Polygonatum cyrtonema transcription factor gene PcWRKY1 as well as encoding protein and application thereof

Abstract

The invention discloses a polygonatum cyrtonema transcription factor gene PcWRKY1 as well as an encoding protein and application thereof. According to the application, a WRKY transcription factor PcWRKY1 which is obviously inhibited by high-temperature stress is screened, the transcription level of the PcWRKY1 transcription factor gene responding to the high-temperature stress is further determined, a tissue expression mode and subcellular sites of the PcWRKY1 transcription factor gene are analyzed, and finally, the regulation effect of the PcWRKY1 transcription factor gene on the high-temperature tolerance of the polygonatum cyrtonema protoplast is determined. The PcWRKY1 transcription factor gene is located in a cell nucleus, codes a protein containing 519 amino acids and is expressed in roots, stems, leaves, tubers and seeds of polygonatum cyrtonema, the expression quantity in the seeds is the highest, and the PcWRKY1 transcription factor gene can inhibit the increase of MDA content and promote the improvement of CAT enzyme activity under high temperature stress. Further, the tolerance of the polygonatum cyrtonema protoplast under high-temperature stress is improved.

Description

Technical Field

[0001] The present invention relates to the field of biological genetic engineering technology, and specifically to the field of transcription factor gene of Polygonatum cyrtonema PcWRKY1 , its encoded protein and its application. Background Art

[0002] As greenhouse gas concentrations rise year by year, the global climate is warming, extreme high temperature events are occurring more frequently, with greater intensity and longer duration, and high temperature has become one of the main abiotic stresses limiting plant growth and plant yield increases.

[0003] Polygonatum multiflorum Polygonatum cyrtonema Hua ) is a perennial herb of the genus Polygonatum in the Liliaceae family. Its dried rhizomes are used as medicine and it is one of the original plants of the Chinese medicine Polygonatum. In recent years, Polygonatum cyrtonema has been very popular as a health food. Its wild resources can no longer meet the market demand, so most of them are produced by artificial cultivation. However, Polygonatum cyrtonema prefers a cool and humid environment. Extreme high temperature events caused by global warming pose a great threat to the Polygonatum cyrtonema cultivation industry. During field cultivation, long-term direct sunlight and high temperature weather in summer will cause high temperature stress damage to the leaves of Polygonatum cyrtonema, resulting in a decrease in the net photosynthetic rate, affecting the formation of its rhizomes and the accumulation of active ingredients. In severe cases, it will cause the above-ground parts to wither and turn yellow, causing Polygonatum cyrtonema to enter the seedling stage prematurely.

[0004] In order to resist the damage caused by high temperature stress, plants have evolved a series of adversity survival mechanisms. Many transcription factors (TFs), such as MYB, AP2 / ERFBP, NAC and WRKY, are involved in the response to abiotic stress. Plant-specific WRKY transcription factors are a gene family that can respond to different abiotic stresses (cold, drought, UV-B, high temperature, salt and alkaline environment). They can act as inhibitors and activators of gene expression and play an important role in key signaling pathways. More and more studies have shown that WRKY transcription factors can actively respond to plant tolerance to high temperatures, thereby improving plant tolerance to high temperatures. For example, in peppers, CaWRKY40 Involved in plant response to high temperature stress, its overexpression reduces tobacco's sensitivity to high temperature treatment, while the deletion mutant has the opposite effect. CaWRKY40 and GhWRKY39 Similar results were also found. In addition, wheat TaWRKY10 and TaWRKY33 , activating downstream stress response-related genes to regulate tolerance to high temperature. TaWRKY10 After that, the content of MDA induced by high temperature stress was reduced, and the activity of antioxidant protection enzymes promoted by high temperature stress was increased, thereby reducing the damage caused by high temperature stress. ZmWRKY106The promoter was analyzed for cis-acting elements and found to have cis-acting elements that respond to high temperature. High temperature induces its gene expression and can induce the activity of antioxidant enzymes in Arabidopsis overexpression lines, thereby improving the heat tolerance of transgenic lines. However, some WRKYs in plants also have a negative effect on high temperature tolerance, for example, in sunflower HaWRKY6 Heterologous expression in Arabidopsis significantly reduces its tolerance to high temperature. However, it is not clear whether there are WRKY transcription factors that respond to high temperature in Polygonatum cyrtonema and whether they have any effect under high temperature stress. Therefore, exploring the WRKY transcription factors that can respond to high temperature stress in Polygonatum cyrtonema will help understand the heat resistance mechanism of Polygonatum cyrtonema and provide important gene resources and related germplasm resources for breeding high temperature resistant Polygonatum cyrtonema varieties.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] The inventors of the present application screened a WRKY transcription factor that was significantly inhibited by high temperature stress. PcWRKY1 ,right PcWRKY1 The transcriptional levels of transcription factor genes in response to high temperature stress were further determined, and their tissue expression patterns and subcellular sites were analyzed. PcWRKY1 The regulation of the transcription factor gene on the high temperature tolerance of the protoplasts of Polygonatum cyrtonema. This gene is located in the cell nucleus and encodes a protein containing 519 amino acids. It is expressed in the roots, stems, leaves, tubers and seeds of Polygonatum cyrtonema, with the highest expression in seeds. PcWRKY1 The transcription factor gene can inhibit the increase of MDA content under high temperature stress, promote the increase of CAT enzyme activity, and thus improve the tolerance of Polygonatum cyrtonema protoplasts under high temperature stress.

