Transcription factor ppe nac33 for regulating plant growth and development and chlorophyll degradation and application thereof

Abstract

The present application provides a kind of transcription factor PpeNAC33 for regulating plant growth and development and chlorophyll degradation, nucleotide sequence is as shown in SEQ.ID.NO.2, amino acid sequence is as shown in SEQ.ID.NO.3.The present application also provides the vector based on the above-mentioned transcription factor, engineering strain.Meanwhile, the application of the above-mentioned transcription factor PpeNAC33 in regulating plant growth and development and chlorophyll degradation is provided.The present application finds a NAC transcriptional regulatory factor PpeNAC33 in the transcriptome analysis of seed abortion peach fruit.Studies have shown that the transcription factor is a key transcription factor for regulating plant growth and development and chlorophyll degradation, and its expression can be accurately regulated by space-time and expression amount, which can be used to realize the accurate control of plant growth process and avoid the occurrence of plant fruit premature aging.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and in particular to a transcription factor PpeNAC33 that regulates plant growth and development and chlorophyll degradation and its applications. Background Technology

[0002] Premature fruit senescence refers to the process by which fruit fails to mature, manifesting as rotting or falling off the tree. While moderate rotting or falling off can selectively remove low-quality fruit and redistribute resources to improve long-term reproductive success, excessive fruit drop or pre-harvest fruit loss poses a significant economic challenge to fruit growers, potentially leading to yield losses of up to 60–80% in some fruit varieties. Therefore, premature fruit senescence is a major problem facing the horticultural industry.

[0003] Premature fruit senescence is a particularly complex process. From a physiological perspective, it manifests as inhibited growth, resulting in smaller fruit size. Later, these fruits undergo senescence and shrink due to water loss. Simultaneously, premature fruit senescence is accompanied by the accumulation or degradation of certain plant hormones, such as chlorophyll degradation and carotenoid accumulation.

[0004] Several plant senescence-related transcription factors have been identified. For example, members of the NAC family, such as KIRA1, ANAC087, ANAC046, and FEZ in Arabidopsis thaliana, are involved in regulating senescence. However, research on the discovery of NAC transcription factors in other plant species is limited, especially in the study of premature senescence of fruit trees. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a transcription factor PpeNAC33 (NAC-type transcription factor) that regulates plant growth and development and chlorophyll degradation, and its application. This transcription factor PpeNAC33 affects premature senescence of plant fruits by regulating plant growth and development and chlorophyll degradation. This invention provides a strong theoretical basis for the study of premature senescence of fruits and the cultivation of better plant varieties.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A transcription factor PpeNAC33 that regulates plant growth, development, and chlorophyll degradation has the nucleotide sequence shown in SEQ.ID.NO.2 and the corresponding encoded amino acid sequence shown in SEQ.ID.NO.3.

[0008] As one of the preferred embodiments of the present invention, the transcript sequence of the transcription factor PpeNAC33, as shown in SEQ.ID.NO.1, is derived from peach fruit tissue that has undergone premature aging.

[0009] As one of the preferred embodiments of the present invention, the sequence shown in SEQ.ID.NO.2 is an open reading frame sequence obtained by predicting and cloning the sequence in SEQ.ID.NO.1.

[0010] As one of the preferred embodiments of the present invention, overexpression of the transcription factor PpeNAC33 can induce chlorophyll degradation in tobacco leaves and inhibit plant growth in Arabidopsis thaliana.

[0011] An overexpression vector pSAK277:PpeNAC33 was obtained by digesting the sequence shown in SEQ.ID.NO.2 with XhoI and XbaI enzymes and then introducing it into the pSAK277 vector.

[0012] An engineered strain containing the overexpression vector pSAK277:PpeNAC33 is prepared by introducing the pSAK277:PpeNAC33 plasmid into Agrobacterium.

[0013] An inducible gene engineering vector, pSAK277:XVE-PpeNAC33, was obtained by digesting the sequence shown in SEQ.ID.NO.2 with KpnI and XhoI enzymes and then introducing it into the pSAK277:XVE vector.

[0014] An engineered strain containing the above-mentioned inducible gene engineering vector pSAK277:XVE-PpeNAC33 is prepared by introducing the pSAK277:XVE-PpeNAC33 plasmid into Agrobacterium.

