-3′。
the PCR amplification system comprises:
1 μl of template DNA; 2×taq buffer 10 μl; dNTP 0.5. Mu.l; forward primer 0.5 μl; reverse primer 0.5. Mu.l; 0.5 μl of taq enzyme; ddH 2 O 7μl。
The PCR amplification procedure was:
pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min,31 cycles; extending at 72℃for 5min.
2. Identification of OsGN11 overexpressing lines
Taking the transgenic positive strain as a material, extracting RNA from sword-leaved leaves in the heading stage, carrying out reverse transcription to obtain cDNA, and then carrying out expression quantity detection, wherein the sequence of an RT-PCR detection primer is as follows:
forward primer (SEQ ID No. 5): 5'-CGGTAACCAAGAGGAAACAAGTG-3';
reverse primer (SEQ ID NO. 6): 5'-CACCACGCACAGTAGCACCTT-3'.
Results: the amount of OsGN11 expression was significantly higher for OsGN11 overexpressing lines than for wild-type, with two representative overexpressing lines OX-1 and OX-2 having OsGN11 expression levels as shown in fig. 1.
The wild type and the over-expression strain are subjected to investigation on the grain number per ear and the grain number per ear in the mature period, as shown in fig. 2, the grain number per ear of the over-expression strain is obviously increased, and the result provides a brand new, simple and effective method for cultivating high-yield rice.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

  1. The application of the OsGN11 gene in improving the grain number per ear character of rice is characterized in that the nucleotide sequence of the OsGN11 gene is shown as SEQ ID NO. 1.
  2. 2. The use according to claim 1, wherein the OsGN11 gene regulates positively the number of grains per ear of rice.
  3. 3. The use according to claim 2, wherein the number of grains per ear of rice is increased by overexpressing OsGN11 gene.
  4. 4. The use according to claim 3, wherein the overexpression of the OsGN11 gene is achieved by transferring the OsGN11 gene or a biological material related thereto into rice, or exogenously applying a substance capable of enhancing expression of the OsGN11 gene.
  5. 5. The use according to claim 3, wherein said OsGN11 gene related biological material comprises an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising said OsGN11 gene.
  6. 6. The use according to claim 1, wherein the OsGN11 gene or expression product thereof is used for the targeted selection or identification of rice grain-per-ear traits.
  7. 7. The application according to claim 6, characterized in that it comprises: and (3) identifying the level of the OsGN11 gene or the expression product thereof in the test rice, and if the level of the OsGN11 gene or the expression product thereof in the test rice is obviously higher than the average value of the rice, obtaining the rice with excellent grain number per spike.
  8. 8. The use according to claim 7, wherein the method for identifying the level of OsGN11 gene in rice comprises fluorescent quantitative PCR using primers as shown in SEQ ID No. 5-6.
  9. 9. The use according to claim 6, wherein the expression product of the OsGN11 gene is OsGN11 protein, and the amino acid sequence is shown in SEQ ID No. 2.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN106967730A (en) * 2017-05-19 2017-07-21 武汉生物工程学院 Application of the OsNPF6.3 genes in rice tillering number is improved
CN107779468A (en) * 2016-08-17 2018-03-09 中国科学院遗传与发育生物学研究所 The application of rice NRT1.1A genes and its encoding proteins in plant products breeding is improved
CN107936103A (en) * 2017-11-27 2018-04-20 武汉生物工程学院 Application of the OsNPF7.11b genes in rice yield is improved
CN108070601A (en) * 2017-12-19 2018-05-25 武汉生物工程学院 Application of the OsNPF8.6b genes in rice yield is improved

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN107779468A (en) * 2016-08-17 2018-03-09 中国科学院遗传与发育生物学研究所 The application of rice NRT1.1A genes and its encoding proteins in plant products breeding is improved
CN106967730A (en) * 2017-05-19 2017-07-21 武汉生物工程学院 Application of the OsNPF6.3 genes in rice tillering number is improved
CN107936103A (en) * 2017-11-27 2018-04-20 武汉生物工程学院 Application of the OsNPF7.11b genes in rice yield is improved
CN108070601A (en) * 2017-12-19 2018-05-25 武汉生物工程学院 Application of the OsNPF8.6b genes in rice yield is improved

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Title
XM_015760038.2: "PREDICTED: Oryza sativa Japonica Group protein NRT1/ PTR FAMILY 4.4 (LOC4350145), mRNA", NCBI, pages 263 - 2143 *

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