CN116064602A - Application of rice OsASN2 gene or protein encoded by same in improving rice yield - Google Patents

Abstract

The invention discloses application of a rice OsASN2 gene or a related sequence thereof in improving rice yield, belonging to the fields of bioengineering and breeding. According to the invention, the OsASN2 is constructed on an over-expression vector and expressed in the rice ZH11, and the effective spike number, the total number of single plants and the single plant yield of the OsASN2 over-expression transgenic plant are obviously increased, so that the rice yield is improved. Therefore, the OsASN2 gene is a potential target point which can be used as a gene engineering to improve the rice yield, and has important application value.

Description

Application of rice OsASN2 gene or protein encoded by same in improving rice yield

Technical Field

The invention belongs to the technical field of bioengineering and breeding, and particularly relates to application of a rice OsASN2 gene or a protein encoded by the rice OsASN2 gene in improving rice yield.

Background

The rice is an important grain crop in the world, is also a main grain crop in China, and has important significance for ensuring grain safety by improving the yield of the rice. The rice yield is determined by the effective spike number, spike grain number, fruiting rate and thousand grain weight. Illumination, temperature, moisture, plant diseases and insect pests, etc. also affect the yield of rice.

Because of the relatively close parental relationships used in traditional breeding, it is becoming increasingly difficult to break through rice yield using traditional breeding techniques. The improvement of rice yield from the viewpoint of genetic engineering has been confirmed in experiments. Although genetic engineering techniques are also used with caution in food crops, the storage of some functional genes related to yield is important for protecting the safety of food.

Amino acid metabolism plays an important role in the growth and development of organisms. Asparagine metabolism is an important process of nitrogen metabolism and carbon metabolism in plants. Asparagine synthase catalyzes the transfer of an amino group from glutamine to aspartic acid, producing glutamic acid and asparagine. AS and its coding gene ASN have been identified in prokaryotes, plants, animals. Two structurally diverse ases exist in prokaryotes for the catalytic synthesis of asparagine, with asnA-like asparagine synthases mainly utilizing free ammonium AS an amino source. The plant AS is homologous to the asnB class AS of prokaryotes, sequence conservation, mainly using glutamine AS an amino source (Gaufichon et al, 2010). Overexpression of ASN-A from E.coli (EscherichiA coli) in plants has A positive effect on plant biomass. At present, the functions of ASN genes of plants are mainly divided into the following main categories:

(1) ASN affects the amino acid content and biomass of a vegetative organ. Overexpression of pissan 1 from peas by Brears et al in tobacco not only increases asparagine levels in leaves, but also increases tobacco plant biomass (Brears et al, 1993); both the T-DNA insertion mutant and CRISPR knockout mutant of rice osan 1 show a phenotype of reduced plant height, shortened root length, reduced tillering, etc. with stunted vegetative growth (Luo et al, 2019); atsn 2 is mainly expressed in phloem companion of leaf, and less is caused after mutation ¹⁵ N flows to the pool organ, plants grow stunted and retard senescence, and the asparagine content of leaves decreases (Gaufichon et al, 2013).

(2) ASN enhances the transfer of amino acids to the reproductive organs, thereby promoting the growth and development of the reproductive organs. The high expression levels of arabidopsis atan 1 resulted in increased free amino acids in flowers and developing pods, increased seed soluble protein and nitrogen content (Lam et al 2003;Gaufichon et al, 2017); the rice OsASN1 over-expressed plant seed has increased amino acid, protein and nitrogen content, unchanged yield compared with wild type, and reduced yield in low nitrogen environment compared with wild type (Lee et al 2020).

(3) ASN has important physiological functions in the seed development process. Gaufichon et al have found, through mutant studies, that AtASN1 plays an important role in the development of sphere center embryos to heart embryos (Gaufichon et al, 2017).

(4) ASN enhances asparagine synthesis in plants in stress and provides nutrition to new organs by accelerating nutrient transfer from old leaves. For example, atan 2 mediates reuse of leaf nitrogen under vegetative stress conditions (Maaroufi-Dguimi et al 2011); wheat seedlings TaASN1 were significantly induced by salt stress, osmotic stress and exogenous abscisic acid (Wang et al, 2005).