[0015] Application of the above-mentioned transcription factor PpeNAC33 in regulating plant growth and development and chlorophyll degradation.

[0016] As one of the preferred embodiments of the present invention, the premature senescence of fruit is improved by regulating plant growth and development and chlorophyll degradation through the transcription factor PpeNAC33.

[0017] The advantages of this invention compared to the prior art are:

[0018] This invention utilizes transcriptomics to compare prematurely senescent peach fruits with normal fruits, identifying a NAC-type transcription factor, PpeNAC33-trans, which is upregulated in prematurely senescent fruits. The predicted and cloned ORF coding sequence is SEQ.ID.NO.2 (named PpeNAC33), and the amino acid sequence is SEQ.ID.NO.3. Functional analysis shows that overexpression of PpeNAC33 induces chlorophyll degradation in tobacco leaves, while overexpression in Arabidopsis inhibits plant growth. Furthermore, regulating PpeNAC33 expression through an estradiol-induced system inhibits taproot elongation. Based on these findings, PpeNAC33 is confirmed as a key transcription factor regulating plant growth and development and chlorophyll degradation. Precise spatiotemporal and quantitative regulation of its expression can be used to achieve precise control of plant growth processes and prevent premature fruit senescence.

[0019] This invention provides a strong theoretical basis for the study of premature fruit senescence and the cultivation of higher-quality plant varieties. Attached Figure Description

[0020] Figure 1 This is a comparison diagram of the normal fruit (SH_NF) and prematurely senescent fruit (SH_AF) in Example 1.

[0021] Figure 2 These are the vector maps of pSAK277 and pSAK277:XVE in Example 2 (left image is vector pSAK277, right image is vector pSAK277:XVE);

[0022] Figure 3 The images show the vector maps of pSAK277:PpeNAC33 and pSAK277:XVE-PpeNAC33 in Example 2 (left image shows vector pSAK277:PpeNAC33, right image shows vector pSAK277:XVE-PpeNAC33);

[0023] Figure 4 The results of the experiment in Example 3 on overexpression of PpeNAC33 in Arabidopsis thaliana (Figure A shows the leaf phenotype of wild-type Col-0 and PpeNAC33-OE Arabidopsis thaliana; Figure B shows the overall phenotype of wild-type Col-0 and PpeNAC33-OE Arabidopsis thaliana; Figure C shows the phenotype of wild-type Col-0 and the two XVE>>PpeNAC33 Arabidopsis thaliana plants after estradiol treatment).

[0024] Figure 5The results of the experiment on overexpression of PpeNAC33 in tobacco leaves in Example 4 are shown in Figure A. The upper empty control group is normal 3 days after injection, while the lower PpeNAC33 experimental group shows leaf chlorosis. Figures B to D show the results of the measurement of leaf SOD activity, relative conductivity and chlorophyll content, respectively. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, the materials, reagents, and experimental methods used in the following embodiments, unless otherwise specified, are all conventional materials, reagents, and methods in the art, and will not be described again.

[0026] Example 1: Discovery and identification of target genes related to premature senescence of peach fruit

[0027] 1. Selection of peach fruit materials

[0028] Two groups of peach fruit materials were selected: the first group consisted of contemporary fruits naturally pollinated and bearing fruit from the existing peach variety "Qingshui," which developed normally and served as the control group (labeled SH_NF); the second group consisted of fruits of the same variety exhibiting natural premature aging, displaying seed abortion characteristics and significantly smaller volume than the control group fruits at the early stage of the second enlargement period (labeled SH_AF). The morphological differences between the two groups are as follows: Figure 1 As shown.

[0029] 2. Transcriptome sequencing and analysis

[0030] Transcriptional library construction and sequencing: First, total RNA was extracted from fruit tissue using the standard Trizol method. RNA concentration was precisely quantified using a Thermo Scientific NanoPhotometer and a Qubit 2.0 fluorometer. RNA integrity was assessed using an Agilent Bioanalyzer 2100 system. The total RNA was then purified using magnetic oligomeric dT beads. Approximately 1 μg of purified total RNA was used to construct a sequencing library. The Trussq RNA sample preparation kit was followed according to the manufacturer's instructions. Fragment purification was performed on the library, selecting cDNA fragments of 300 base pairs in length. First-strand cDNA was synthesized using reverse transcriptase and random hexamer primers, followed by second-strand cDNA synthesis according to the base pairing principle. End repair was performed on the newly generated double-stranded cDNA, with a single adenine nucleotide added to the 3' end. PCR amplification was performed using NEB's Phusion high-fidelity DNA polymerase. The PCR products were purified and quality-tested using an Agilent Bioanalyzer 2100 system. Following the instrument manufacturer's instructions, clusters were generated for the indexed samples using the cBot Cluster Generation system. Finally, the library was sequenced using the Hiseq X-ten platform.

