Promoter for regulating premature senility character of Chinese cabbage leaves and application thereof

Abstract

The invention discloses a promoter for regulating premature senility characters of Chinese cabbage leaves and application of the promoter. The premature senility gene is a newly identified control gene, a promoter of the premature senility gene is responsible for regulating and controlling premature senility characters, and a new method is provided for premature senility molecular mechanism research and breeding.

Description

Technical Field

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

[0002] Chinese cabbage belongs to the Brassica rapa species of the Brassica family. It is an important vegetable crop with leaves as its product organ. The leaves are an important indicator of the yield and quality of Chinese cabbage. Leaf senescence is the last stage of leaf development, which belongs to post-mitotic senescence. It is a functional transformation process from nutrient assimilation to nutrient transport, which is crucial to the survival of plants.

[0003] Leaf senescence is regulated by complex genetic programs. The applicant previously used EMS chemical mutagenesis to germinate seeds of the Chinese cabbage DH line 'FT' to create a Chinese cabbage mutant library with rich genetic resources. This experiment screened out a leaf premature senescence mutant lpsm (leaf premature senescence mutant). Based on the phenotypic and genetic characteristics of the mutant traits, the candidate mutant gene was cloned by forward genetics and its expression characteristics were 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 regulating 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 in 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 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 a promoter as 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 a promoter as 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, the transformation or transfection is performed on day 1-7 of the onset of the progeria trait. In some specific embodiments, the transformation or transfection is performed on day 1, 2, 3, 4, 5, 6, or 7. Preferably, the transformation or transfection is performed on day 1, 2, or 3. More preferably, the transformation or transfection is performed on day 1 to achieve reversal of the progeria trait.

[0021] In some embodiments, the 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, using transgenic technology to cause a base mutation in the promoter region of the BrATG5 gene in an early senescence mutant at 51 bp upstream of the start codon.

[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 senescence 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 Chinese cabbage premature aging model and cultivating anti-aging or reversing aging plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are 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 The growth characteristics of wild type 'FT' and mutant lpsm at seedling stage. (2a, 2b) Changes in length and width of the third true leaf (2c) Changes in plant width; the horizontal axis in the figure indicates the number of days measured.

[0036] Figure 3 The agronomic traits of the wild type 'FT' and the mutant lpsm at the ball stage. (3a) Leaf ball longitudinal diameter (3b) Leaf ball transverse diameter (3c) Leaf ball weight (3d) Leaf ball index (ratio of leaf ball longitudinal diameter to transverse diameter); The data in the figure were subjected to independent sample T test, and different lowercase letters indicate significant differences at P < 0.05.

[0037] Figure 4 The results of the determination of photosynthetic pigment content in wild type 'FT' and mutant lpsm. The figures show the contents of Chla, Chlb and Car from left to right; the data in the figure were subjected to independent sample T test, and different lowercase letters indicate significant differences with P<0.05. DETAILED DESCRIPTION

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

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

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

[0041] A Chinese cabbage mutant library was created by treating the germinating seeds of the Chinese cabbage DH line 'FT' with 0.8% EMS solution. A stable early leaf senescence mutant lpsm was screened and identified in the mutant library as the experimental material of this study. All the materials used were cultivated in the vegetable genetic breeding experimental base of Shenyang Agricultural University.

[0042] Wild-type 'FT' and leaf senescence mutant lpsm seedlings were selected for routine determination of growth parameters and photosynthesis. Specifically:

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

[0044] 2) During the cabbage head maturity period, 5 wild-type 'FT' and early-senescence mutant lpsm with the same growth were selected, and the head was weighed and the horizontal and vertical diameters of the head were measured to calculate the head index. 3) During the seedling stage, 3 plants with the same growth of wild-type 'FT' and mutant lpsm were selected, and the outer leaves of the plants were taken for photosynthetic pigment measurement. 4) On a sunny morning, Li-6800 photosynthetic instrument (LI-COR, America) was selected to measure the photosynthetic characteristics of the outer leaves of 10 wild-type 'FT' and early-senescence mutant lpsm in the seedling stage. After the instrument was turned on, the instrument self-checked, and the photosynthetic parameters were recorded after the photosynthesis index readings stabilized. Three repetitions were set for each measurement. The net photosynthetic rate is the apparent photosynthesis measured without considering the mitochondrial respiration and photorespiration of the plant leaves. The transpiration rate is the amount of water transpired per unit leaf area within a certain period of time. Stomatal conductance can show the degree of stomatal opening.

