Application of OsLPS1 gene and mutant thereof in response to exogenous hormone

文档序号：501559 发布日期：2021-05-28 浏览：16次中文

阅读说明：本技术 OsLPS1基因及其突变体在响应外源激素中的应用 (Application of OsLPS1 gene and mutant thereof in response to exogenous hormone ) 是由饶玉春褚晓洁杨窑龙林晗王盛叶涵斐芦涛严钢于 2021-01-28 设计创作，主要内容包括：本发明公开了OsLPS1基因及其突变体在响应外源激素中的应用,属于植物生物技术领域。OsLPS1基因核苷酸序列如SEQ ID NO.1所示,所编码的氨基酸序列如SEQ ID NO.3所示；突变体核苷酸序列如SEQ ID NO.2所示,所编码的氨基酸序列如SEQ ID NO.4所示。本发明提供的水稻早衰OsLPS1基因及其突变体在影响水稻衰老和响应外源激素方面具有广阔的应用前景,通过研究激素在水稻衰老中的遗传调控机制,为培育耐早衰优质高产的农作物提供基础。(The invention discloses an application of OsLPS1 gene and mutant thereof in response to exogenous hormone, and belongs to the technical field of plant biology. The OsLPS1 gene has a nucleotide sequence shown as SEQ ID NO.1, and a coded amino acid sequence shown as SEQ ID NO. 3; the mutant nucleotide sequence is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ ID NO. 4. The rice premature senility OsLPS1 gene and the mutant thereof provided by the invention have wide application prospects in the aspects of influencing rice senescence and responding to exogenous hormones, and provide a foundation for cultivating premature senility-resistant high-quality and high-yield crops by researching the genetic regulation mechanism of hormones in rice senescence.)

1. The application of the rice senilis OsLPS1 gene and the mutant thereof in responding exogenous hormone is characterized in that,

the OsLPS1 gene has a nucleotide sequence shown as SEQ ID NO.1, and a coded amino acid sequence shown as SEQ ID NO. 3;

the nucleotide sequence of the OsLPS1 gene mutant is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ ID NO. 4.

2. The use according to claim 1,

the response exogenous hormone is response exogenous hormone stress.

3. The use according to claim 1,

the exogenous hormone is methyl jasmonate or abscisic acid.

4. The use according to claim 1,

responding to exogenous hormonal stress by at least one of:

(1) inhibit or promote aerial part length;

(2) inhibiting or promoting a length of the subterranean portion;

(3) up-regulating or down-regulating the expression level of abscisic acid synthesis and metabolism related genes;

(4) up-regulating or down-regulating the expression level of the OsLPS1 gene before and after stress.

5. The use according to claim 4,

the abscisic acid synthesis and metabolism related genes comprise OsABA1, OsABA2, OsABA3, OsNCED1, OsNCED2, OsNCED3, OsNCED5, OsZEP, OsZDS, OsABA80x1, OsABA80x2 or OsABA80x 3.

6. The application of the response action of the early senescence OsLPS1 gene of rice and the mutant thereof to exogenous hormone in the breeding of rice varieties is characterized in that,

the OsLPS1 gene and its mutant are as described in claim 1.

Technical Field

The invention relates to the technical field of plant biology, in particular to application of an OsLPS1 gene and a mutant thereof in response to exogenous hormones.

Background

The process of rice growth and development is a process of continuously resisting and adapting to adverse environments such as biological stress, abiotic stress and the like, plant hormones have indispensable functions in the aspects of regulating growth and development and participating in plant stress resistance, can be induced by external environmental stress, and often regulate various physiological reactions at low level in a plant body, thereby having extremely important significance for ensuring the survival, growth and development of rice.

Plant senescence is a programmed cell death process initiated spontaneously by plants at the final stage of growth and development, which is controlled by genes and is influenced and induced by internal and external environmental factors; the method comprises the influences of plant endogenous hormones, heredity and gene regulation, external environment and the like, and the factors are mutually related and mutually restricted to form a complex regulation network.

At present, the research on rice senescence-related genes mostly focuses on the aspects of positioning, functions and the like, and the molecular mechanism of senescence and the regulation mechanism of hormone participating in rice senescence are not clear. Therefore, further intensive research is needed to reveal the regulation and control mechanism of hormone in rice senescence, and lay the foundation for the breeding of high-quality rice varieties.

