A promoter for regulating the premature senescence of Chinese cabbage leaves and its application

Abstract

The present invention discloses a promoter for regulating the premature aging trait of Chinese cabbage leaves and its application. The premature aging gene of the present invention is a newly identified control gene, and its promoter is responsible for regulating the occurrence of premature aging traits, providing a new method for studying the molecular mechanism of premature aging and breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant trait regulation, and in particular to a promoter that can be used to regulate plant phenotypes, a regulatory gene thereof, and applications thereof. Background Art

[0002] Chinese cabbage, a Brassica species in the genus Brassica of the family Cruciferae, is an important vegetable crop whose leaves are its main product organ. Leaf senescence is a key indicator of Chinese cabbage yield and quality. Leaf senescence is the final stage of leaf development, a post-mitotic senescence process that transitions from nutrient assimilation to nutrient transport and is crucial to plant survival.

[0003] Leaf senescence is regulated by a complex genetic program. The applicant previously established a rich genetic resource library of Chinese cabbage mutants using EMS chemical mutagenesis of germinating seeds of the DH line 'FT'. This study identified a leaf premature senescence mutant (lpsm). Based on phenotypic and genetic characterization of the mutant, the candidate mutant gene was positionally cloned using forward genetics and its expression profile was analyzed. Summary of the Invention

[0004] The purpose of the present invention is to provide a promoter for regulating the premature senescence trait of Chinese cabbage leaves, a regulatory gene thereof and an application thereof, especially an application for regulating the premature senescence of plant leaves.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A promoter for regulating the early senescence trait of Chinese cabbage leaves, wherein the promoter is shown as SEQ ID NO: 1, 2, 3 or 4.

[0007] In some embodiments, the promoter is as shown in SEQ ID NO: 2 or 4.

[0008] In some embodiments, the promoter regulates the expression of BrATG5, BraA10g022760.3.5C, in Chinese cabbage.

[0009] In some embodiments, the promoter is as shown in SEQ ID NO: 2. In some specific embodiments, it downregulates the expression of BraA10g022760.3.5C in Chinese cabbage leaves.

[0010] In some embodiments, the promoter is as shown in SEQ ID NO: 4. In some specific embodiments, it upregulates the expression of BraA10g022760.3.5C in Chinese cabbage leaves.

[0011] A gene construct comprises any one of the aforementioned promoters for regulating the early senescence trait of Chinese cabbage leaves.

[0012] In some embodiments, the genetic construct further comprises BraA10g022760.3.5C.

[0013] In some embodiments, the BraA10g022760.3.5C is the BrATG5 gene. In some specific embodiments, it is the CDS (or ORF) region of the BrATG5 gene.

[0014] A method for regulating plant leaf senescence comprises expressing any of the aforementioned gene constructs in a plant, wherein the expression pathway comprises transformation or transfection.

[0015] In some embodiments, a plant is transformed or transfected with a gene construct containing the promoter shown in SEQ ID NO: 2. In some specific embodiments, premature senescence of plant leaves is induced.

[0016] In some embodiments, a plant is transformed or transfected with a gene construct containing the promoter shown in SEQ ID NO: 4. In some embodiments, premature leaf senescence in a plant is prevented, inhibited, or reversed.

[0017] In some embodiments, the plant is Chinese cabbage.

[0018] In some embodiments, plant seeds or plant leaves are transformed or transfected.

[0019] In some embodiments, the transformation or transfection is performed before the progeria trait appears.

[0020] In some embodiments, transformation or transfection is performed on day 1-7 after the onset of progeria traits. In some specific embodiments, transformation or transfection is performed on day 1, 2, 3, 4, 5, 6, or 7. Preferably, transformation or transfection is performed on day 1, 2, or 3. More preferably, transformation or transfection is performed on day 1 to achieve reversal of progeria traits.

[0021] In some embodiments, methods for transformation or transfection in plants are routine methods in the art.

