A mutant Arabidopsis protein and gene that promotes chloroplast development and its applications

Abstract

This invention relates to the field of agricultural technology, specifically to a mutant Arabidopsis protein and gene that promotes chloroplast development, and their applications. The mutant Arabidopsis protein is the AtMurE protein in wild-type Arabidopsis Sig6 factor-deficient mutants, with mutations occurring at positions 89, 97, 106, 125, 126, 130, or 140. The mutant protein or gene provided by this invention, when introduced into wild-type Arabidopsis Sig6 factor-deficient mutants, can restore chlorophyll content and resolve the cotyledon yellowing problem in wild-type Arabidopsis Sig6 factor-deficient mutants.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a mutant Arabidopsis protein and gene that promotes chloroplast development and its application. Background Technology

[0002] Chloroplast development is fundamental to plant photosynthesis, and its process is regulated by the coordinated action of both the nuclear genome and the chloroplast genome. Transcription of plastid genes is accomplished by two types of RNA polymerases: nucleus-encoded RNA polymerase (NEP) and plastid-encoded RNA polymerase (PEP). PEP is responsible for transcribing most photosynthesis-related genes, and its complex contains σ factors and PEP-related proteins (PAPs). Arabidopsis thaliana has six σ factors (SIG1-SIG6), and the absence of SIG6 results in an early cotyledon yellowing phenotype followed by later greening, indicating that SIG6 plays a crucial role in early chloroplast development, but the mechanism remains unclear.

[0003] AtMurE (AT1G63680) encodes the PAP11 subunit of the PEP complex, and its bacterial homolog is a cell wall peptidoglycan synthase. The albino lethality of the Arabidopsis atmure deletion mutant indicates that AtMurE is indispensable for chloroplast development, but the mechanism is unknown. Currently, there are no reports on the genetic interaction between SIG6 and AtMurE, and a repressor that can specifically restore the early etiolation phenotype of SIG6 and its molecular mechanism are also lacking. Therefore, elucidating the molecular mechanism by which AtMurE regulates SIG6 function has significant theoretical and applied value. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a mutant Arabidopsis protein and gene that promote chloroplast development, and their applications. When this mutant protein or gene is introduced into a wild-type Arabidopsis Sig6 factor-deficient mutant, it can restore chlorophyll content and resolve the cotyledon yellowing problem in the wild-type Arabidopsis Sig6 factor-deficient mutant.

[0005] Therefore, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides, in optional embodiments, an Arabidopsis mutant protein that promotes chloroplast development, wherein the Arabidopsis mutant protein is the AtMurE protein of a wild-type Arabidopsis Sig6 factor deletion mutant with mutations at positions 89, 97, 106, 125, 126, 130, or 140. The amino acid sequence of the AtMurE protein of the wild-type Arabidopsis Sig6 factor deletion mutant is shown in SEQ ID NO. 15.

[0007] Preferably, the Arabidopsis mutant protein is the AtMurE protein of the wild-type Arabidopsis Sig6 factor deletion mutant, which has a mutation at position 89 from aspartic acid to asparagine, at position 97 from lysine to alanine, at position 106 from aspartic acid to asparagine, at position 125 from isoleucine to alanine, at position 126 from aspartic acid to alanine, at position 130 from serine to phenylalanine, or at position 140 from glycine to arginine.

[0008] Preferably, the amino acid sequence of the Arabidopsis mutant protein is selected from one of SEQ ID NO.1-7.

[0009] Secondly, in an optional embodiment, the present invention provides an Arabidopsis mutant gene that promotes chloroplast development. The Arabidopsis mutant gene is the MurE gene on a wild-type Arabidopsis Sig6 factor deletion mutant, wherein the mutation occurs at positions 265, 290 and 291, 316, 373 and 374, 377, 389, or 418. The nucleotide sequence of the MurE gene on the wild-type Arabidopsis Sig6 factor deletion mutant is shown in SEQ ID NO. 16.