[0045] Experimental results:

[0046] Compared with the wild type 'FT', the leaf senescence mutant lpsm begins to senescent early in the seedling stage, with the outer whorl of leaves gradually chlorotic from the tip and edge to the base; then the number of leaves with senescent phenotype increases in the rosette stage, and the symptoms worsen and become white; finally, the outer whorl of leaves shows overall senescence at the head stage, and the symptoms worsen (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 leaf premature senescence mutant lpsm showed a gradual increase first and then a gradual decrease; the plant width showed a gradual increase 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 the measurement on the 10th day, the plant width of the wild-type 'FT' and the leaf premature senescence mutant lpsm showed a faster growth rate; the third true leaf of the leaf premature senescence mutant lpsm began to show premature senescence after the measurement on the 7th day, and the leaf fell off on the 19th day (see Figure 2 ).

[0048] During the cabbage heading period, the cabbage head yield was measured, the longitudinal diameter, transverse diameter and weight of the cabbage head were measured, and the cabbage head index was calculated. Figure 3 The analysis showed that compared with the wild type 'FT', the leaf head longitudinal diameter of the leaf premature senescence mutant lpsm was not significantly different, the leaf head transverse diameter was significantly smaller, the leaf head weight was significantly lighter, the yield was reduced, and the leaf head index was significantly reduced as the leaf head transverse diameter decreased.

[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 leaf premature 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 with the wild type 'FT', the transpiration rate and net photosynthetic rate of the outer leaves of the leaf premature senescence mutant lpsm were significantly decreased, the stomatal conductance was significantly reduced, and the intercellular CO 2 The concentration increased significantly (see Table 1). 2 The increase in concentration may be due to the weakening of photosynthesis in the outer leaves of the leaf senescence mutant lpsm, and the decrease in stomatal conductance and transpiration rate, which in turn leads to the fixation of CO 2 The ability to enter the cell is weakened. 2 Decreased intercellular CO 2 Increased concentration.

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

[0052]

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

[0054] The wild type 'FT' was crossed with the mutant lpsm to obtain F 1 Seeds, after sowing, plant phenotypes were identified and counted at the seedling stage. During the reproductive growth stage, F 1 Self-fertilization to obtain F 2 Seeds, then sow to obtain F 2 The phenotypes of the plants were observed and the separation of the traits was statistically analyzed. 1 The wild type 'FT' was backcrossed with the lpsm mutant to obtain BC 1 The phenotypes of the plants were observed and the segregation of the traits was statistically analyzed.

[0055] Experimental results: The leaf premature senescence mutant lpsm was crossed with the wild type 'FT'. 1 All plants showed wild-type phenotype. 1 The plants were backcrossed with the wild-type 'FT' parent, BC 1 All plants showed wild-type phenotype; 1 When the plants were backcrossed with the leaf senescence mutant parent lpsm, 51 plants showed the wild-type phenotype and 45 plants showed the leaf senescence phenotype. The segregation ratio of the wild-type phenotype to the leaf senescence mutant phenotype was 1.12:1, which was close to the theoretical ratio of 1:1. 1 Plants were self-pollinated to obtain F 2 Of the plants, 309 showed a wild-type phenotype and 99 showed a leaf senescence phenotype. The segregation ratio of the wild-type phenotype to the leaf senescence mutant phenotype was 3.12:1, close to the theoretical ratio of 3:1. Genetic analysis results showed that the leaf senescence trait of the lpsm mutant was controlled by a recessive nuclear gene.

