CN118440955A - Application of over-expressed rice P3A gene in improving plant abiotic stress resistance - Google Patents

Abstract

The invention discloses application of a rice P3A gene and a coding protein thereof in improving plant abiotic stress resistance and yield. The invention also discloses a method for improving the resistance of a plant to abiotic stress, comprising: the transgenic plant is obtained by over-expressing the rice P3A gene in the plant, and the obtained transgenic plant has obviously enhanced resistance to low temperature stress and obviously increased yield. The invention has application prospect in improving and enhancing the stress resistance of rice, accelerating the breeding process of stress-resistant molecules and the like.

Description

Application of over-expressed rice P3A gene in improving plant abiotic stress resistance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of over-expressed rice P3A genes in improving abiotic stress resistance of plants.

Background

Rice (Oryza sativa l.) is one of the most prominent food crops in the world, providing staple food to more than half of the world's population. Rice originates from tropical and subtropical regions, belongs to the field of crops with happiness temperature and is very sensitive to low temperature. The low-temperature cold injury not only limits the geographical distribution of the rice, but also seriously affects the growth and development of the rice. As rice planting areas expand from tropical and subtropical areas to high-altitude areas, the probability of suffering from low-temperature cold injury in each period of rice growth is continuously increased. The rice seedling stage can cause seedling dysplasia, yellowing or withering of leaves and reduced tillering after suffering from low-temperature cold injury, and finally the yield and quality of the rice are reduced. Therefore, the key genes involved in regulating and controlling the cold tolerance of the rice in the seedling stage are mined, and the action mechanism of the key genes is explained to have important significance for preventing and controlling the low-temperature cold damage of the rice and cultivating the cold-resistant rice varieties.

The rice-specific P3 subfamily P3A (Ribosomal P3 protein) encodes an acidic Ribosomal protein. Ribosomes are mainly composed of ribosomal RNA (rRNA) and ribosomal proteins (Ribosomalprotein, RP), and are sites for translation of biological proteins. The cold resistance of the arabidopsis and sweet potatoes can be obviously improved by over-expressing the arabidopsis P3 gene. It has been found that low temperature affects not only the expression of ribosome-associated proteins, but also ribosome-mediated protein translation. Early studies in tomato have found that low temperatures can inhibit protein translation by affecting the function of ribosomes in the plastid. Recently, studies have been made in Arabidopsis thaliana to find that protein translation can be inhibited in a short period of time at low temperatures. The above studies demonstrate that there is a close link between ribosomes and plant cold tolerance. However, it is not clear how the ribosomes regulate plant cold tolerance.

Disclosure of Invention

The main purpose of the invention is to provide the application of the over-expressed rice P3A gene in improving the abiotic stress resistance of plants, and meanwhile, the over-expressed rice P3A gene can also obviously improve the maturing rate and tillering number of rice.

To achieve the above object, the first aspect of the present invention provides an isolated nucleic acid encoding a rice P3A protein, said P3A protein comprising the amino acid sequence as set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

As a preferred embodiment of the invention, the nucleic acid comprises SEQ ID NO:1 or a degenerate sequence thereof.

As a preferred embodiment of the invention, the nucleic acid comprises SEQ ID NO:3 or a degenerate sequence thereof

In a second aspect the invention provides a vector comprising a nucleic acid according to the first aspect of the invention.

Wherein, the coding gene of the rice P3A protein is connected with an expression regulatory element to obtain a recombinant plant expression vector; the recombinant plant expression vector can consist of a rice P3A coding region; the promoter may be a constitutive promoter, an inducible promoter, an enhanced promoter, a tissue or organ specific promoter. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells, for selection of transformed cells or tissues. The marker gene includes: genes encoding antibiotic resistance, hygromycin, herbicide genes, and the like. In addition, the marker genes include phenotypic markers such as green fluorescent protein and the like.

In a third aspect, the invention provides the use of a rice P3A protein or a nucleic acid encoding the same for increasing plant resistance to abiotic stress and/or increasing plant yield.

As a preferred embodiment of the invention, the use comprises overexpressing a nucleic acid encoding a P3A protein of rice in a plant to obtain a transgenic plant; preferably, the amino acid sequence of the P3A protein is shown in SEQ ID NO:2 is shown in the figure; more preferably, the nucleic acid encoding the P3A protein is as described in the first aspect of the invention.