[0031] Bioinformatics analysis: Raw sequencing data (FastQ format) underwent quality control using FastQC software, and data filtering was performed using SeqPrep software. Quality-controlled data were aligned to the peach genome reference sequence (Lovell-v2.0) using HISAT2 software. Aligned reads were assigned to genomic features and quantitatively analyzed using the R language package Rsubread (featureCounts module, version 1.6.1). Differentially expressed genes (DEGs) were screened using the Deseq2 package, with the following parameters: a log2 fold change greater than 1 and a corrected p-value (qvalue) less than 0.05.

[0032] In this step, three biological replicates of comparative RNA sequencing were performed on normal fruits (SH_NF) and prematurely senescent fruits (SH_AF). In-depth analysis of the comparative transcriptome data revealed 226 differentially expressed transcription factors between normal fruits (SH_NF) and prematurely senescent fruits (SH_AF), of which 160 were upregulated and 66 were downregulated. One NAC transcription factor showed significant difference, with marked upregulation in prematurely senescent fruits; its sequence is shown in SEQ.ID.NO.1, and its transcript is named PpeNAC33-trans (NAC-type transcription factor).

[0033] 3. Determine the target gene sequence.

[0034] The PpeNAC33-trans transcript sequence (SEQ.ID.NO.1) was analyzed using DNASTAR's EditSeq 5.01 software. Its complete open reading frame coding sequence (SEQ.ID.NO.2) was predicted and ultimately cloned, and the gene was formally named PpeNAC33. The amino acid sequence encoded by this gene is shown in SEQ.ID.NO.3.

[0035] Example 2: Construction of expression vector

[0036] 1. Construction of the basic vector for the inducible expression system

[0037] First, a DNA fragment containing multiple functional elements was designed and synthesized. This fragment includes the following components: (1) an XVE sequence encoding a chimeric transcription factor composed of three functional regions: amino acid sequences 1-87 of the LexA protein, amino acid sequences 403-479 of the VP16 protein, and amino acid sequences 282-595 of the human estrogen receptor; (2) a transcription terminator sequence of the rbcs gene; (3) eight tandem LexA operator sequences; and (4) a 35S minimal promoter sequence at position -59. The synthesized DNA fragment was cloned into the multiple cloning site of the binary vector pSAK277 to construct the basic inducible vector pSAK277:XVE (all sequences mentioned above are publicly available sequences). The specific maps of vectors pSAK277 and pSAK277:XVE are shown below. Figure 2 As shown.

[0038] 2. Construction of the target gene expression vector

[0039] Construction of the conventional expression vector: cDNA was prepared using RNA extracted from premature senescent fruit (AF) as a template, and the full-length coding sequence of the PpeNAC33 gene (SEQ.ID.NO.2) was amplified using primer pair Primers-No.1. The PCR amplification product was double-digested with XhoI and XbaI, and then inserted into the binary vector pSAK277 to obtain the conventional expression vector pSAK277:PpeNAC33.

[0040] Construction of the inducible expression vector: The full-length coding sequence of the PpeNAC33 gene (SEQ.ID.NO.2) was amplified using primer pair Primers-No.2. Simultaneously, the pSAK277:XVE vector was double-digested with KpnI and XhoI. The target gene fragment was ligated to the treated vector using the one-step seamless cloning kit (product number SC612) from Jinsha Biotechnology Co., Ltd., to obtain the inducible expression vector pSAK277:XVE-PpeNAC33.

[0041] The two expression vector structures constructed in this step are as follows: Figure 3 As shown.

[0042] The primers involved in this embodiment are shown in Table 1.

[0043] Table 1 Primers used to construct the expression vector.