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

[0057] 1.1 Experimental methods

[0058] MutMap Positioning

[0059] The improved MutMap method was used to screen candidate genes for the leaf premature senescence mutant lpsm. 50 F 2Leaves of plants with early leaf senescence mutation phenotype in the population were used to construct DNA pools (W-pool), and DNA from leaves of the two parents, 'FT' and lpsm, was extracted according to the operation steps of the centrifugal column DNAsecurePlant Kit (TIANGEN, China). The sequencing library was prepared by Illumina NovaSeq steps such as DNA sequence end repair, adding A base to the 3' end of the DNA sequence, and adding library-specific tags to the 5' end to complete high-throughput sequencing. The raw data of high-throughput sequencing was initially filtered, and reads with adapters and low quality were removed from the data to generate high-quality sequence data. The sequence information of the reference genome (Brara Chiifu V3.5genome) was sorted and counted. The high-quality data was aligned to the reference genome using the bwa mem program, and the alignment information was sorted and counted. The GATK software was selected for SNP detection, and the SNP sites were filtered by removing the sites with deletions and other standards, and the distribution map of SNP variant sites was drawn. Detect and filter InDel (small Insertion and Deletion) mutation sites, draw a distribution map of mutation sites, and use ANNOVAR software to annotate SNP and INDEL sites. In order to screen candidate regions associated with trait genes, the population SNPs are further filtered by screening SNP sites with EMS mutagenesis characteristics, SNP-index is calculated, and a SNP-index chromosome distribution map is drawn. The SNP-index value is positively correlated with the correlation degree of the target trait. The chromosome distribution region with a SNP-index greater than 95% is selected as the trait-related candidate region, the SNPs in the candidate region are screened, and the SNP sites are annotated. GO (Gene Ontology) annotations are made for the corresponding candidate genes.

[0060] KASP genotyping

[0061] KASP technology was used to verify the genotyping of the SNP sites of candidate genes. 187 F2 plants with early leaf senescence mutation phenotypes were selected, and the wild-type parent 'FT', the mutant parent lpsm and the F1 heterozygous plants were used as controls. Fresh leaf materials were taken and leaf DNA was extracted according to the improved CTAB method. The DNA samples were detected by an ELISA instrument, and specific primers were designed based on the reference gene sequence containing the SNP site with reference to the Chinese cabbage reference genome database (Brara Chiifu V 3.5genome). 187 F 2 Plants with leaf premature senescence mutation phenotype were selected from wild-type parent 'FT', mutant parent lpsm and F 1 The DNA of heterozygous plants was used for preliminary experiments to verify the quality of specific primers, and then the F 2 The DNA of plants with leaf premature senescence mutation phenotype was used for genotype detection.

[0062] 1.2 Experimental Results

[0063] The wild-type parent 'FT', the leaf premature senescence mutant phenotype parent lpsm and F 2 The plants with leaf premature senescence mutant phenotype were mixed and subjected to high-throughput sequencing. The reads with adapters and low quality in the obtained raw data were filtered to obtain high-quality data. The results showed that the wild-type parent 'FT', the mutant phenotype parents lpsm and F 2 The percentage of high-quality reads in the mutant phenotype plants to the original reads was higher than 95%, and the percentage of high-quality read bases to the total number of original bases was higher than 90%. The filtered high-quality data were aligned to the reference genome (Brara Chiifu V3.5 genome) for analysis. The results showed that the percentage of reads aligned to the reference genome (i.e., alignment rate) of the mutant phenotype parent lpsm was 98.05%, and the percentage of high-quality read bases to the total number of original bases was 90%. 2 The comparison rates between the mutant phenotype plants and the wild-type parent 'FT' were higher than 99%.

[0064] Single nucleotide polymorphism (SNP) sites were detected, SNP sites were filtered according to relevant standards, and SNP sites were annotated. In order to screen candidate regions associated with trait genes, SNP polymorphism markers were screened, and the SNP-index of each polymorphic site was calculated respectively. Then, the population SNP was further filtered according to the standards, and finally the SNP-index results were obtained, and the SNP-index chromosome distribution map was drawn. 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 in the exon region of BraA10g0196403.5C, and BraA10g0196403.5C were determined. There are 11 candidate genes in total, including BraA10g021620.3.5C, 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 12 candidate SNP loci, including SNP14363364, SNP14365863, SNP15905282, and SNP17014295, using the parents and F1 plants as references and 187 plants with early leaf senescence phenotypes in the F2 population. Theoretically, the candidate SNP loci co-segregated with the early leaf senescence mutant phenotypes of the 187 plants in the F2 population, and the genotypes of the plants with early leaf senescence mutant phenotypes in the F2 population were all T:T. The results showed that the genotype of SNP15905282 in BraA10g022760.3.5C was T:T in the F2 population leaf early senescence mutant phenotype plants, while T:T and C:T genotypes in the other 11 candidate SNP sites such as SNP14363364 and SNP14365863 were detected in the F2 population leaf early senescence mutant phenotype plants. The results showed that the candidate SNP15905282 of BraA10g022760.3.5C was co-segregated with the leaf early senescence mutant phenotype, and the other candidate SNP sites were not co-segregated with the leaf early senescence mutant phenotype. Therefore, the BraA10g022760.3.5C gene where SNP15905282 was located was speculated to be the mutant candidate gene of the leaf early senescence mutant lpsm. According to the homologous gene comparison, 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 sequence of the candidate gene BrATG5 in the wild type 'FT' and the leaf premature senescence mutant lpsm were cloned and sequence comparison analysis was performed. The cloning results showed that there was no difference in the full-length gene sequence and CDS sequence of the BrATG5 gene in the wild type 'FT' and the leaf premature senescence mutant lpsm; compared with the wild type 'FT', the promoter region of the BrATG5 gene in the leaf premature senescence mutant lpsm had a C→T base mutation 51bp upstream of the start codon, which was consistent with the MutMap positioning result.

