Transcription factor PpeNAC33 for regulating plant growth and development and chlorophyll degradation and application thereof

Abstract

The invention provides a transcription factor PpeNAC33 for regulating and controlling plant growth and development and chlorophyll degradation, the nucleotide sequence of the transcription factor PpeNAC33 is as shown in SEQ.ID.NO.2, and the amino acid sequence of the transcription factor PpeNAC33 is as shown in SEQ.ID.NO.3. The invention also provides a vector and an engineering strain based on the transcription factor. Meanwhile, the invention also provides application of the transcription factor PpeNAC33 in regulation and control of plant growth and development and chlorophyll degradation. According to the invention, an NAC type transcriptional regulatory factor PpeNAC33 is found in transcriptomic analysis of seed abortion peach fruits. Researches show that the transcription factor is a key transcription factor for regulating and controlling plant growth and development and chlorophyll degradation, and the expression of the transcription factor can be accurately regulated and controlled in time, space and expression quantity, so that the transcription factor can be used for accurately controlling the plant growth process, and the premature senility phenomenon of plant fruits is avoided.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and in particular to a transcription factor PpeNAC33 for regulating plant growth and development and chlorophyll degradation and an application thereof. Background Art

[0002] Premature fruit senescence is the process by which fruit fails to mature, manifested by fruit rotting or falling off the tree. While moderate rotting or falling can selectively remove low-quality fruit and reallocate resources to improve long-term reproductive success, excessive fruit drop or fruit shedding before harvest can pose a huge economic challenge to fruit growers, resulting in up to 60-80% yield loss in some fruit tree varieties. Therefore, the phenomenon of premature fruit senescence is a major problem facing the horticultural industry.

[0003] Early fruit senescence is a particularly complex process. From a physiological point of view, early fruit senescence manifests itself as growth inhibition, resulting in smaller fruit size. In the later stages, these fruits undergo a senescence process and shrink due to water loss. At the same time, early fruit senescence is also accompanied by the accumulation or degradation of some plant hormones, such as chlorophyll degradation and carotenoid accumulation.

[0004] At present, some plant aging-related transcription factors have been discovered. Taking the NAC family as an example, its members, such as KIRA1, ANAC087, ANAC046 and FEZ in Arabidopsis, are involved in regulating aging. However, there are few studies on NAC transcription factors in other plant species, especially in the early aging of fruit trees. Summary of the invention

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

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A transcription factor PpeNAC33 that regulates plant growth and development and chlorophyll degradation, the nucleotide sequence of which is shown in SEQ.ID.NO.2, and the corresponding encoded amino acid sequence is 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 is shown in SEQ.ID.NO.1, and 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 of 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.

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

[0012] An engineered strain contains the above-mentioned overexpression vector pSAK277:PpeNAC33, and the preparation method is: introducing the pSAK277:PpeNAC33 plasmid into Agrobacterium.

[0013] An inducible genetic engineering vector pSAK277:XVE-PpeNAC33 is obtained by cutting the sequence shown in SEQ.ID.NO.2 with KpnI and XhoI and then introducing it into the pSAK277:XVE vector.

[0014] An engineering strain contains the above-mentioned inducible genetic engineering vector pSAK277:XVE-PpeNAC33, and the preparation method is: introducing the pSAK277:XVE-PpeNAC33 plasmid into Agrobacterium.

[0015] An application of the above transcription factor PpeNAC33 in regulating plant growth and development and chlorophyll degradation.

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

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

[0018] The present invention uses transcriptomics to compare and analyze peach premature aging fruits and normal fruits, and discovers a NAC-type transcription factor PpeNAC33-trans that is upregulated in premature aging 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 can induce chlorophyll degradation in tobacco leaves, overexpression in Arabidopsis inhibits plant growth, and regulating PpeNAC33 expression through an estradiol induction system can inhibit taproot elongation. Based on this, PpeNAC33 has been confirmed to be a key transcription factor that regulates plant growth and development and chlorophyll degradation. By precisely regulating its expression in time, space and expression, it can be used to achieve precise control of plant growth processes and avoid the occurrence of premature aging of plant fruits.

[0019] The invention provides a strong theoretical basis for the research on premature senescence of fruits and the cultivation of better quality plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a comparison diagram of the fruit morphology of the normal fruit (SH_NF) and the premature senescence fruit (SH_AF) in Example 1;

[0021] Figure 2 is the vector map of the vectors pSAK277 and pSAK277:XVE in Example 2 (the left picture is the vector pSAK277, the right picture is the vector pSAK277:XVE;

[0022] Figure 3 is the vector map of the vectors pSAK277:PpeNAC33 and pSAK277:XVE-PpeNAC33 in Example 2 (the left picture is the vector pSAK277:PpeNAC33, and the right picture is the vector pSAK277:XVE-PpeNAC33);

[0023] Figure 4 It is the experimental result of overexpressing PpeNAC33 in Arabidopsis in Example 3 (in the figure, Figure A is the leaf phenotype of wild-type Col-0 and PpeNAC33-OE Arabidopsis; Figure B is the overall phenotype of wild-type Col-0 and PpeNAC33-OE Arabidopsis; Figure C is the phenotype of wild-type Col-0 and two XVE>>PpeNAC33 Arabidopsis plants after estradiol treatment);

[0024] Figure 5These are the experimental results of overexpressing PpeNAC33 in tobacco leaves in Example 4 (in the figure, Figure A shows the result of the empty control group at the top being normal 3 days after injection, while the PpeNAC33 experimental group at the bottom showed leaf chlorosis; Figures B to D are the measurement results of leaf SOD activity, relative conductivity and chlorophyll content, respectively). DETAILED DESCRIPTION

[0025] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the materials, reagents and experimental methods used in the following embodiments are conventional materials, reagents and methods in the art unless otherwise specified, and will not be repeated.

