Arabidopsis beta-ketoacyl-ACP reductase gene promoter and use thereof

Abstract

The application discloses an Arabidopsis beta-ketoacyl-ACP reductase gene promoter and application thereof. The promoter has the nucleotide sequence shown in SEQ ID No. 1, and controls the Arabidopsis beta-ketoacyl-ACP reductase gene to be specifically expressed in the hypocotyl and root of the Arabidopsis seedling, the leaf and pistil stigma of the seedling, and the fungal infection site of the seedling leaf after inoculation of powdery mildew (Erysiphe cichoracearum, UCSC1). G. cichoracerum The application provides a new tissue-specific and white-powder-fungus-infection-specifically-induced expression promoter, and provides a new regulating element for improving the genetic engineering breeding of the plant resistance to the white-powder fungus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering technology; in particular to a KAR (Ketoacyl-ACP reductase) gene promoter specifically expressed in response to powdery mildew infection of Arabidopsis thaliana and application thereof. BACKGROUND

[0002] Powdery mildew caused by fungi of the order Erysiphales in the subphylum Ascomycotina is one of the most common plant fungal diseases, which can infect nearly 10,000 species of angiosperms, and occurs commonly on many important crops such as wheat, Solanaceae and Leguminosae, causing huge losses to agricultural production (Celio and Hausbeck, 1998; Byrne et al., 2000). Screening and identifying plant genes related to powdery mildew resistance and applying them to production are research hotspots in the field.

[0003] Fatty acids and their derivatives lipids are key components of cells, mainly providing biological membrane skeletons for cells and subcellular organelles; they are one of the energy sources necessary for cell life activities and the best energy storage mode for organisms. There have been a large number of reports on the role of fatty acids in plant-microbe interactions. The de novo biosynthesis process (FAS) of fatty acids includes four stages of condensation, reduction, dehydration and re-reduction, which is completed by seven enzymes, including: pyruvate kinase (PK), malonyl CoA-ACP transacylase (MCAT), ketoacyl-ACP synthase (KAS), ketoacyl-ACP reductase (KAR), enoyl-ACP reductase (ENR), hydroxyacyl-ACP dehydratase (HAD) and acyl-ACP thioesterase B (FatB) (Gah-Hyun Lim et al. 2017).

[0004] The chip expression data in the TAIR database shows that the KAR gene encoding beta-ketoacyl-ACP reductase is highly specifically expressed in response to the Blumeria infection, and previous studies have shown that the T-DNA insertion mutant kar-1 (SALK_011081) of the Arabidopsis KAR (At1g24360) gene has a significantly enhanced resistance phenotype compared with the wild type Col-0 after inoculation with Blumeria, indicating that the fatty acid synthesis process is involved in the regulation of plant resistance to powdery mildew (Yina Jiang et al., 2017), and the KAR gene plays an important role in the process of plant resistance to powdery mildew. In addition, the KAR homologous gene in corn is also specifically induced to express in the transcriptome data of U. maydis-infected host, suggesting that the fatty acid synthesis process may be widely involved in the regulation of plant resistance to biotrophic fungi (Doehlemann et al., 2008).

[0005] The promoter is a specific nucleotide sequence located upstream of the 5' end of the structural gene, which contains cis-acting elements that can be recognized and combined by transcription factors, and plays a key role in the regulation of gene expression. The promoter of a plant can be divided into three categories: constitutive promoter, tissue-specific promoter and inducible promoter. In the genetic engineering breeding of plants, the constitutive promoter such as CaMV35S of cauliflower virus can drive the expression of foreign genes in all plant tissues and organs, but it will also cause excessive consumption of intracellular substances and energy, and cause negative effects on the normal growth and development of plants. Cloning and identifying the promoter specifically induced by Blumeria in plants can be applied to the genetic manipulation of plants, which can precisely regulate the expression of plant disease resistance genes in response to pathogen infection, and does not affect the growth and development of plants, and lays a foundation for the cultivation of new varieties of disease-resistant transgenic crops. SUMMARY

[0006] In order to make up for the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide an Arabidopsis beta-ketoacyl-ACP reductase gene promoter and its application.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] An Arabidopsis beta-ketoacyl-ACP reductase gene KAR promoter, which is a nucleotide sequence shown in SEQ ID NO. 1.