[0007] The first aspect disclosed in the present application relates to a polyantha cyrtonema transcription factor gene PcWRKY1 , whose DNA sequence is shown in SEQ ID NO.1.

[0008] The second aspect disclosed in the present application relates to a transcription factor gene of Polygonatum cyrtonema PcWRKY1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.2.

[0009] In the third aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 Or the protein encoded by it is used to regulate the high temperature resistance of Polygonatum cyrtonema.

[0010] In a fourth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 Or the protein encoded by the same is used in the preparation of reagents for regulating the high temperature resistance of Polygonatum cyrtonema.

[0011] In a fifth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 Or the protein encoded by the same is used in the preparation of reagents for regulating the MDA content and / or CAT enzyme activity of Polygonatum cyrtonema.

[0012] In a sixth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 Or the protein encoded by the same is used in regulating the MDA content and / or CAT enzyme activity of Polygonatum cyrtonema.

[0013] In the seventh aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 It is used in the breeding / identification of varieties / lines of Polygonatum cymbidium with high temperature stress resistance traits.

[0014] In an eighth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 The method is used in the preparation of reagents for breeding / identification of the high temperature stress resistance traits of Polygonatum cymbopogon.

[0015] In a ninth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 It is used to improve the high temperature tolerance of Polygonatum cyrtonema protoplasts.

[0016] In the tenth aspect disclosed in the present application, the polygonatum cyrtonema transcription factor gene PcWRKY1 The invention is applied in the preparation of reagents for improving the high temperature tolerance of protoplasts of Polygonatum cyrtonema.

[0017] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: Polygonatum cyrtonema transcription factor gene PcWRKY1 It is located in the cell nucleus and encodes a protein containing 519 amino acids. It is expressed in roots, stems, leaves, tubers and seeds, with the highest expression in seeds. PcWRKY1 It can inhibit the increase of MDA content in high temperature, promote the increase of CAT enzyme activity, and thus improve the tolerance of Polygonatum cyrtonema protoplasts in high temperature. The analysis and utilization of the gene function will help to understand the heat resistance mechanism of Polygonatum cyrtonema, provide important gene resources and related germplasm resources for the cultivation of heat-resistant Polygonatum cyrtonema varieties, and provide important technical support for its molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In one embodiment of the present application, high temperature stress is PcWRKY1A: RT-qPCR detection of the effects of high temperature stress at different times on the expression of PcWRKY1 B: Effect of simulated field high temperature stress on the expression of PcWRKY1 The impact of expression.

[0019] Figure 2 In one embodiment of this application PcWRKY1 Analysis of expression patterns in different tissues of Polygonatum cymbopogon.