The AS of rice has 2 isozymes which are respectively encoded by OsASN1 and OsASN 2. OsASN1 plays an important role in root asparagine synthesis, and OsASN1 is mainly expressed in epidermis, exodermis, thick-walled tissue of roots, and is up-regulated by external ammonium induction (Ohashi et al, 2015). The expression of ostn 2 was not induced by ammonium (Ohashi et al, 2015), and ostn 2 was expressed in leaf phloem companion, in the ear of the grouted period, seed phloem parenchyma cells, dorsal vascular bundle bulbil epidermis, and bulbil processes (Nakano et al, 2000), suggesting that ostn 2 plays an important role in aerial parts. Although the effects on OsASN1 have been reported in rice, the biological functions of OsASN2 are still lacking. No report is made on the application of OsASN2 protein in improving rice yield.

Disclosure of Invention

In order to solve the related problems, the primary aim of the invention is to provide the application of the rice OsASN2 gene or the encoded protein thereof in improving the rice yield. Rice OsASN2 gene (accession number LOC_Os06g15420 in MSU database/accession number Os06g0265000 in RAP-DB database), the whole length of the gene is shown as SEQ ID NO.1, the cDNA sequence is shown as SEQ ID NO.2, the CDS sequence is shown as SEQ ID NO.3, 876 amino acids are coded altogether, and shown as SEQ ID NO. 4. According to the invention, the OsASN2 is constructed on an over-expression vector and expressed in rice ZH11, and the effective spike number, the total number of single plants and the single plant yield of the OsASN2 over-expression transgenic plant are found to be obviously increased. Therefore, the OsASN2 gene is a potential target point which can be used as a gene engineering to improve the rice yield, and has important application value. The homologous gene OsASN1 of the gene has the effect of maintaining the yield under the condition of reducing nitrogen, and the invention discovers that the OsASN2 gene has the remarkable yield-increasing effect under the condition of normally applying nitrogen for the first time.

In order to achieve the above object, the present invention adopts the following technical scheme:

application of rice OsASN2 gene or coded protein thereof in improving rice yield.

Further, the base sequence of the OsASN2 gene is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3; the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4.

Further, the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the effective spike number of rice.

Further, the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the total grain number of a single rice plant.

Further, the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the yield of a single rice plant.

Further, the application is in the cultivation of high-yield rice.

Further, the application comprises the following steps:

(1) Constructing an OsASN2 gene overexpression vector;

(2) Introducing the over-expression vector into receptor rice cells, and over-expressing the OsASN2 gene to obtain transgenic plants.

Further, the basic vector of the over-expression vector in the step (1) is a binary plant expression vector.

Further, the binary plant expression vector is pOx.

Further, the recipient rice described in step (2) includes, but is not limited to, ZH11 varieties.

Further, the introduction described in step (2) includes, but is not limited to, transgenic approaches to Agrobacterium infection.

Further, the agrobacterium-infected cells are derived from rice seed-induced callus.

Still further, the agrobacterium includes, but is not limited to, agrobacterium EHA105 strain.

A preparation for regulating rice yield contains protein encoded by rice OsASN2 gene or element or carrier for over-expressing OsASN2 gene as main active component.

Further, the amino acid sequence of the protein is shown as SEQ ID NO. 4; the base sequence of the OsASN2 gene is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

Compared with the prior art, the invention has the following advantages and effects:

according to the invention, the OsASN2 is constructed on an over-expression vector and expressed in the rice ZH11, and the effective spike number, the total number of single plants and the single plant yield of the OsASN2 over-expression transgenic plant are obviously increased, so that the rice yield is improved. Therefore, the OsASN2 gene is a potential target point which can be used as a gene engineering to improve the rice yield, and has important application value.