Disclosure of Invention

In view of the above, the invention researches the rice senilis OsLPS1 gene and the mutant thereof, and shows obvious influence on rice senescence and responding to exogenous hormone stress.

The invention adopts the following technical scheme:

the application of the rice senilis OsLPS1 gene and the mutant thereof in responding exogenous hormone,

the OsLPS1 gene has a nucleotide sequence shown as SEQ ID NO.1, and a coded amino acid sequence shown as SEQ ID NO. 3;

the nucleotide sequence of the OsLPS1 gene mutant is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ ID NO. 4.

Compared with the OsLPS1 gene, the mutant has the mutation of nucleotide 566G to A, which leads to the premature termination of the coded amino acid sequence. The mutant enables the rice to show a yellow leaf premature senescence phenotype from the three-leaf stage, and also shows the phenomena of plant height, effective tillering, seed setting rate and thousand-grain weight remarkably reduced in the mature stage; it shows a significant difference from the OsLPS1 gene in responding to exogenous hormones.

Further, responding to exogenous hormones is responding to exogenous hormone stress.

Preferably, the exogenous hormone is Methyl Jasmonate (MeJA) or Abscisic Acid (abasic Acid, ABA).

Further, responding to exogenous hormonal stress by at least one of:

(1) inhibit or promote aerial part length;

(2) inhibiting or promoting a length of the subterranean portion;

(3) up-regulating or down-regulating the expression level of abscisic acid synthesis and metabolism related genes;

(4) up-regulating or down-regulating the expression level of the OsLPS1 gene before and after stress.

Further, the abscisic acid synthesis and metabolism-related genes include OsABA1, OsABA2, OsABA3, OsNCED1, OsNCED2, OsNCED3, OsNCED5, OsZEP, OsZDS, OsABA80x1, OsABA80x2 or OsABA80x 3.

The application of the response action of the early senescence OsLPS1 gene of rice and the mutant thereof on exogenous hormones in rice variety breeding is disclosed, and the OsLPS1 gene and the mutant thereof are as described above.

Furthermore, a rice sample to be screened, rice containing the OsLPS1 gene and rice containing the OsLPS1 gene mutant can be subjected to tissue culture, exogenous hormone stress is carried out in the culture process, and response results of the rice to the exogenous hormone stress are compared to screen the premature senility resistant high-quality high-yield rice variety.

Because various factors such as rice yield, stress resistance and the like need to be considered in the screening process of high-quality rice varieties, and the experiment is relatively complicated, the response effect of the early-senescence OsLPS1 gene of rice and the mutant thereof on exogenous hormones is fully utilized for tissue culture screening, the screening period is favorably shortened, and the screening efficiency is improved.

Furthermore, the rice premature senility OsLPS1 gene and the mutant thereof can be transformed or the expression level thereof can be regulated by utilizing a genetic engineering method, and then the rice variety with high quality and high yield of resisting premature senility is screened through the response effect of the rice variety on exogenous hormones.

In conclusion, the rice senilism OsLPS1 gene and the mutant thereof provided by the invention have wide application prospects in the aspects of influencing rice senescence and responding to exogenous hormones.

Drawings

FIG. 1 shows a schematic diagram of the mutation sites of mutant lps1 used in the present invention;

FIG. 2 shows the phenotypic characteristics of mutant lps 1;

a: wild type "Longjing 31" and mutant lps1 trilobate phenotype;

b: wild type Longjing 31 and mutant lps1 three-leaf stage leaf phenotype;

c: the tillering phenotype of the wild type Longjing 31 and the mutant lps 1;

d, mature phenotype of wild type Longjing 31 and mutant lps 1;

FIG. 3 shows the growth conditions (A) of wild type "Longjing 31" and mutant lps1 materials under MeJA stress and the influence on the plant height (B) and root length (C) of the wild type "Longjing 31" and mutant lps1 before and after the stress;

FIG. 4 shows the growth conditions (A) of wild type "Longjing 31" and mutant lps1 materials under ABA stress and the influence on the plant height (B) and root length (C) of the wild type "Longjing 31" and the mutant lps1 before and after the stress;

FIG. 5 shows ABA synthesis and metabolism related gene expression levels in wild type "Longjing 31" and mutant lps1 before and after ABA stress;

a: before stress; b: after the stress;