[0026] Example 1: Discovery and determination of target genes associated with premature senescence of peach fruit

[0027] 1. Selection of peach fruit materials

[0028] Two groups of peach fruit materials were selected: the first group was the contemporary fruits of the existing peach variety "Qingshui" that were naturally pollinated and fertilized. These fruits developed normally and served as the control group (labeled as SH_NF); the second group was the fruits of the same variety that showed natural premature aging. These fruits showed seed abortion characteristics and were significantly smaller in size than the control group fruits (labeled as SH_AF) at the beginning of the second expansion period. The morphological differences between the two groups of samples are as follows: Figure 1 shown.

[0029] 2. Transcriptome sequencing and analysis

[0030] Transcriptome library sequencing: First, total RNA was extracted from fruit tissue using the standard Trizol method. RNA concentration was accurately quantified using a Thermo Scientific NanoPhotometer photometer and a Qubit 2.0 fluorometer. RNA integrity was assessed using an Agilent Bioanalyzer 2100 system. Total RNA was then purified using magnetic oligo dT beads. Approximately 1 μg of purified total RNA was used to construct sequencing libraries. The Truseq RNA sample preparation kit instructions provided by the manufacturer were followed. The library was fragment purified and cDNA fragments with a length of 300 base pairs were selected. The first-strand cDNA was synthesized using reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized according to the principle of complementary base pairing. The newly generated double-stranded cDNA was end-repaired and a single adenine nucleotide was added to the 3' end. PCR amplification reactions were performed using Phusion high-fidelity DNA polymerase from NEB. PCR products were purified and quality tested on an Agilent Bioanalyzer 2100 system. According to the instrument manufacturer's instructions, cluster generation was performed on the cBot Cluster Generation system for samples with index markers. Finally, the library was sequenced using the HiSeq X-ten platform.

[0031] Bioinformatics analysis: The raw sequencing data (fastq format) were quality controlled using fastqc software, and data were filtered using SeqPrep software. The qualified data were aligned to the peach genome reference sequence (Lovell-v2.0 version) using HISAT2 software. The aligned reads were assigned to genomic features and quantitatively analyzed using the R language package Rsubread (featureCounts module, version 1.6.1). The Deseq2 software package was used to screen differentially expressed genes (DEGs), and the screening parameters were set as follows: log2 fold change greater than 1 and the corrected p value (qvalue) less than 0.05.

[0032] In this step, three biological replicates of normal fruit (SH_NF) and premature senescent fruit (SH_AF) were compared by RNA sequencing. In-depth analysis of the comparative transcriptome data showed that there were 226 differentially expressed transcription factors between normal fruit (SH_NF) and premature senescent fruit (SH_AF), of which 160 were upregulated and 66 were downregulated. One of the NAC transcription factors was very different, with significant upregulation in premature senescent fruit. The sequence is shown in SEQ.ID.NO.1, and its transcript was 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, and its complete open reading frame coding sequence (SEQ.ID.NO.2) was obtained through prediction and final cloning, and the gene was formally named PpeNAC33. The amino acid sequence encoded by the gene is shown in SEQ.ID.NO.3.

[0035] Example 2: Construction of expression vector

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

[0037] First, a DNA fragment containing multiple functional elements is designed and synthesized. The fragment contains the following components: (1) XVE sequence, which encodes a chimeric transcription factor composed of the following three functional regions: the amino acid sequence of positions 1 to 87 of LexA protein, the amino acid sequence of positions 403 to 479 of VP16 protein and the amino acid sequence of positions 282 to 595 of human estrogen receptor; (2) the transcription terminator sequence of rbcs gene; (3) eight tandem LexA operator sequences; (4) -59 position 35S minimal promoter sequence. The above synthesized DNA fragment is cloned into the multiple cloning site of binary vector pSAK277 to construct the basic induction vector pSAK277:XVE (the sequences involved above are all existing public sequences). The specific maps of vectors pSAK277 and pSAK277:XVE are shown in the figure. Figure 2 shown.

[0038] 2. Construction of target gene expression vector

[0039] Construction of conventional expression vector: RNA extracted from premature senescent fruit (AF) was used as a template to prepare cDNA, 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 inducible expression vector: The full-length coding sequence of the PpeNAC33 gene (SEQ.ID.NO.2) was amplified using primer pair Primers-No.2. At the same time, the pSAK277:XVE vector was double-digested with KpnI and XhoI. The target gene fragment was connected to the treated vector using the one-step seamless cloning kit (product number SC612) of Jinsha Biotechnology to obtain the inducible expression vector pSAK277:XVE-PpeNAC33.