Drawings

FIG. 1 is a graph showing the increase in the expression level of gene OsASN2 in an overexpressed transgenic line;

FIG. 2 is a statistical chart of effective spike number, total grain number of single plant and single plant yield of OsASN2 over-expression strain; wherein A is the effective spike number, B is the total grain number of a single plant, and C is the yield of the single plant;

FIG. 3 is a graph showing the statistics of seed setting rate and thousand kernel weight of OsASN2 overexpressing lines; wherein A is the setting percentage and B is the thousand grain weight.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The following experimental methods are all conventional methods without specific description.

Vector pOx in the following experimental method is disclosed in "a MYB21 gene for regulating and controlling bacterial leaf blight resistance of rice and its application-CN 112250745A" in Chinese patent application.

Example 1

1. Cloning of rice OsASN2 gene and construction of super-expression vector

The following primers were designed according to the OsASN2 sequence for amplification of OsASN2CDS, the primer sequences were as follows:

ASN2OE-F:5’-tggtgttacttctgcagggtaccATGTGTGGCATCCTCGCC-3’(SEQ ID NO.5)

ASN2OE-R:5’-gatccataacgcgtactagtaagcttTTAAACGGCAGTGGCTGAAG-3’(SEQ ID NO.6)

extracting leaf RNA of rice ZH11 or Japanese sunny leaf by conventional method, making reverse transcription into cDNA, using the cDNA as template, using the above-mentioned primer, adopting high-fidelity PCR polymerase to make amplification, after electrophoresis cutting out band, making DNA fragment recovery, using an infusion method to make the recovered fragment pass through homologous recombination and construct on KpnI & HindIII double-enzyme tangential vector pOx (rice genetic transformation binary vector whose promoter is ubi), transforming colibacillus DH5 alpha, culturing on LB screening culture containing 50 mug/mL kanamycin, picking monoclonal for 14 hr, making sequencing verification, and making OsASN2CDS sequence completely coincide with database so as to obtain the OsASN2 over-expression vector.

2. Agrobacterium tumefaciens transformed rice

The over-expression vector is transformed into an agrobacterium tumefaciens EHA105 strain, monoclonal is selected for sequencing verification for 14 hours, and the EHA105 strain which is successfully transformed is transformed into rice ZH11 callus according to the following method.

Callus induction: after the seeds are shelled, washing the surfaces of the seeds with 70% alcohol for 1min, then washing the seeds with 5% sodium hypochlorite (stock solution is 10%) for 45min under the condition of shaking culture at 100rpm, washing the seeds with sterile water for 6 times, and sucking the water on the surfaces of the seeds with sterilized filter paper; culturing in solid N6D culture medium at 26-34 deg.C for about 10-15 days to remove small callus particles, and selecting light yellow callus for subculture to infect agrobacterium in about 7 days.

Transformation of callus: activating the EHA105 strain which is successfully transformed, culturing for about 4 hours to OD600 to 0.1-0.8, centrifugally collecting thalli, re-suspending by using an AAM culture medium, adding the thalli into the collected calli, immersing the calli into the AAM liquid culture medium, and co-culturing for 6 minutes; draining bacterial liquid, sucking the bacterial liquid by using filter paper, transferring to a culture dish paved with the filter paper, and drying for about 1-2 hours until the calluses are dried and dispersed; transfer to a N6D+AS solid plate with filter paper and dark culture for 48-72h.

Resistant callus screening: collecting co-cultured callus, washing with sterile water until the callus is clear, adding Cef, and standing for 20min; cleaning with sterile water for 3 times, spreading on a culture dish with filter paper, and blow-drying for about 2 hours until the callus is scattered; the dried calli are inoculated on a N6D+Hyg+Cef plate and cultivated at the constant temperature of 28-34 ℃ by a double-layer sealing film;

callus differentiation and rooting: and screening for about 20-30 days, and then growing new white callus from white to yellow, namely picking the callus, inoculating the callus to a differentiation medium (RE-III), and transferring the seedling to an HF medium for strengthening after the seedling grows to about 2 cm.