[0022] In some embodiments, transformation or transfection is achieved by Agrobacterium transformation, electroporation, chemical induction, or physical delivery.

[0023] A method for constructing transgenic plants, wherein a base mutation occurs in the promoter region of the BrATG5 gene in a premature aging mutant 51 bp upstream of the start codon through transgenic technology.

[0024] In some embodiments, the mutation is specifically a T→C mutation.

[0025] In some embodiments, the mutation is specifically a T→G mutation.

[0026] In some embodiments, the mutation is specifically a C→G mutation.

[0027] In some embodiments, the mutation is specifically an A→G mutation.

[0028] In some embodiments, the transgenic plants exhibit suppression or reversal of premature aging traits.

[0029] In some embodiments, the transgenic plant does not exhibit premature senescence traits.

[0030] In some embodiments, the plant is Chinese cabbage.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] First, this is the first time that BrATG5 has been cloned and proposed to be related to premature leaf senescence in Chinese cabbage, which is conducive to further research on the molecular mechanism of premature leaf senescence.

[0033] Second, the discovery of the promoter element that regulates the expression of the BrATG5 gene provides feasibility for constructing a premature aging model of Chinese cabbage and cultivating anti-aging or reverse aging plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 1 shows the morphological characteristics of the wild type 'FT' and the mutant lpsm. (1a) Phenotypes of 'FT' and lpsm at the seedling stage (1b) Phenotypes of 'FT' and lpsm at the rosette stage (1c) Phenotype of 'FT' at the heading stage (1d) Phenotype of lpsm at the heading stage.

[0035] Figure 2 Growth characteristics of wild-type 'FT' and the lpsm mutant at the seedling stage. (2a, 2b) Changes in length and width of the third true leaf (2c) Changes in plant width; the horizontal axis indicates the number of days measured.

[0036] Figure 3 Agronomic traits of the wild-type 'FT' and the mutant lpsm at the bulb stage. (3a) Head diameter (3b) Head diameter (3c) Head weight (3d) Head index (ratio of head diameter to head diameter). Data in the figures were analyzed by independent-samples T-test; different lowercase letters indicate significant differences at P < 0.05.

[0037] Figure 4 The results of photosynthetic pigment content determination in the wild-type 'FT' and the mutant lpsm are shown. From left to right, the figures show the contents of Chla, Chlb, and Car. Data were analyzed using an independent-samples T-test. Different lowercase letters indicate significant differences at P < 0.05. DETAILED DESCRIPTION

[0038] The present invention will be described in detail below with reference to examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available.

[0040] Example 1: Phenotypic identification of premature leaf senescence mutants

[0041] A Chinese cabbage mutant library was created by treating germinating seeds of the DH line 'FT' with 0.8% EMS solution. A stable early leaf senescence mutant, lpsm, was identified from the mutant library and used as the experimental material for this study. All materials were cultivated at the Vegetable Genetics and Breeding Experimental Base of Shenyang Agricultural University.

[0042] Wild-type 'FT' and the leaf premature senescence mutant lpsm seedlings were selected for routine growth parameter and photosynthesis measurements, including:

[0043] 1) At the seedling stage, select five wild-type 'FT' and leaf senescence mutant lpsm seedlings with consistent growth, and measure the length and width of the third true leaf and the plant width with a ruler.

[0044] 2) During the mature stage of Chinese cabbage heads, five plants of the wild-type 'FT' and the premature senescence mutant (lpsm) with identical growth were selected. The heads were weighed, their transverse and longitudinal diameters measured, and the head index was calculated. 3) During the seedling stage, three plants of the wild-type 'FT' and the mutant (lpsm) with identical growth were selected, and the outer leaves of the plants were used for photosynthetic pigment measurement. 4) On a clear morning, a Li-6800 photosynthesis meter (LI-COR, America) was used to measure the photosynthetic characteristics of the outer leaves of ten wild-type 'FT' and premature senescence mutant (lpsm) seedlings. After turning on the meter, a self-check was performed. After the photosynthesis readings stabilized, the photosynthetic parameters were recorded. Three replicates were set for each measurement. The net photosynthetic rate is the apparent photosynthesis measured without taking into account mitochondrial respiration and photorespiration in the plant leaves. The transpiration rate is the amount of water transpired per unit leaf area over a given period of time. Stomatal conductance indicates the degree of stomatal opening.