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

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

[0043] Table 1 Primers used to construct expression vectors

[0044]

[0045]

[0046] Example 3: Overexpression of PpeNAC33 in Arabidopsis

[0047] The vector pSAK277:PpeNAC33 constructed above was transformed into Arabidopsis thaliana by the floral dipping method. However, after two rounds of screening, only three surviving offspring were screened (the positive rate of screening other gene overexpression plants at the same time was much higher than this test), indicating that the stable overexpression of PpeNAC33 has a great impact on the survival rate of plants. Morphological observation of the three surviving seedlings revealed that: compared with the wild type (Col-0), the plants overexpressing PpeNAC33 (PpeNAC33-OE) were short, and the growth of leaves, stems and roots was significantly inhibited ( Figure 4 (as shown in A and B).

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

[0049] The above results show that PpeNAC33 has the function of hindering normal organ growth.

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

[0051] To investigate whether PpeNAC33 has the ability to induce other symptoms associated with senescence, transient overexpression experiments of PpeNAC33 were performed in Nicotiana benthamiana leaves.

[0052] Agrobacterium tumefaciens GV3101 strain carrying empty vector pSAK277 or pSAK277:PpeNAC33 was cultured at 28°C for 2 days. The culture was diluted with a buffer containing 10mM MgCl2, 200μM acetosyringone and 10mM MES (pH 5.7) and allowed to stand at 21°C for 2 hours for infection. Subsequently, the Agrobacterium tumefaciens suspension was injected into 3-week-old tobacco leaves by needleless syringe. Three sets of biological replicates were set up in the experiment, and photos were taken on the 3rd day after infection.

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

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

[0055] To further test cell mortality, 0.6 g leaf discs were taken from 3-day-infected tobacco leaves and incubated overnight in double distilled water. The initial conductivity of the solution was measured, and then the leaf discs and solution were boiled at 100°C for 1 hour and then cooled to room temperature for the 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. Higher percentages indicate more severe cell damage and death, and the results showed that there was no significant difference in the conductivity activity between the two ( Figure 5 C), indicating that PpeNAC33 did not induce cell death.

[0056] The chlorophyll content was further tested by 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 it completely faded. The absorbance at 665 nm and 649 nm was then measured on a microplate reader. The test results showed that transient overexpression of PpeNAC33 significantly reduced the chlorophyll content of leaves ( Figure 5 D).

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

[0058] In summary, PpeNAC33 of the present invention is a key transcription factor that regulates plant growth and development and chlorophyll degradation. By precisely regulating its expression in time, space and expression level, it can be used to achieve precise control of the plant growth process and improve premature aging of fruits.

[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 in the protection scope of the present invention.

Claims

1. A transcription factor PpeNAC33 that regulates plant growth and development and chlorophyll degradation, characterized in that: Its nucleotide sequence is shown in SEQ.ID.NO.2, and the corresponding encoded amino acid sequence is shown in SEQ.ID.NO.

3.

2. The transcription factor PpeNAC33 for regulating plant growth and chlorophyll degradation according to claim 1, characterized in that: The transcript sequence of the transcription factor PpeNAC33 is shown in SEQ.ID.NO.1, and is derived from peach fruit tissue that has undergone premature aging.

3. The transcription factor PpeNAC33 for regulating plant growth and chlorophyll degradation according to claim 2, characterized in that: The sequence shown in SEQ.ID.NO.2 is an open reading frame sequence obtained by predicting and cloning the sequence of SEQ.ID.NO.

1.

4. The transcription factor PpeNAC33 for regulating plant growth and chlorophyll degradation according to claim 1, characterized in that: Overexpression of the transcription factor PpeNAC33 can induce chlorophyll degradation in tobacco leaves and inhibit plant growth in Arabidopsis.

5. An overexpression vector pSAK277:PpeNAC33, characterized in that: The sequence shown in SEQ.ID.NO.2 was digested with XhoI and XbaI and then introduced into the pSAK277 vector to obtain it.

6. An engineered strain, characterized in that: The overexpression vector pSAK277:PpeNAC33 according to claim 5 is prepared by introducing the pSAK277:PpeNAC33 plasmid into Agrobacterium.

7. An inducible genetic engineering vector pSAK277:XVE-PpeNAC33, characterized in that: The sequence shown in SEQ.ID.NO.2 was digested with KpnI and XhoI and then introduced into the pSAK277:XVE vector to obtain it.

8. An engineered strain, characterized in that: The inducible genetic engineering vector pSAK277:XVE-PpeNAC33 according to claim 7 is prepared by: introducing the pSAK277:XVE-PpeNAC33 plasmid into Agrobacterium.

9. Use of the transcription factor PpeNAC33 as claimed in any one of claims 1 to 4 in regulating plant growth and development and chlorophyll degradation.

10. The use according to claim 9, characterized in that: The transcription factor PpeNAC33 regulates plant growth and development and chlorophyll degradation, thereby improving premature senescence of fruits.

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