3. Detection of over-expressed positive plants

DNA level detection:

primers were designed with the backbone and insert of the vector, and the primer sequences were as follows:

ASN2POX-tF：5’-TTGGGAACTGTATGTGTGTGTC-3’(SEQ ID NO.7)；

ASN2POX-tR：5’-ATCAACAATAGCCAACCGCT-3’(SEQ ID NO.8)。

ensuring that the primer cannot amplify the product in the wild type. Extracting the DNA of the transgenic single plant for PCR identification, and selecting the offspring isolation ratio to accord with 3:1, a strain of 1.

RNA level detection:

extracting RNA of a transgenic single plant, reversely transcribing the RNA into cDNA, carrying out fluorescent quantitative PCR reaction by taking the cDNA as a template, repeating three times of each system, and carrying out primers of a target gene OsASN2 as follows:

OsASN2-QF：5’-AAAAGCTTCATTGCAGCCCG-3’(SEQ ID NO.9)；

OsASN2-QR：5’-AACCGAACCATCAAGACCCC-3’(SEQ ID NO.10)。

the reference gene selected OsUBC (LOC_Os02g42314.2), the primers were as follows:

OsUBC-QF：5’-CCGTTTGTAGAGCCATAATTGCA-3’(SEQ ID NO.11)；

OsUBC-QR：5’-AGGTTGCCTGAGTCACAGTTAAGTG-3’(SEQ ID NO.12)。

and (5) performing seed test on the overexpressing strain:

transgenic lines were planted in Guangzhou in early 2022 season with a total nitrogen application of 150kg N/hm ² Phosphate fertilizer 45kg P ₂ O ₅ /hm ² Potash fertilizer 120kg K ₂ O/hm ² The planting specification is 20cm multiplied by 20cm, and the conventional field water and fertilizer management is carried out. In the mature period, the indexes such as effective spike number, total grain number of single plant, single plant yield, setting rate, thousand grain weight and the like of wild type and over-expressed strain (OsASN 2 gene relative expression quantity is improved by about 10 times compared with the wild type) through DNA and RNA level detection (shown in figure 1) are examined. The results show that the OsASN2 overexpressing strain has significant spike number, total grain number and yield per plant higher than that of the wild-type strain (as shown in FIG. 2, which represents a significant difference between the corresponding overexpressing strain and the wild-type strain (p<0.05 And) the seed setting rate and thousand kernel weight have no obvious difference (as shown in figure 3).

The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.

Claims (10)

1. The application of the rice OsASN2 gene or the coded protein thereof in improving the rice yield is characterized in that:

the base sequence of the OsASN2 gene is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3; the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4.

2. The use according to claim 1, characterized in that:

the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the effective spike number of rice.

3. The use according to claim 1, characterized in that:

the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the total grain number of a single rice plant.

4. The use according to claim 1, characterized in that:

the application is the application of the rice OsASN2 gene or the coded protein thereof in improving the yield of a single rice plant.

5. The use according to claim 1, characterized in that:

the application is the application in cultivating high-yield rice.

6. The use according to claim 5, characterized in that: the application comprises the following steps:

(1) Constructing an OsASN2 gene overexpression vector;

(2) Introducing the over-expression vector into receptor rice cells, and over-expressing the OsASN2 gene to obtain transgenic plants.

7. The use according to claim 6, characterized in that:

the basic vector of the over-expression vector in the step (1) is a binary plant expression vector.

8. The use according to claim 7, characterized in that:

the introduction in the step (2) is a transgenic mode of agrobacterium infection.

9. The use according to claim 8, characterized in that:

the binary plant expression vector is pOx;

the acceptor rice is ZH 11;

the agrobacterium-transfected cells are derived from callus induced by rice seeds;

the agrobacterium is agrobacterium EHA105 strain.

10. A formulation for controlling rice yield, characterized in that:

the main active component is protein coded by rice OsASN2 gene, or element or carrier for over-expressing OsASN2 gene;

the amino acid sequence of the protein is shown as SEQ ID NO. 4; the base sequence of the OsASN2 gene is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

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