[0045] Experimental results:

[0046] Compared with the wild type 'FT', the leaf premature senescence mutant lpsm begins to show leaf premature senescence at the seedling stage, with the outer whorl of leaves gradually chlorosing from the tip and edge to the base of the leaf. Then, at the rosette stage, the number of leaves with premature senescence phenotype increases, and the symptoms become more severe, showing whitening. Finally, at the head stage, the outer whorl of leaves shows premature senescence as a whole, and the symptoms become more severe (see Figure 1 ).

[0047] During the measurement of plant growth characteristics at the seedling stage, the length and width of the third true leaf of the wild-type 'FT' and the plant width all showed a gradual increase; the length and width of the third true leaf of the premature leaf senescence mutant lpsm showed a gradual increase at first and then a gradual decrease; the plant width showed a gradual increase at first, then a temporary decrease with the premature senescence of the largest leaf of the plant, and then a gradual increase with the growth of the leaf. After measurement on the 10th day, the plant width of both the wild-type 'FT' and the premature leaf senescence mutant lpsm showed a faster growth rate; the third true leaf of the premature leaf senescence mutant lpsm began to show premature senescence after measurement on the 7th day, and the leaf fell off on the 19th day (see Figure 2 ).

[0048] During the cabbage heading period, the leaf head yield was measured, and the agronomic traits related to the leaf head diameter, transverse diameter and weight were measured, and the leaf head index was calculated. The results are as follows: Figure 3 The analysis showed that compared with the wild type 'FT', the leaf bulb longitudinal diameter of the leaf early senescence mutant lpsm had no significant difference, but the leaf bulb transverse diameter was significantly smaller, the leaf bulb weight was significantly lighter, the yield decreased, and the leaf bulb index decreased significantly with the decrease in leaf bulb transverse diameter.

[0049] The outer leaves of wild-type 'FT' and mutant lpsm plants at the seedling stage were taken for photosynthetic pigment extraction. The experimental results showed that the content of Chla, Chlb and Car in the outer leaves of the premature leaf senescence mutant lpsm were significantly lower than those of wild-type 'FT' (see Figure 4 ), indicating that chlorophyll degradation may have occurred in the outer leaves of the leaf premature senescence mutant lpsm, biological macromolecules such as proteins began to degrade, and chloroplasts began to degenerate, resulting in a decrease in the content of photosynthetic pigments.

[0050] Compared to the wild-type 'FT', the transpiration rate and net photosynthetic rate of the outer leaves of the premature leaf senescence mutant lpsm were significantly decreased, the stomatal conductance was significantly reduced, and the intercellular CO2 concentration was significantly increased (Table 1). The increased intercellular CO2 concentration may be due to the reduced photosynthesis in the outer leaves of the premature leaf senescence mutant lpsm, which also decreases the stomatal conductance and transpiration rate, weakening the ability to fix CO2, reducing CO2 entry into the cells, and increasing the intercellular CO2 concentration.

[0051] Table 1. Photosynthesis parameters of wild type 'FT' and mutant lpsm

[0052]

[0053] Example 2: Analysis of genetic characteristics of premature leaf senescence mutants

[0054] F1 seeds were obtained by crossing the wild-type 'FT' with the mutant lpsm. Plant phenotypes were analyzed and analyzed at the seedling stage after sowing. During the reproductive growth stage, the F1 lines were selfed to obtain F2 seeds, which were then sown to obtain an F2 population. Plant phenotypes were observed and trait segregation was analyzed. The F1 generation was then backcrossed with the wild-type 'FT' and the mutant lpsm to obtain the BC1 generation. Plant phenotypes were also observed and trait segregation was analyzed.