[0010] Preferably, the Arabidopsis mutant gene is the MurE gene of the wild-type Arabidopsis Sig6 factor deletion mutant, in which a mutation occurs at position 265 (G to A), positions 289 and 290 (TA to GC), position 316 (G to A), positions 373 and 374 (AT to GC), position 377 (A to C), position 389 (C to T), or position 418 (G to A).

[0011] Preferably, the nucleotide sequence of the Arabidopsis mutant gene is selected from one of SEQ ID NO. 8-14.

[0012] Thirdly, in an optional embodiment, the present invention provides an expression cassette comprising the aforementioned Arabidopsis mutant gene.

[0013] Fourthly, in optional embodiments, the present invention provides a recombinant vector or recombinant cell containing the aforementioned Arabidopsis mutant gene.

[0014] Fifthly, in optional embodiments, the present invention provides the application of the above-mentioned Arabidopsis mutant protein, the above-mentioned Arabidopsis mutant gene, the above-mentioned expression cassette, or the above-mentioned recombinant vector or recombinant cell in promoting chloroplast development.

[0015] Preferably, the Arabidopsis mutant protein, Arabidopsis mutant gene, expression cassette, recombinant vector, or recombinant cell is introduced into the wild-type Arabidopsis Sig6 factor-deficient mutant to restore the chlorophyll content of the wild-type Arabidopsis Sig6 factor-deficient mutant and solve the problem of cotyledon yellowing in the wild-type Arabidopsis Sig6 factor-deficient mutant.

[0016] Compared with the prior art, the present invention has one of the following beneficial effects:

[0017] 1. The mutant protein or mutant gene provided by this invention can be introduced into wild-type Arabidopsis thaliana Sig6 factor-deficient mutants to restore chlorophyll content and solve the problem of cotyledon yellowing in wild-type Arabidopsis thaliana Sig6 factor-deficient mutants.

[0018] 2. This invention can utilize the obtained mutation sites as tools for molecular marker-assisted breeding or genetic improvement. This includes using these sites as functional markers to screen or identify germplasm resources with superior chloroplast development traits; it can also introduce corresponding mutations into important crops through gene editing (such as CRISPR / Cas9) to improve cotyledon greening ability in the seedling stage and subsequent photosynthetic efficiency, thereby increasing crop yield or biomass. Attached Figure Description

[0019] Figure 1 The wild-type Arabidopsis thaliana and the repressor sig6 atmure in Example 1 of this invention S130F Schematic diagram showing the cotyledon phenotype and chlorophyll content detection results of the single mutant sig6 after 5 days of growth under normal conditions;

[0020] Figure 2 The wild-type Arabidopsis thaliana and the repressor sig6 atmure in Example 1 of this invention S130F Schematic diagram showing the results of maximum photochemical activity of photosystem II in the single mutant sig6;

[0021] Figure 3 The wild-type Arabidopsis thaliana and the repressor sig6 atmure in Example 1 of this invention S130FA schematic diagram of the transcription of chloroplast genome-encoding genes in the single mutant sig6;

[0022] Figure 4 This is a schematic diagram showing the cotyledon phenotype and chlorophyll content detection results of wild-type Arabidopsis thaliana, Line1 strain, Line2 strain and single mutant sig6 under normal conditions for 5 days in Example 1 of the present invention.

[0023] Figure 5 The suppressor sig6 atmure in Embodiment 1 of the present invention FD89N , repressor sig6 atmure F106N and the repressor sig6 atmurer G140RN A schematic diagram of mutation sites;

[0024] Figure 6 The wild-type Arabidopsis thaliana and the repressor sig6 atmure in Example 1 of this invention S130F Single mutant sig6, sig6 atmure FD89N , repressor sig6 atmure F106N and the repressor sig6 atmurer G140RN A schematic diagram showing the results of cotyledon phenotype and chlorophyll content detection after 5 days of growth under normal conditions;

[0025] Figure 7 The MurE gene promoter sequence and atmure gene sequence in Example 2 of this invention Y97A atmure I125A atmure D126A mutated sequences;