As a preferred embodiment of the invention, the application comprises in particular: (1) Constructing a recombinant plant expression vector containing rice P3A protein coding nucleic acid; (2) Transforming the constructed recombinant plant expression vector into plant tissue or plant cells; (3) Culturing and screening to obtain transgenic plants with improved resistance to abiotic stress; preferably, the expression vector is as described in the second aspect of the invention.

As a preferred embodiment of the present invention, the abiotic stress comprises low temperature stress.

As a preferred embodiment of the present invention, the abiotic stress comprises low temperature stress. Preferably, the low temperature is below 10 ℃,9 ℃,8 ℃,7 ℃,6 ℃,5 ℃,4 ℃,3 ℃,2 ℃,1 ℃,0 ℃.

As a preferred embodiment of the invention, the yield indicators include fruiting rate, effective tiller number and individual yield.

As a preferred embodiment of the present invention, the plant includes, but is not limited to, a monocot or dicot; more preferably, the plant includes crops, vegetables or ornamental plants, fruit trees and the like, and for example, rice, cotton, maize, sorghum, wheat, soybean, potato, barley, tomato, sugarcane or arabidopsis and the like, preferably rice.

Compared with the prior art, the invention has the following technical effects:

According to the invention, through cloning the homologous gene P3A of the Arabidopsis RPP3 in rice, and further through over-expression and CRISPR knocking out the phenotype change of rice plants at extremely low temperature of 6 ℃ and relatively low temperature of 20 ℃, gene cloning and functional analysis are carried out, and the relationship between candidate genes and abiotic stress response of rice seedling stage and booting stage is analyzed, so that the over-expression P3A gene in rice can obviously improve the capability of the rice for resisting low temperature stress, and the seed setting rate and yield of the over-expression P3A plants are increased. The invention has very important theoretical and practical significance for improving and enhancing the stress resistance of rice, cultivating high-yield stress-resistant varieties and accelerating the breeding process of stress-resistant molecules.

Drawings

FIG. 1 shows the knockout of individual lines of the P3A mutant of rice. Fig. 1A: the coding region base of the P3A of the mutation type 1 (P3A-1) is deleted by 25bp (336 bp to 360 bp), so that the P3A protein shifts in the process of translation and finally translation is terminated in advance. Mutation type 2 (P3A-2) is that the coding region base of P3A is respectively deleted by 135bp (201 bp to 335 bp), so that 45 amino acids are deleted in the P3A protein translation process; FIG. 1B, the expression levels of p3a-1 and p3a-2 mutant mRNA were determined by RT-PCR. P3A produced a new transcript in the P3A-1 mutant and expressed in a similar amount as in the wild type; in the p3a-2 mutant, transcription was unstable and no expression was detected. It was demonstrated that p3a-1 and p3a-2 are two different types of loss-of-function mutants.

FIG. 2 is a PCR positive identification of hygromycin in transgenic plants over-expressed and knocked-out. Wherein lanes 1-6 are P3A mutant, lanes 7-12 are P3A over-expression, lane 13 is negative control, and lane 14 is negative control of added double distilled water.

FIG. 3 shows the expression level of each strain of the rice P3A gene overexpression material. Over-expression each strain expressed an average 15-20 fold higher level than the wild type.

FIG. 4 shows P3A overexpression and the cold tolerance phenotype of CRISPR knockout transgenic plants and wild type plants at 6 ℃. A is a phenotype picture of the mutant after 3 days of treatment at 6℃and 7 days of recovery. B is the statistical result of survival rate of the mutant before and after treatment at 6 ℃. C is a phenotype picture after 3 days of treatment and 7 days of recovery of the overexpressed P3A at 6 ℃. D is the statistical result of survival rate before and after treatment at 6 ℃ of the over-expression P3A.

Fig. 5 is an agronomic trait for P3A over-expression and CRISPR knockout transgenic plants and wild type plants under normal conditions. The indexes are effective tillering number, fruiting rate and yield.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

Definition of terms in connection with the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoroamidites, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. I.e. the description for polypeptides applies equally to the description of peptides and to the description of proteins and vice versa. The term applies to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens) in which the amino acid residues are linked via covalent peptide bonds.