[0055] Experimental results: When the leaf premature senescence mutant lpsm was crossed with the wild-type 'FT' plant, all F1 plants exhibited a wild-type phenotype. When the F1 plants were backcrossed with the wild-type 'FT' parent, all BC1 plants exhibited a wild-type phenotype. When the F1 plants were backcrossed with the leaf premature senescence mutant parent, lpsm, 51 plants exhibited a wild-type phenotype and 45 plants exhibited a leaf premature senescence phenotype. The segregation ratio of the wild-type phenotype to the leaf premature senescence mutant phenotype was 1.12:1, close to the theoretical ratio of 1:1. When the F1 plants were selfed, 309 F2 plants exhibited a wild-type phenotype and 99 plants exhibited a leaf premature senescence phenotype. The segregation ratio of the wild-type phenotype to the leaf premature senescence mutant phenotype was 3.12:1, close to the theoretical ratio of 3:1. Genetic analysis showed that the leaf premature senescence trait of the lpsm mutant is controlled by a recessive nuclear gene.

[0056] Example 3: Localization of genes controlling premature leaf senescence mutants

[0057] 1.1 Experimental methods

[0058] MutMap Positioning

[0059] A modified MutMap method was used to screen candidate genes for the premature leaf senescence mutant lpsm. Leaves from 50 F2 plants with premature leaf senescence phenotypes were selected for DNA pool construction (W-pool). DNA from leaves of the two parental lines, 'FT' and lpsm, was extracted using the spin-column DNAsecurePlant Kit (TIANGEN, China). Sequencing libraries were prepared using Illumina NovaSeq procedures, including DNA end repair, 3'-end A addition, and 5'-end library-specific tags, for high-throughput sequencing. The raw data were initially filtered to remove adapters and low-quality reads to generate high-quality sequence data. Sequence information was then compiled and statistically analyzed against the reference genome (Brara Chiifu V3.5 genome). The high-quality data were aligned to the reference genome using the bwa mem program, and the alignment information was statistically analyzed. Single-nucleotide polymorphisms (SNPs) were detected using GATK software. SNPs were filtered using criteria such as removing deletions, and the SNP distribution map was constructed. InDel (small insertion and deletion) mutation sites were detected and filtered, and a distribution map of the mutation sites was drawn. ANNOVAR software was used to annotate SNPs and INDEL sites. To identify candidate regions associated with trait genes, population SNPs were further filtered by screening for SNP sites with EMS-induced mutation characteristics. The SNP-index was calculated and a chromosomal distribution map of the SNP-index was drawn. The SNP-index value showed a positive correlation with the degree of association with the target trait. Chromosomal distribution regions with a SNP-index greater than 95% were selected as candidate trait-associated regions. SNPs in these candidate regions were screened and the SNP sites were annotated. Gene Ontology (GO) annotations were performed for the corresponding candidate genes.

[0060] KASP genotyping

[0061] KASP technology was used to genotype and verify the SNP loci of candidate genes. 187 F2 plants with premature leaf senescence mutations were selected, with the wild-type parent 'FT', the mutant parent 'lpsm', and F1 heterozygous plants used as controls. Fresh leaf material was extracted, and leaf DNA was extracted using a modified CTAB method. DNA samples were analyzed using a microplate reader. Specific primers were designed based on the reference gene sequence containing the SNP loci, using the Chinese cabbage reference genome database (Brara Chiifu V 3.5 genome). Preliminary experiments were conducted to verify the quality of the specific primers using DNA from the wild-type parent 'FT', the mutant parent 'lpsm', and F1 heterozygous plants. Genotyping of DNA from F2 plants with premature leaf senescence mutations was then performed.