FIG. 6 shows the expression levels of OsLPS1 genes in wild type "Longjing 31" and mutant lps1 before and after ABA stress.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 acquisition of Rice mutant lps1 Material

Taking a wild type of a japonica rice variety 'Longjing 31' as a raw material, and carrying out EMS (ethyl methane sulfonate) chemical mutagenesis: soaking the wild type Longjing 31 dry seeds to be treated for 8-10 hours by using clean water, draining, soaking the seeds to be treated for 10 hours by using 1.5 percent EMS solution at the temperature of 28 ℃, washing for 10-12 hours by using tap water, and finally putting the seeds into a 37 ℃ oven for accelerating germination.

The mutagenized material was selfed to obtain M2 generations.

A premature senility mutant is screened from an M2 generation mutation library, and then the premature senility mutant is discovered to be stably inherited through multi-generation selfing, and a homozygous mutant strain is obtained and named as lps 1.

Genomic DNA of mutant lps1 was extracted and the primers lps1-867F (cggccaaattatagctgga) and lps1-2260R (atgggcaggaactggtag), lps1-2162F (gactgcatgacagcagggaa) and lps1-3642R (acaagcatgtttcaggt), lps1-3461F (tcggtcaacaacaacaacaacaataatcc) and lps1-4868R (gattgcgagtctgcttg), lps 7-4572F ccgcgcgcgctcgctcacattat) and lps1-6020R (acacaggaggagtccagaggcaggctagg), lps 2-5762F (ttgcaggcaggcatgctaggcttag) and lps1-6020R (acaggaggcaggcaggcaggctagg) and the primers cgs 70839-843-gcaggcaggcaggcaggcaggcaggctagg and gcaggcaggcaggcatcg, and PCR were performed, respectively (gcaggcaggcaggcaggcaggcaggcatgcatgctag) and (gcaggcaggcaggctag) and (gcaggcaggctag) and gctag, gctaaggcps 1-6020R 3-8427 g, gcaggcaggcaggcaggcaggcaggcaggcaggcaggcaggctaaggctaaggctag, and gctaaggctag, and gctaaggctaaggctaaggctag, and gctag, and gctaaggctaaggc.

As shown in FIG. 1, compared with the genomic DNA of the wild type "Longjing 31", the mutant lps1 has only a single-base substitution in the first exon, i.e., the 566bp G of OsLPS1 gene is replaced by A, resulting in premature termination of the encoded amino acid sequence. Wherein, the nucleotide sequence of OsLPS1 gene of wild type Longjing 31 is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 3; the nucleotide sequence of the OsLPS1 gene mutant of the mutant lps1 is shown as SEQ ID NO.2, and the coded amino acid sequence is shown as SEQ ID NO. 4.

Example 2 phenotypic analysis of Rice plants

Comparing the phenotype of wild type "Longjing 31" and mutant lps1 rice plants, the phenotype of yellowing of leaves appeared from the beginning of the three-leaf stage (FIGS. 2A and 2B), the yellowing area of leaves gradually increased as the plants continued to grow and develop, but the bases of leaves remained green (FIG. 2C), and the phenotype continued until the maturity stage (FIG. 2D). In addition, the mature stage showed significant reductions in plant height, effective tillering, seed set rate, thousand-kernel weight, and the like (table 1).

TABLE 1 comparison of major agronomic traits for wild type and mutant lps1

Example 3 exogenous hormone stress assay

Selecting rice seeds with plump seeds (wild type 'Longjing 31' and mutant lps1), shelling, sterilizing with 70% alcohol for 2min, sterilizing with 30% sodium hypochlorite solution for 30min, washing with distilled water, inoculating on 1/2MS culture medium containing 0, 2.5 μ M, 5 μ M MeJA or 0.2 μ M, 0.25 μ MABA, culturing in artificial climate incubator for 10d, respectively counting plant height and root length, and repeating for 3 groups. The incubator is set under the conditions of light/dark (14h/10h), temperature of 30 ℃ and humidity of 70%.

After 10 days of culture, sampling the rice seedlings, cleaning the residual culture medium with sterile water, and measuring the plant height and the root length of the rice seedlings, wherein each group has 6 repetitions.