[0026] Figure 8 The wild-type Arabidopsis thaliana, single mutant sig6, and atmure in Example 2 of this invention Y97A atmure I125A and atmure D126A A schematic diagram showing the results of cotyledon phenotype and chlorophyll content detection after 5 days of growth under normal conditions;

[0027] Figure 9 This is a schematic diagram of the amino acids of the seven Arabidopsis mutant proteins that promote chloroplast development in this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] In the following examples, Arabidopsis thaliana was used as the Col-0 background, and the T-DNA insertion mutant was purchased from the Arabidopsis SALK seed bank (https: / / abrc.osu.edu / stocks). Arabidopsis thaliana solid medium was 1 / 2 MS, with the following formulation: 1% sucrose, 2.15 g / L Murashige and Skoog basal salt mixtures (Sigma), 0.7% Phytol agar (Duchefa Biochemie), pH = 5.7. Plates containing the seeds were placed on a light rack for cultivation. Plates used to observe root length were vertically cultured on the light rack (using square plates).

[0032] The soil cultivation conditions were black soil:vermiculite:perlite = 5:4:1, and the greenhouse cultivation conditions were 20℃, 16 h light / 8 h dark.

[0033] Escherichia coli TOP10 strains were purchased from Thermo Fisher Scientific (Invitrogen); Agrobacterium strain GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0034] Chlorophyll content determination: Select uniformly germinating seeds in good physiological condition, sterilize them, and prepare three biological replicates. Sow the seeds on 1 / 2 MS medium. Take a certain weight or number (approximately 20 seedlings) of cotyledons that are in good growth and uniform development, absorb excess water, place them in 1.5 mL centrifuge tubes, add 80% acetone, mix well, ensuring that the cotyledons in the centrifuge tubes are completely immersed in 80% acetone, and place overnight at 4°C in the dark. After the cotyledon leaves have completely decolorized and turned white, centrifuge at 5000 rpm for 5 min at 4°C, and determine the chlorophyll content using a Nano-drop UV spectrophotometer. Use 80% acetone as a blank control to detect the absorbance (A) of the sample extract at wavelengths of 645 nm and 663 nm. Calculate the chlorophyll content according to the following formula:

[0035] Total chlorophyll (ng / μL) = A645 × 20.29 + A663 × 8.04

[0036] Chlorophyll content (mg / g) = Total chlorophyll (ng / μL) × V80% acetone (μL) / Fresh weight of leaf (g)

[0037] Real-time PCR using Hieff ® qPCR was performed using SYBR Green Master Mix (High Rox Plus) (Yeasen) on a QuantStudio 3 (Applied Biosystems) Real-time PCR instrument. The 20 μl reaction mixture consisted of: 10 μl Hieff ® qPCR was performed using SYBR Green Master Mix (High Rox Plus), 0.4 μl each of forward and reverse primers (10 μm), 7.2 μl of ultrapure water, and 2 μl of cDNA. The two-step PCR program was: 95 °C for 5 s, 60 °C for 25 s, 40 cycles. The Melt curve stage was: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. ACTIN2 was used as an internal control gene.

[0038] Melting curve analysis: A single peak in the melting curve indicates good reaction specificity, allowing for quantitative analysis. Relative quantification was used to represent differences in gene changes, using 2... -ΔΔCt This represents the expression level of the target gene in the experimental group relative to the control group. Where ΔCt = (Ct...) 目的基因 -Ct actin Experimental group - (Ct) 目的基因 -Ct actin Control group.

[0039] Example 1

[0040] To elucidate the molecular mechanism by which Arabidopsis thaliana SIG6 regulates cotyledon chloroplast development, 50,000 wild-type Arabidopsis thaliana SIG6-deficient mutant seeds (hereinafter referred to as single mutant SIG6) were induced to mutate using ethyl methanesulfonate (EMS). In the M2 population, suppressors of green cotyledon development were screened, resulting in four lines. Genetic analysis and map-based cloning revealed that the suppression of SIG6 cotyledon yellowing in this line was due to a cytosine (C) to thymine (T) change at position 389 of the AT1G63680 gene, resulting in a change of serine (S) to phenylalanine (F) at position 130 of the encoded protein (named SIG6 atmure). S130F To investigate the extent to which this mutation restores etiolated cotyledons in the sig6 mutant, the sig6atmure site was analyzed. S130F The basic characteristics of repressors are analyzed, including: Figure 1 As shown, the repressor sig6 atmure, which grows for 5 days under normal conditions, S130FThe cotyledons of the wild type are green, while those of the single mutant sig6 are yellow. Its chlorophyll content is also not significantly different from the wild type, but is significantly higher than that of the single mutant sig6.