[0068] >pBrATG5-wt promoter nucleotide sequence

[0069]

[0070] >pBrATG5CTT promoter nucleotide sequence

[0071]

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

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

[0074] A new promoter sequence was designed, and the promoter sequence of BrATG5 such as SEQ ID NO: 1-4 was cloned respectively, and the driving pBWA(V)HG-pBrATG5-GUS gene expression vector (groups A, B, C, and D, respectively) was constructed to further verify the gene expression pattern of BrATG5. Wild-type Arabidopsis plants were infected multiple times using the floral dipping method, Arabidopsis seeds were harvested and sown, and the surviving Arabidopsis seedlings were transplanted, Arabidopsis genomic DNA was extracted, and PCR detection was performed to screen and obtain Arabidopsis positive plants (corresponding to groups A, B, C, and D, respectively).

[0075] >pBrATG5CTA promoter nucleotide sequence

[0076]

[0077] >pBrATG5CTG promoter nucleotide sequence

[0078]

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

[0080] 1) In groups A, B, C, and D, the roots, stems, leaves, and flower organs of Arabidopsis plants were all stained blue, but the staining degree of leaves was the strongest. This indicates that the GUS gene is expressed in the roots, stems, leaves, and flower organs of positive Arabidopsis plants, and is most strongly expressed in leaves. 2) In groups A and C, there was no significant difference in the staining level of each organ; in groups B and D, the staining degree of leaves was significantly weaker and stronger than that of groups A and C. This indicates that the gene regulatory functions of different promoter sequences are significantly different.

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

[0082] The results showed that the plants transfected before the premature senescence trait appeared did not show premature senescence trait as determined in Example 1 during the whole growth process; the plants transfected on the first day after the premature senescence trait appeared showed significant improvement in premature senescence trait and recovered to normal phenotype on the 7th day after transfection; the plants transfected on the 7th day after the premature senescence trait appeared showed improvement in premature senescence trait and did not completely recover to normal phenotype within the 21-day test period. This indicates that the BrATG5 gene is one of the key genes controlling premature senescence trait in Chinese cabbage, and premature senescence trait caused by promoter C to T mutation is reversible in the early stage of premature senescence.

[0083] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the 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: 2 or 4. 2 . The promoter according to claim 1 , wherein the promoter regulates the expression of the BrATG5 gene in Chinese cabbage, the BrATG5 gene being BraA10g022760.3.5C. 3 . The promoter according to claim 2 , which is as shown in SEQ ID NO: 2 and down-regulates the expression of BrATG5 gene in Chinese cabbage leaves. 4 . The promoter according to claim 2 , which is as shown in SEQ ID NO: 4 and upregulates the expression of BrATG5 gene in Chinese cabbage leaves.

5. A gene construct comprising the promoter for regulating the early senescence trait of Chinese cabbage leaves as claimed in any one of claims 1 to 4. The gene construct according to claim 5 , further comprising a BrATG5 gene.

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

8. The method according to claim 7, wherein a gene construct containing the promoter shown in SEQ ID NO: 2 is transformed or transfected in a plant to induce premature senescence of plant leaves; Alternatively, 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.

9. The method according to any one of claims 7-8, wherein the plant is Chinese cabbage.

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