[0009] A plant expression vector containing the beta-ketoacyl-ACP reductase gene KAR promoter.

[0010] The plant expression vector is pBGWFS7.

[0011] The application of the promoter of the Arabidopsis beta-ketoacyl-ACP reductase gene KAR or the expression vector in improving the powdery mildew resistance of plants.

[0012] The application of the promoter of the Arabidopsis beta-ketoacyl-ACP reductase gene KAR or the expression vector in plant breeding.

[0013] The application of the promoter of the Arabidopsis beta-ketoacyl-ACP reductase gene KAR or the expression vector in cultivating transgenic plants.

[0014] The plants include Arabidopsis, tomato, rice, wheat and corn.

[0015] The application has the following advantages and effects relative to the prior art:

[0016] The application clones the promoter sequence of the Arabidopsis beta-ketoacyl-ACP reductase KAR gene, and obtains a homozygous transgenic material pKAR::GUS by using a plant expression vector and connecting the KAR gene promoter and a GUS reporter gene. 1-708bp GUS staining tests are performed on plant tissue and organ materials at different growth and development stages of seedlings, mature plants and the reproductive stage, and it is detected that the KAR promoter drives the GUS reporter gene to be specifically expressed in the hypocotyl, root, leaf and stigma of Arabidopsis. After the leaves of the transgenic material grown under short-day conditions for four weeks are inoculated with powdery mildew, GUS staining and WGA staining tests are performed, and it is found that the KAR promoter drives the GUS reporter gene to be specifically induced and expressed at the site of powdery mildew infection. The KAR gene promoter sequence provided by the application can be used not only for the research on the specific expression of genes in seedlings, leaves and female reproductive development of plants, but also for the research on the powdery mildew resistance of plants, and provides a research foundation and scientific basis for improving the powdery mildew resistance of crops by using biological strengthening means. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a schematic diagram for the bioinformatics analysis of cis-acting elements in the KAR promoter;

[0018] Figure 2 It is a schematic diagram for the construction of the KAR promoter expression vector Promoter-pBGWFS7;

[0019] Figure 3 It is a GUS staining result analysis of the KAR promoter responding to the growth and development process;

[0020] Figure 4 It is a GUS staining and WGA staining result analysis of the KAR promoter responding to powdery mildew infection. DETAILED DESCRIPTION

[0021] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Unless otherwise defined, the experimental methods in the following examples without specific conditions are generally in accordance with the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available. The primer synthesis and sequencing were completed by Shenguo (Shanghai) Biotechnology Co., Ltd.

[0022] All the professional and scientific terms used in the present application have the same meanings as those familiar to the skilled in the art. In addition, any method and material similar or equivalent to those described can be applied in the present application. The preferred implementation methods and materials described in the present application are only for demonstration.

[0023] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. Unless otherwise defined, the experimental methods in the following examples without specific conditions are generally in accordance with the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press), or the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available. The primer synthesis and sequencing were completed by Shenguo (Shanghai) Biotechnology Co., Ltd.

[0024] Example 1. Cloning of Arabidopsis KAR gene promoter

[0025] According to the Arabidopsis genome sequence provided by NCBI (https: / / www.ncbi.nlm.nih.gov / ), the range of 708 bp upstream of the transcription start site of the KAR gene was selected as the candidate promoter region. The genomic DNA of Arabidopsis wild type Col-0 leaves was extracted by CTAB method, and the gDNA was used as a template to amplify the PCR forward and reverse primers using Hieff Gold High Fidelity DNA Polymerase, and the gDNA sequence PKAR 1-708bp of the KAR promoter was cloned. Then Hieff Plus OneStep Cloning Kit was used to insert the KAR gene promoter sequence into the pENTR intermediate vector by recombination exchange method, and the positive clones were screened after transformation of E. coli DH5α. The plasmid was extracted and sequenced, and the results are shown in SEQ ID NO. 1.