[0062] 1.2 Experimental Results

[0063] High-throughput sequencing was performed on a pool of wild-type parent 'FT', the parent with premature leaf senescence mutant phenotype lpsm, and F2 plants with premature leaf senescence mutant phenotype. The raw data were filtered for adapter-containing and low-quality reads to generate high-quality data. The results showed that the percentage of high-quality reads to the total raw reads for the wild-type parent 'FT', the mutant phenotype parent 'lpsm', and the F2 mutant phenotype plants was greater than 95%, and the percentage of high-quality read bases to the total raw bases was greater than 90%. The filtered high-quality data were then aligned to the reference genome (Brara Chiifu V3.5 genome) and analyzed. The results showed that the percentage of reads from the mutant phenotype parent 'lpsm' that mapped to the reference genome (i.e., the alignment rate) was 98.05%, while the alignment rates for the F2 mutant phenotype plants and the wild-type parent 'FT' were both greater than 99%.

[0064] Single nucleotide polymorphism (SNP) sites were detected, filtered according to relevant standards, and annotated. To identify candidate regions associated with trait genes, SNP polymorphic markers were screened and the SNP-index of each polymorphic site was calculated. The population SNPs were further filtered according to the standards to obtain the SNP-index results and draw a chromosome distribution map of the SNP-index. On chromosome A10, candidate regions with SNP-index exceeding 95% were selected and aligned to the reference genome. Combined with functional annotation analysis, a total of 12 candidate SNP sites were screened, including SNP14363364, SNP14365863, SNP15905282, and SNP17014295. Among them, SNP14363364 and SNP14365863 were both located in the exon region of BraA10g0196403.5C, and BraA10g0196403.5C was determined. There are 11 candidate genes in total, including 0g021620.3.5C, BraA10g021720.3.5C, BraA10g022460.3.5C, BraA10g022760.3.5C, BraA10g022850.3.5C, BraA10g023270.3.5C, BraA10g023750.3.5C, BraA10g024000.3.5C, BraA10g025260.3.5C, and BraA10g0252803.5C.

[0065] To identify candidate genes, KASP genotyping was performed on 187 plants from the F2 population with an early leaf senescence phenotype, using the parents and F1 plants as references. The candidate SNPs, including SNP14363364, SNP14365863, SNP15905282, and SNP17014295, were validated. Theoretically, the candidate SNPs co-segregated with the early leaf senescence mutant phenotype in the 187 F2 plants, and the genotype of the plants with the early leaf senescence mutant phenotype in the F2 population was all T:T. The results showed that SNP15905282 in BraA10g022760.3.5C had a T:T genotype in all F2 plants with the early leaf senescence mutant phenotype. However, T:T and C:T genotypes were detected in all 11 other candidate SNPs, including SNP14363364 and SNP14365863, in F2 plants with the early leaf senescence mutant phenotype. These results suggest that candidate SNP15905282 in BraA10g022760.3.5C cosegregates with the early leaf senescence mutant phenotype, while the other candidate SNPs do not cosegregate with the early leaf senescence mutant phenotype. Therefore, the BraA10g022760.3.5C gene, where SNP15905282 resides, was hypothesized to be a candidate gene for the early leaf senescence mutant lpsm. Based on homologous gene alignment, BraA10g022760.3.5C was named BrATG5.

[0066] Example 4: BrATG5 gene cloning and analysis

[0067] The full-length gene sequence, CDS sequence, and promoter region of the candidate gene BrATG5 from the wild-type 'FT' and the premature leaf senescence mutant lpsm were cloned and aligned. The cloning results showed no differences in the full-length gene sequence or CDS sequence between the wild-type 'FT' and the premature leaf senescence mutant lpsm. Furthermore, the promoter region of the BrATG5 gene in the premature leaf senescence mutant lpsm exhibited a C→T mutation 51 bp upstream of the start codon compared to the wild-type 'FT', consistent with the MutMap mapping results.