The term "recombinant plant expression vector" means one or more DNA vectors for effecting transformation of a plant; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "transformation" as used herein refers to genetic transformation of a polynucleotide or polypeptide into a plant in such a way that the gene encoding the P3A protein of rice is introduced into the interior of a plant cell. Methods of introducing the polynucleotide or polypeptide into a plant are well known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and the like. "stable transformation" refers to integration of an introduced polynucleotide construct into the genome of a plant cell and inheritance by its progeny; "transient transformation" refers to the introduction of a polynucleotide into a plant but only temporary expression or presence in the plant.

The term "effective tillering number" refers to tillering called effective tillering that can be finally set out in tillering of rice. "set percentage" refers to the proportion of filled kernels to total kernels. EXAMPLE 1 construction of P3A Gene overexpression vector

Cloning homologous gene P3A of Arabidopsis RPP3 in Nippon Rice, and determining that the sequence is SEQ ID NO:3.

The pCAMBIA1300s vector (addgene, # KM 1033157) was digested with two restriction enzymes KpnI and SacI, linearized and recovered.

The primer was designed based on the full length of the P3A sequence of the Japanese sunny gene (SEQ ID NO: 3), the full length of the gene coding region of the P3A protein was obtained by amplification using the cDNA of Japanese sunny as a template (SEQ ID NO: 1), the stop codon (TGA) was removed, and the adaptor linearized with the pCAMBIA1300s vector was added to the 5 'and 3' ends, and the amplification primers were as shown in Table 1.

TABLE 1P3A amplification primers

Wherein the lower case portion is a linker sequence.

The target DNA fragment (360 bp) was amplified and recovered by PCR. Carrying out homologous recombination on a target fragment and a linearization vector by ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, code No. C115-01), carrying out PCR and sequencing verification on a positive cloning plasmid, and obtaining a recombinant vector named P1300S-P3A (an over-expression strain named P3A-OE) by inserting a P3A gene fragment shown by SEQ ID NO.1 between two enzyme cutting sites of the vector P1300S according to a sequencing result.

Example 2P3A Gene CRISPR knockout vector construction

Target site 1 and target site 2 (table 2) were designed for knockout based on the P3A gene cDNA sequence using the website: http:// www.genome.arizona.edu/crispr/CRISPRsearch. Html;

TABLE 2 knockout target sites

Target site 1	5′-ACATGGGCGTGTACACCTTC-3′(SEQ ID NO:6)
		Target site 2	5′-CGATTGGTGGAGGTGCTGCT-3′(SEQ ID NO:7)

Primers P3A target1-BsF, P3A target1-F0, P3A target2-R0 and P3A target2-BsR (Table 3) are respectively designed according to targets, and the pCBC-MT1T2 plasmid diluted 100 times is used as a template for four-primer PCR amplification. And purifying and recovering the PCR product, and constructing a final vector by using an enzyme digestion-connection system, wherein the vector is named as CRISPR-P3A.

TABLE 3 Gene knockout primers

FIG. 1A shows the knockout of individual lines of the P3A mutant of rice. The coding region base of the P3A of the mutation type 1 (P3A-1) is deleted by 25bp (336 bp to 360 bp), so that the P3A protein shifts in the process of translation and finally translation is terminated in advance. Mutation type 2 (P3A-2) is that the coding region base of P3A is deleted by 135bp (201 bp to 335 bp) respectively, resulting in deletion of 45 amino acids in the P3A protein translation process.

EXAMPLE 3 Agrobacterium transformation

The expression vector P1300S-P3A and the knockout vector CRISPR-P3A are transferred into competent cells of agrobacterium EHA105 (competent purchased from Shanghai Biotechnology Co.) by freeze thawing method, and the experimental method refers to the molecular cloning experimental guideline.

EXAMPLE 4 genetic transformation

1) And (3) sterilization: removing shell of healthy and plump Japanese seed, soaking in 70% ethanol for 1-2min, adding 50% bleachs, standing on a shaker (200 rpm) for about 1-1.5h, washing with sterile water for 4-6 times, standing on sterile filter paper to absorb water, standing in NBD medium, and culturing in dark at 28deg.C. All the steps are operated on an ultra-clean workbench.