[0026] The PCR primer sequences used are as follows:

[0027] PKAR 1-708bp -F: 5'-GGAGCCCTTCACCGATTGAGGAACCAGGCA-3';

[0028] PKAR1-708bp -R: 5′-CGCGCCCACCCTTTGCCTGGTTCCTCAATC-3′;

[0029] The PCR reaction system is as follows:

[0030]

[0031]

[0032] PCR amplification conditions are as follows:

[0033]

[0034] Example 2. Bioinformatics analysis of the Arabidopsis thaliana KAR gene promoter

[0035] The KAR promoter sequences were input into the PLACE database, and the cis-elements contained in the sequences were predicted. For example... Figure 1 As shown, the KAR promoter region is enriched with various types of cis-regulatory elements, including those related to growth, development, and hormone signaling responses, such as: AW-box elements at bases 557-570 and 658-671 related to fatty acid synthesis; TGA-motif at bases 270-275 related to auxin signaling; E-box elements at bases 545-550 and 674-679 related to brassinosteroid response; MeJARE elements at bases 115-119 related to methyl jasmonate response; GARE elements at bases 697-703 related to gibberellins response; ERE elements at bases 575-581 related to ethylene signaling; and elements at bases 640-649 related to salicylic acid. TCA-motif elements related to acid responses, and W-box elements related to defense and stress responses located at bases 367-372.

[0036] The sequence of the aforementioned cis-acting elements is shown in the table below:

[0037]

[0038]

[0039] The above results indicate that the KAR gene promoter may be induced by environmental stress and plant hormones such as auxin, salicylic acid, abscisic acid and ethylene.

[0040] Example 3. Obtaining transgenic lines of Arabidopsis thaliana KAR gene promoter fusion GUS tag

[0041] Gateway LR reaction was used to construct the KAR gene promoter from pENTR intermediate vector to pBGWFS7 plant expression vector (as shown in Figure 2 After positive clones were screened, plasmid was extracted and sequenced, and the correct pBGWFS7-PKAR was verified by freeze-thaw method. 1-708bp The vector plasmid was transformed into Agrobacterium tumefaciens strain GV3101, and then transformed into Arabidopsis thaliana wild type Col-0 (wild type Arabidopsis thaliana Col-0 was purchased from Arabidopsis Biological Resource Center) background by Agrobacterium-mediated flower dipping method. A 1 / 2MS medium solution containing transformation aid silwet-77 (purchased from Biotopped company, CAS.NO: 27306-78-1, and the concentration used was 200 μL / L) and high concentration sucrose (50 g / L) was prepared, and the Agrobacterium was diluted to OD600 about 1.0. Arabidopsis thaliana with 10-15 cm inflorescences was selected, and the siliques and fully opened flowers were cut off, leaving only the just whitening and young flower buds for dipping. The dipped Arabidopsis thaliana was wrapped with a black plastic bag, kept away from light and moisture at 22°C overnight. The transformed plants were collected for seeds after about 4 weeks. The collected T1 generation transgenic seeds were spread on sterilized soil, and when the seedlings grew to the first pair of true leaves, an appropriate amount of 0.05% Basta solution was sprayed, and the spraying was repeated every three days for three times. The healthy and well-developed seedlings were transplanted and single seedlings were collected, and the T2 generation seeds were screened by Basta solution. The seedlings resistant to Basta corresponded to homozygous lines. Among them, the homozygous line obtained by transforming the Col-0 with the pBGWFS7 empty vector was denoted as Empty Vector, abbreviated as EV.