[0068] >pBrATG5-wt promoter nucleotide sequence

[0069]

[0070] >pBrATG5CTT promoter nucleotide sequence

[0071]

[0072] Note: The bold underlined part indicates the mutation position relative to the wild-type promoter.

[0073] Example 5: Study on BrATG5 gene expression regulation

[0074] New promoter sequences were designed, and the promoter sequences of BrATG5, such as SEQ ID NOs: 1-4, were cloned to construct pBWA(V)HG-pBrATG5-GUS gene expression vectors (Groups A, B, C, and D, respectively). The gene expression pattern of BrATG5 was further verified. Wild-type Arabidopsis plants were infected multiple times using the floral dip method. Arabidopsis seeds were harvested and sown. Surviving Arabidopsis seedlings were transplanted, and Arabidopsis genomic DNA was extracted. PCR testing was performed to screen for positive Arabidopsis plants (Groups A, B, C, and D, respectively).

[0075] >pBrATG5CTA promoter nucleotide sequence

[0076]

[0077] >pBrATG5CTG promoter nucleotide sequence

[0078]

[0079] After identifying the positive Arabidopsis plants, GUS histochemical staining was performed on the positive Arabidopsis plants to verify the activity of the BrATG5 promoter. The GUS staining results showed that:

[0080] 1) In groups A, B, C, and D, the roots, stems, leaves, and flowers of Arabidopsis plants all stained blue, with the leaves showing the strongest staining. This indicates that the GUS gene is expressed in the roots, stems, leaves, and flowers of the positive Arabidopsis plants, with the strongest expression in the leaves. 2) In groups A and C, there was no significant difference in staining levels among the organs; in groups B and D, the leaf staining was significantly weaker and stronger than in groups A and C, respectively. This suggests that different promoter sequences have distinct gene regulatory functions.

[0081] The promoter sequence of BrATG5CTG as shown in SEQ ID NO: 4 was cloned to construct the pBWA(V)HS-pBrATG5CTG-BrATG5 gene expression vector, which was then transformed into the premature senescence mutant lpsm plant.

[0082] The results showed that plants transfected before the onset of premature aging traits did not show premature aging traits as determined in Example 1 throughout their growth. Plants transfected one day after the onset of premature aging traits showed significant improvement in premature aging traits and returned to normal phenotype seven days after transfection. Plants transfected seven days after the onset of premature aging traits showed improvement in premature aging traits but did not fully return to normal phenotype within the 21-day experimental period. This indicates that the BrATG5 gene is one of the key genes controlling premature aging traits in Chinese cabbage, and that premature aging traits caused by the C to T mutation in the promoter are reversible in the early stages of premature aging.

[0083] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A promoter for regulating the premature senescence trait of Chinese cabbage leaves, characterized in that: The promoter is shown in SEQ ID NO:

4. 2 . The promoter according to claim 1 , which regulates the expression of the BrATG5 gene in Chinese cabbage, wherein the BrATG5 gene is BraA10g022760.3.5C. 3 . The promoter according to claim 2 , as shown in SEQ ID NO: 4, which upregulates the expression of the BrATG5 gene in Chinese cabbage leaves.

4. A gene construct comprising the promoter for regulating the early senescence trait of Chinese cabbage leaves according to any one of claims 1 to 3. The gene construct according to claim 4 , further comprising a BrATG5 gene.

6. A method for regulating plant leaf senescence, characterized in that: The gene construct according to claim 4 or 5 is expressed in a plant, wherein the expression pathway comprises transformation or transfection; and the plant is Chinese cabbage.

7. The method according to claim 6, wherein a gene construct containing the promoter shown in SEQ ID NO: 4 is transformed or transfected into a plant to prevent, inhibit or reverse premature senescence of plant leaves.

Images