2) Subculture: after dark culture for about 10-15 days, peeling rice seed buds, transferring the rice seed buds to a secondary culture medium NBD, and continuing dark culture at 28 ℃; after 10 days, the seeds were stripped from the calli and the calli transferred to new subculture medium NBD, after which Agrobacterium transformation was performed after dark culture at 28℃for about 4-5 days.

3) During this period, agrobacterium containing the plasmid (P1300S-P3A, CRISPR-P3A) was streaked on LB medium containing kanamycin and rifampicin, and after 2 days, the monoclonal was picked up, streaked again, and cultured for 1 day.

4) The cells were collected from the medium, and the cells were vortexed and suspended in NBC1 medium containing Acetosyringone (AS) to adjust the OD=about 0.1 to 0.2.

5) Conversion: healthy calli were additionally selected in sterile triangular flasks, the suspension with the above-mentioned adjusted concentration was added, gently shaken at room temperature for about 10min, the bacterial solution was discarded, the calli were placed on sterile filter paper, the excess bacterial solution was aspirated and placed in an ultra clean bench for blowing until the calli were slightly whitened, the calli were transferred onto NBC2 medium with a layer of sterile filter paper laid on it, and co-cultured in darkness at 22℃for 2 days.

6) Screening: co-cultured calli were transferred to NBS1 medium containing the corresponding antibiotic, transferred to NBS2 medium after 10-12 days of dark culture at 28℃and continued dark culture at 28℃for 10-12 days.

7) Differentiation: after transferring the callus to NBR1 culture medium, culturing at 28deg.C in darkness for 6 days, transferring to a 15h light/9 h darkness artificial climate incubator, culturing at 28deg.C for 15-20 days, transferring the callus with green spots to NBR2 culture medium, and culturing until differentiating into seedling.

8) Transgenic seedlings with the height of about 5cm are cut into roots and leaves, transferred into rooting culture medium and cultured in a 12h light/12 h dark artificial climate incubator at 28 ℃.

9) Hardening seedlings: after the root system of the transgenic seedling is sufficiently developed, the culture bottle cap is opened for about 2 days, the culture medium is washed away, and the seedling is placed in water for culture for 1 week and then transferred to soil for planting.

The culture medium formula adopted in the genetic transformation process is shown in table 4; the preparation of hormone and antibiotic stock solutions in the medium is shown in Table 5.

TABLE 4 genetic transformation Medium

TABLE 5 preparation of hormone and antibiotic stock solutions

Auxin/antibiotic	Concentration of stock solution (solvent)	Preservation conditions
			NAA	1Mg/ml (ethanol primary solution)	4℃
IAA	1Mg/ml (ethanol primary solution)	-20℃
			KT	1Mg/ml (NaOH primary solution)	-20℃
6-BA	2Mg/ml (NaOH primary solution)	-20℃
			2,4-D	2Mg/ml (95% ethanol primary solution)	4℃
Rifampicin	50Mg/ml (dissolved in methanol)	-20℃
			Kanamycin	50mg/ml	-20℃
Temeitin	50mg/ml	4℃
			Hygromycin	50mg/ml	4℃
Acetosyringone	100mM/L(DMSO)	-20℃

And carrying out PCR verification and planting seed reproduction on the generated T0 generation transgenic seedlings, harvesting T1 generation transgenic seeds, carrying out positive verification and sequencing to obtain corresponding homozygous mutant materials, continuously planting seed reproduction on the obtained homozygous mutant materials, and screening seeds by using hygromycin (50 mg/L) in offspring to obtain the homozygous mutant materials of the corresponding Cas9 free.

PCR positive identification of transgenic plants over-expressed and knocked-out using primers for hygromycin is shown in Table 6.

TABLE 6 hygromycin primers

Example 5 identification of transgenic plant molecules and identification of stress resistance

(1) And selecting T2 generation P3A transgenic overexpression, T3 generation P3A knockout mutant and wild type Japanese seed.

(2) Soil cultivation of rice: after dormancy breaking, the newly harvested seeds are sowed after soaking the seeds in a 28 ℃ incubator for 3 days until germination. Selecting seeds with consistent germination, and uniformly sowing the seeds in the nutrient soil according to the following steps: vermiculite = 3:1, after a layer of vermiculite is covered on the surface, the vermiculite is normally grown in an incubator at 28 ℃, and watering is carried out 1 time every 2-3 days.