[0041] like Figure 2 As shown, the repressor sig6 atmure S130F The maximum photochemical activity of the mid-light system II (F v / F m The levels were also restored, showing no significant difference from the wild type, and were significantly higher than the single mutant sig6.

[0042] like Figure 3 As shown, mutations in sig6 lead to transcriptional abnormalities in chloroplast genome-encoded genes, including PEP-dependent genes ( Figure 3 a) Downregulation of transcriptional genes such as PsaB, PsbA, and rbcL; while NEP-dependent genes ( Figure 3 b) Upregulation of rpoC1 and clpP transcription. S130F The mutation can restore the transcription of these PEP-dependent and NEP-dependent genes in the sig6 mutant to the wild-type level.

[0043] To verify that the cotyledon yellowing in the sig6 repressor is caused by a point mutation at amino acid 130 of AtMurE, a vector containing the AtMurE genome sequence with the S→F mutation at position 389, driven by the AtMurE promoter itself, was constructed and introduced into the sig6 mutant. The results are as follows: Figure 4 As shown, the sig6 mutant expresses AtMurE containing the mutation site. S130F Both lines can complement each other's cotyledon yellowing phenotype. The chlorophyll content in both Line 1 and Line 2 lines recovered to wild-type levels.

[0044] Furthermore, genetic analysis and whole-genome sequencing revealed that the remaining three repressors were all caused by single-base mutations in the MurE gene, leading to amino acid changes. Mutation types include... Figure 5 As shown, the mutation at position 265, from G to A, causes the amino acid at position 89 of the encoded protein to change from aspartic acid (D) to asparagine (N) (named sig6atmure). FD89N The mutation of G to A at position 316 causes the encoded protein to change from aspartic acid (D) to asparagine (N) at position 106 (named sig6 atmure). F106N The mutation of G to A at position 418 causes the amino acid at position 140 of the encoded protein to change from glycine (G) to arginine (R) (named sig6 atmurer). G140RN ).

[0045] Mutations at these sites all restored the cotyledon yellowing phenotype of the sig6 mutant. Analysis of their chlorophyll content revealed that sig6 atmure FD106N and sig6 atmure S130F Similarly, there was no significant difference from the wild type. And sig6 atmure FD89N with sig6 atmure G140RN Although the chlorophyll content of the phase was slightly lower than that of the wild type, it was significantly higher than that of the sig6 mutant (e.g., Figure 6 ).

[0046] Example 2

[0047] The MurE protein expressing a single amino acid alteration can also restore the etiolated cotyledon phenotype of the sig6 mutant.

[0048] The sig6 cotyledon yellowing suppressors obtained by chemical mutagenesis screening were all caused by mutations in the MurE gene, indicating that this gene plays an important role in regulating chloroplast development in sig6 cotyledons. To further confirm that other sites on the MurE gene also have this function, three versions of the MurE genome sequence containing mutation sites were synthesized, with nucleotide changes at positions 97, 125, and 126, resulting in the amino acids encoded at these sites being mutated to alanine (A). Then, the MurE gene promoter sequence itself was analyzed as follows: Figure 7 a and the three mutated versions of the MurE genome sequence (e.g. Figure 7 b) The mixture was constructed together into the expression vector pGWB1, and Agrobacterium-mediated transformation was performed on Arabidopsis thaliana sig6 mutant plants. T0 generation seeds were harvested. T0 generation seeds were screened on Invitrogen-resistant medium. After transplanting and maturing, leaf RNA was extracted from the plants, and the reverse-transcribed cDNA was sequenced for identification. The harvested T1 generation seeds were cultured together with wild-type and sig6 mutant plants, and phenotypes were observed. Results are as follows: Figure 8 The expression atmure was shown to be overexpressed. I125A It can completely restore the cotyledon phenotype and chlorophyll content of the sig6 mutant to wild-type levels. Overexpression of atmure... Y97A and atmure D126A It can basically restore the cotyledon yellowing and chlorophyll content of the sig6 mutant.