[0044]

[0045]

[0046] Example 3: Overexpression of PpeNAC33 in Arabidopsis thaliana

[0047] The constructed vector pSAK277:PpeNAC33 was transformed into Arabidopsis thaliana using the flower-dipping method. However, after two rounds of screening, only three surviving progeny plants were obtained (the positive rate of other gene-overexpressing plants during the same period was much higher than in this experiment), indicating that stable overexpression of PpeNAC33 has a significant impact on plant survival. Morphological observation of the three surviving seedlings revealed that, compared with the wild type (Col-0), the PpeNAC33-overexpressing plants (PpeNAC33-OE) were dwarfed, and the growth of leaves, stems, and roots was significantly inhibited. Figure 4 (As shown in A and B).

[0048] To avoid the potential lethal effects of PpeNAC33, the estradiol-induced XVE expression system was integrated into the pSAK277 vector (constructing the vector pSAK277:XVE) to achieve precise regulation of PpeNAC33 expression. After culturing for 5 days in MS medium containing 10 μM estradiol, Arabidopsis seedlings transformed with pSAK277:XVE-PpeNAC33 (XVE>>PpeNAC33) showed a significant cessation of taproot growth (e.g., ...). Figure 4 (as shown in C).

[0049] The above results indicate that PpeNAC33 has the function of inhibiting the growth of normal organs.

[0050] Example 4: Transient overexpression of PpeNAC33 in tobacco leaves

[0051] To investigate whether PpeNAC33 has the ability to induce other symptoms related to aging, a transient overexpression experiment of PpeNAC33 was conducted in tobacco leaves.

[0052] Agrobacterium tumefaciens strain GV3101 carrying the empty vector pSAK277 or pSAK277:PpeNAC33 was cultured at 28°C for 2 days. The culture was diluted with a buffer solution containing 10 mM MgCl2, 200 μM acetylsylgenone, and 10 mM MES (pH 5.7) and incubated at 21°C for 2 hours in preparation for infection. Subsequently, the Agrobacterium tumefaciens suspension was injected into 3-week-old tobacco leaves using a needle-free syringe. Three biological replicates were set up, and photographs were taken on day 3 post-infection.

[0053] The results are as follows Figure 5 As shown in Figure A: When the full-length sequence of PpeNAC33 was inserted into the multiple cloning site of the binary vector pSAK277, obvious leaf chlorosis was observed in the injected area 3 days after the injection of Agrobacterium tumefaciens containing pSAK277:PpeNAC33, while these phenomena were not observed in the empty vector control group.

[0054] In addition, to quantify free radical scavenging ability, SOD activity was measured using a plant SOD detection kit developed in Nanjing. The results showed no significant difference in SOD activity between the two methods. Figure 5 B).

[0055] To further assess cell death rate, 0.6 g leaf discs were taken from tobacco leaves infected for 3 days and incubated overnight in double-distilled water. The initial conductivity of the solution was measured. Subsequently, the leaf discs and solution were boiled at 100°C for 1 hour, then cooled to room temperature for final conductivity measurement, representing 100% cell death. The percentage of electrolyte leakage was calculated by dividing the initial conductivity by the total conductivity after boiling. A higher percentage indicated more severe cell damage and death; however, no significant difference in conductivity activity was found between the two methods. Figure 5 C) indicates that PpeNAC33 did not induce cell death.

[0056] Further analysis of chlorophyll content was conducted using an ethanol extraction method. 0.1 g of leaf tissue was placed in 10 mL of 95% ethanol and incubated in the dark for 12 hours until complete fading. The absorbance at 665 nm and 649 nm was then measured using a microplate reader. The results showed that transient overexpression of PpeNAC33 significantly reduced leaf chlorophyll content. Figure 5 D).

[0057] The above findings indicate that PpeNAC33 has the ability to induce chlorophyll degradation.

[0058] In summary, PpeNAC33 of this invention is a key transcription factor that regulates plant growth and development as well as chlorophyll degradation. By precisely regulating its expression in terms of time, space, and level, it can be used to achieve precise control of plant growth processes and improve premature fruit senescence.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A transcription factor PpeNAC33 Its application in regulating chlorophyll degradation is characterized by, The transcription factor PpeNAC33 The nucleotide sequence is shown in SEQ.ID.NO.2, and the corresponding encoded amino acid sequence is shown in SEQ.ID.NO.3; the regulation is to overexpress the transcription factor. PpeNAC33 The specific application is: overexpression of the transcription factor. PpeNAC33 To induce chlorophyll degradation in tobacco leaves.

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