[0042] Example 4. Analysis of KAR promoter expression in response to growth and development

[0043] T3 generation pBGWFS7 empty vector transformed Arabidopsis thaliana and T3 generation pBGWFS7-PKAR 1-708bp Arabidopsis thaliana seedlings 7 days old, four-week-old seedlings rosette leaves and inflorescences after bolting, were placed in 10 ml centrifuge tubes on ice. An appropriate amount of GUS staining solution was poured into the tubes until the material was covered, vacuumed for 15 minutes, wrapped with tin foil paper, and placed in a 37°C shaking bed at 220 rpm for 5 hours to overnight. The GUS staining solution was discarded, and 95% ethanol was used for decolorization for 2-4 hours, followed by 75% ethanol for decolorization until the negative control material was white. The material was observed under a body dissecting microscope and photographed.

[0044] The pBGWFS7 empty vector Arabidopsis was used as control, and the T3 generation of pBGWFS7-PKAR 1-708bp The GUS staining results of different organs of the plants in the vegetative and reproductive stages are shown in Fig. 2. Figure 3 The results show that in the 7-day-old transgenic Arabidopsis seedlings, the KAR promoter drives the specific expression of the reporter gene in the hypocotyl and pericycle; in the short-day grown four-week-old seedling rosette leaves, the KAR promoter drives the very low expression of the reporter gene only in the stomata position of the leaf margin and a few leaf veins; after flowering, the KAR promoter drives the specific expression of the reporter gene in the mature pistil stigma. The above results show that the promoter of the KAR gene exhibits obvious tissue-specific expression characteristics during the growth and development of Arabidopsis.

[0045] Example 5. Analysis of the response of the KAR promoter to the process of powdery mildew infection

[0046] The powdery mildew (G. cichoracerum, UCSC1) was inoculated on the T3 generation of pBGWFS7 empty vector Arabidopsis and the T3 generation of pBGWFS7-PKAR 1-708bp The GUS staining results of different organs of the plants in the vegetative and reproductive stages are shown in Fig. 2.

[0047] The results show that on the leaves 1-4 days after powdery mildew infection, only the WGA green fluorescent signal showing the structure of powdery mildew infection can be seen, and the KAR promoter-driven GUS reporter gene always maintains the expression pattern only in the stomata position of the leaf margin, and no other GUS signal is observed. However, on the 5th day, the GUS blue signal highly overlaps with the green fluorescent staining of powdery mildew is detected on the leaves Figure 4), indicating that KAR drives the reporter gene to express significantly at the position of the S. sclerotiorum infection. The results confirm that the mode of KAR promoter induction expression in the process of Arabidopsis responding to S. sclerotiorum infection has high spatial and temporal specificity. In summary, the application illustrated in the present application can be implemented appropriately without any element, limitation not specifically disclosed herein. Therefore, for example, the terms "comprise" and "include" and the like should be understood as open and not limited. In addition, the terms and expressions used in the present application are used as descriptive terms and not as limiting terms, and such terms and expressions are not intended to exclude any equivalent features shown and described from the scope claimed by the present application, but it should be recognized that various modifications can be made within the scope claimed by the present application. Therefore, it should be understood that although the present application has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can employ modifications and variations of the present application embodied in the present application, and such modifications and variations are considered within the scope of the present application.

Claims

1. An Arabidopsis β-ketoacyl-ACP reductase gene KAR The application of the promoter of KAR in improving resistance to powdery mildew fungus in Arabidopsis thaliana, the nucleotide sequence of the promoter of KAR is shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The Arabidopsis β-ketoacyl-ACP reductase gene was constructed through genetic transformation. KAR The promoter sequence was obtained using a plant expression vector. KAR Homozygous transgenic material with a gene promoter linked to the GUS reporter gene.

3. Use according to claim 2, characterized in that, The plant expression vector is pBGWFS7.

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