(3) Water planting of rice: seeds with consistent germination are selected and sown on 96-hole PCR plates with the bottom removed, the seeds are placed in a 28 ℃ incubator for growth, water is changed every 2-3 days during the growth, a proper amount of nutrient solution is added when the third leaf is just extracted, and the third leaf is completely unfolded and then changed into clean water.

(4) The sterilized rice seeds are germinated at normal temperature, then sowed, each experimental group is provided with at least 3 repetitions, and are treated for 2-4 days (determined according to actual conditions) at 6 ℃ after being cultivated for 2 weeks under the illumination of 28 ℃ under the condition, and then are transferred to the 28 ℃ for 1 week of growth recovery.

(5) For survival, the study judged whether new leaves were grown and whether new She Ze was present to consider the plant to be viable, and vice versa. According to FIG. 4, it can be seen that over-expression of the rice P3A gene in rice can significantly improve the capability of the rice to resist low temperature stress, and mutation or knockout of the P3A gene in rice can significantly reduce the capability of the rice to resist low temperature stress, so that the rice P3A protein, the coding gene and the recombinant vector thereof can be applied to enhancing the capability of crops to resist abiotic stress.

(6) The expression level of transgenic rice at RNA level was measured by fluorescence quantitative PCR (AceQ qPCR SYBR GREEN MASTER Mix (vazyme)) and sequencing (primers shown in Table 7).

FIG. 1B shows the expression level of the P3A knockout line. As can be seen from FIG. 1B, P3A produced a new transcript in the P3A-1 mutant and expressed in a similar amount as in the wild type; in the p3a-2 mutant, transcription was unstable and no expression was detected. It was demonstrated that p3a-1 and p3a-2 are two different types of loss-of-function mutants.

FIG. 3 shows the expression levels of the individual strains of the P3A gene overexpression material. As can be seen from FIG. 3, the expression level of the P3A gene in the P3A gene overexpression material was improved to a different extent as compared with the wild-type material.

The following over-expression and mutant strain primers were used for subsequent material identification as to whether homozygous use:

TABLE 7 primer for detecting expression quantity of transgenic rice at RNA level by fluorescent quantitative PCR

Example 6 identification of transgenic plant yield

The statistics of the over-expression and knockout mutant plants is carried out at a low temperature of 20 ℃ in the booting stage, the phenotype is shown in figure 5, and the result shows that the over-expression of the rice P3A gene in the rice can obviously improve the rice setting rate and tillering number, and the mutation or knockout of the P3A gene in the rice can obviously reduce the rice setting rate and tillering number, so that the rice P3A protein, the coding gene and the recombinant vector thereof can be applied to the enhancement of the crop yield.

The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.

Claims (10)

1. An isolated nucleic acid encoding a rice P3A protein, said P3A protein comprising the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.

2. The nucleic acid of claim 1, wherein the nucleic acid comprises the sequence of SEQ id no:1 or a degenerate sequence thereof.

3. The nucleic acid of claim 2, wherein the nucleic acid comprises the sequence of SEQ id no:3 or a degenerate sequence thereof.

4. A vector comprising the nucleic acid of any one of claims 1-3.

5. Use of a rice P3A protein or a nucleic acid encoding the same for increasing plant resistance to abiotic stress and/or increasing plant yield.

6. The use according to claim 5, comprising overexpressing a nucleic acid encoding a rice P3A protein in a plant to obtain a transgenic plant; preferably, the amino acid sequence of the P3A protein is shown in SEQ ID NO:2 is shown in the figure; more preferably, the nucleic acid encoding the P3A protein is as defined in any one of claims 1 to 3.

7. The use according to claim 6, characterized in that it comprises in particular: (1) Constructing a recombinant plant expression vector containing rice P3A protein coding nucleic acid; (2) Transforming the constructed recombinant plant expression vector into plant tissue or plant cells; (3) Culturing and screening to obtain transgenic plants with improved resistance to abiotic stress; preferably, the expression vector is as claimed in claim 4.

8. The use of claim 5, wherein the abiotic stress comprises a low temperature stress.

9. The use according to claim 5, wherein the yield indicators include fruiting rate, effective tiller number and individual yield.

10. The use according to claim 5, wherein the plant is rice.