In addition, total RNA of the sampled rice was extracted using a plant total RNA extraction kit (Axygen), and 1. mu.g of the total RNA was reverse-transcribed into cDNA using a cDNA reverse transcription kit (TOYOBO). SYBR Green Realtime PCR Master Mix (TOYOBO) and primers in Table 2 are used for real-time fluorescent quantitative PCR to detect the expression levels of ABA related genes and OsLPS1 genes, and rice Actin is used as an internal reference gene.

TABLE 2 real-time quantitative primer sequence Listing

And (3) test results:

1. physiological response to MeJA stress

As shown in fig. 3, in the absence of MeJA stress, mutant lps1 did not significantly differ in plant height and root length from wild type; while the wild type strain height and root length under 2.5 μ M MeJA stress were 85% and 54% of MeJA stress free, mutant lps1 strain height and root length were 61% and 37% of MeJA stress free, respectively; while the plant height and root length of the wild type under 5 μ M MeJA stress were 66% and 42% of the MeJA stress free, the plant height and root length of mutant lps1 were 50% and 43% of the MeJA stress free, respectively; under MeJA stress conditions of different concentrations, the plant height and root length of the mutant lps1 were significantly inhibited, and a significant hypersensitivity phenotype to MeJA was exhibited. The result shows that the inhibition of the expression of the OsLPS1 gene can enhance the intolerance of seedlings to MeJA.

2. Physiological response to ABA stress

As shown in fig. 4, the plant height and root length of wild type under 0.2 μ MABA stress were 52% and 90% of ABA-stress free, while the plant height and root length of mutant lps1 were 41% and 160% of ABA-stress free, respectively; the plant height and root length of the wild type under 0.25. mu.M ABA stress are 47% and 76% of those without ABA stress, while the plant height and root length of the mutant lps1 are 34% and 93% of those without ABA stress, respectively; ABA stress of different concentrations obviously inhibits the plant height of the mutant lps1, but under the stress of 0.2 mu MABA, the root length of the mutant lps1 is obviously elongated; with the increase of the stress concentration of the ABA, the root length of the ABA is remarkably inhibited. The fact that the OsLPS1 gene mutation can enhance the ABA tolerance of rice seedling roots under certain conditions is shown.

ABA biosynthesis and metabolism related Gene Change in mutant lps1 under ABA stress

As shown in fig. 5, before ABA stress, compared with wild type, the expression levels of the mutant lps1 and ABA synthesis-related genes OsABA1, OsABA3, OsNCED1, OsNCED3, OsNCED5, OsZEP and OsZDS were significantly reduced, and the expression levels of the ABA metabolism-related genes OsABA80x1 and OsABA80x2 were significantly reduced; after ABA stress, the expression levels of the ABA synthesis related genes OsABA3, OsNCED1 and OsZEP in the mutant lps1 are obviously up-regulated, while the expression level of OsNCED3 is down-regulated, and the expression level of the ABA metabolism related gene OsABA80x1 is also obviously down-regulated.

The results confirm that the expression of ABA synthesis and metabolism related genes in the mutant lps1 is inhibited before ABA stress, and the expression level of ABA synthesis related genes (OsABA3, OsNCED1 and OsZEP) is remarkably increased after ABA stress, which indicates that the lps1 mutant can respond to the treatment of exogenous ABA by influencing the ABA synthesis pathway after the OsLPS1 gene mutation.

Expression level of OsLPS1 gene under ABA stress

As shown in fig. 6, the expression level of OsLPS1 gene in mutant lps1 was significantly reduced before ABA stress, which was 0.3 times higher than the expression level of OsLPS1 gene in wild type; after ABA stress, the expression level of OsLPS1 gene in the mutant lps1 is obviously up-regulated and is 5.7 times of the expression level of OsLPS1 gene in wild type. The results confirmed that the expression of the OsLPS1 gene in the lps1 mutant was significantly induced under ABA stress.

In conclusion, the lps1 mutant responds to exogenous MeJA treatment, and also responds to exogenous ABA treatment by influencing an ABA synthetic pathway; meanwhile, under the stress of ABA, the expression level of the OsLPS1 gene in the lps1 mutant is also remarkably induced. Therefore, further research on the function and action mechanism of the OsLPS1 gene has important significance for resisting the aging of rice leaves and improving the stress resistance of rice, and provides reference for breeding new rice varieties with high stress resistance.

The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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