[0049] In summary, it was found that independent amino acid changes at seven sites at the N-terminus of the Arabidopsis AtMurE protein could restore the development of cotyledon chloroplasts in the sig6 mutant. Figure 9 As shown, these 7 sites are amino acids at positions 89, 97, 106, 125, 126, 130, and 140.

[0050] This invention can utilize the obtained mutation sites as tools for molecular marker-assisted breeding or genetic improvement. This includes using these sites as functional markers to screen or identify germplasm resources with superior chloroplast development traits; it can also introduce corresponding mutations into important crops through gene editing (such as CRISPR / Cas9) to improve cotyledon greening ability in the seedling stage and subsequent photosynthetic efficiency, thereby increasing crop yield or biomass.

[0051] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A mutant Arabidopsis protein that promotes chloroplast development, characterized in that, The Arabidopsis mutant protein is the AtMurE protein in the wild-type Arabidopsis Sig6 factor deletion mutant with mutations at positions 89, 97, 106, 125, 126, 130, or 140.

2. The Arabidopsis mutant protein for promoting chloroplast development according to claim 1, characterized in that, The Arabidopsis mutant protein is the AtMurE protein of the wild-type Arabidopsis Sig6 factor deletion mutant, which has a mutation at position 89 from aspartic acid to asparagine, at position 97 from lysine to alanine, at position 106 from aspartic acid to asparagine, at position 125 from isoleucine to alanine, at position 126 from aspartic acid to alanine, at position 130 from serine to phenylalanine, or at position 140 from glycine to arginine.

3. The Arabidopsis mutant protein for promoting chloroplast development according to claim 1, characterized in that, The amino acid sequence of the Arabidopsis mutant protein is selected from one of SEQ ID NO.1-7.

4. An Arabidopsis mutant gene that promotes chloroplast development, characterized in that, The Arabidopsis mutant gene is the MurE gene on the wild-type Arabidopsis Sig6 factor deletion mutant, with mutations at positions 265, 290 and 291, 316, 373 and 374, 377, 389, or 418.

5. The Arabidopsis mutant gene for promoting chloroplast development according to claim 4, characterized in that, The Arabidopsis mutant gene is the MurE gene of the wild-type Arabidopsis Sig6 factor deletion mutant, which has a mutation from G to A at position 265, a mutation from TA to GC at positions 289 and 290, a mutation from G to A at position 316, a mutation from AT to GC at positions 373 and 374, a mutation from A to C at position 377, a mutation from C to T at position 389, or a mutation from G to A at position 418.

6. The Arabidopsis mutant gene for promoting chloroplast development according to claim 4, characterized in that, The nucleotide sequence of the Arabidopsis mutant gene is selected from one of SEQ ID NO. 8-14.

7. An expression cassette comprising the Arabidopsis mutant gene as described in any one of claims 4-6.

8. A recombinant vector or recombinant cell comprising the Arabidopsis mutant gene as described in any one of claims 4-6.

9. The use of the Arabidopsis mutant protein of claims 1-3, the Arabidopsis mutant gene of any one of claims 4-6, the expression cassette of claim 7, or the recombinant vector or recombinant cell of claim 8 in promoting chloroplast development.

10. The application according to claim 9, characterized in that, The Arabidopsis mutant protein, Arabidopsis mutant gene, expression cassette, recombinant vector, or recombinant cell were introduced into wild-type Arabidopsis Sig6 factor-deficient mutants to restore chlorophyll content and solve the problem of cotyledon yellowing in wild-type Arabidopsis Sig6 factor-deficient mutants.

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