[0020] Figure 3 In one embodiment of this application PcWRKY1 And analysis of its subcellular locations under high temperature stress; scale bar, 10 μm; Bright: bright field; mCherry: cherry red fluorescence; GFP: green fluorescence; Merged: overlay image.

[0021] Figure 4 In one embodiment of the present application, transient overexpression in protoplasts of Polygonatum cyrtonema PcWRKY1 Effects on CAT activity and MDA content under high temperature stress; OE-PcWRKY1: transient overexpression of PcWRKY1 in maize protoplasts; H: high temperature stress treatment; CK: control; Data represent mean ± SD (3 biological replicates); Different letters indicate significant differences among the groups (Duncan's test, P < 0.05). DETAILED DESCRIPTION

[0022] The specific implementation methods of the present invention are described below in conjunction with examples, but the following examples are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0023] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and materials involved are all conventional commercial products unless otherwise specified; the tests and detection methods involved are all conventional methods unless otherwise specified.

[0024] Example 1: WRKY transcription factors in Polygonatum cyrtonema responding to high temperature stress PcWRKY1 Identification This case is based on the previous laboratory screening of a WRKY transcription factor that was significantly inhibited by high temperature stress from the high temperature transcriptome of Polygonatum cyrtonema PcWRKY1 Based on PcWRKY1To investigate whether the transcription level of Polygonatum cyrtonema was affected by high temperature, seedlings cultivated from rhizomes of Polygonatum cyrtonema (2-year-old) with one complete terminal bud were subjected to high temperature treatment (40℃ for 10 h, sampling at 0, 0.5, 1, 2, 4, 8 and 10 h) and simulated field high temperature treatment (based on 28℃, increasing by 2℃ for 1 h until it reached 40℃, and sampling was taken after maintaining it for 2 h). Total RNA was extracted from mature leaves in the middle and upper parts of the leaves, and analyzed by RT-qPCR. PcWRKY1 The results are shown in Figure 2. Figure 1 As shown, PcWRKY1 The expression of was obviously inhibited by high temperature stress, and its expression level was the lowest when the high temperature was treated for 2 h.

[0025] Embodiment 2 PcWRKY1 Tissue expression pattern analysis To test PcWRKY1 The gene expression in various tissues of Polygonatum cyrtonema was analyzed by RT-qPCR. PcWRKY1 The expression of 2-year-old Polygonatum cyrtonema in roots, stems, leaves, tubers and seeds was as follows Figure 2 As shown, PcWRKY1 The gene was expressed in the roots, stems, leaves, tubers and seeds of Polygonatum cyrtonema. The expression levels in the tissues from high to low were seeds, tubers, roots, stems and leaves. There was no significant difference in the expression levels in roots and tubers, indicating that PcWRKY1 It may play an important role in the seeds of Polygonatum cyrtonema.

[0026] Embodiment 3 PcWRKY1 Subcellular localization analysis in protoplasts of Polygonatum cyrtonema To further determine PcWRKY1 The subcellular sites of the plant protein subcellular localization prediction website (http: / / www.csbio.sjtu.edu.cn / bioinf / plant-multi / ) were used for prediction. The results showed PcWRKY1 localized in the cell nucleus. Subsequently, this example used homologous recombination to construct a 35S:PcWRKY1-GFP (Green fluorescent protein-tagged fusion vector), a fusion vector tagged with a known nuclear localization cherry red protein 35S:AtIMP4- mCherry (Cherry Red Fluorescent Protein-labeled Fusion Vector) was used as a control and co-expressed in the PEG-mediated transient expression system of Polygonatum cyrtonema protoplasts. The co-expressed protoplasts were cultured at 25°C for 16 h in the dark and then observed for fluorescence under a laser confocal microscope. Figure 3 As shown, under normal conditions, PcWRKY1 Localized in the cell nucleus.

[0027] In order to further determine whether high temperature stress would affect its subcellular localization, the cultured protoplasts were subjected to high temperature treatment at 38°C for 30 min. Figure 3 As shown, after high temperature treatment, PcWRKY1 The subcellular locations of PcWRKY1 The subcellular location of the protein is consistent with the predicted results and is located in the cell nucleus. Its subcellular location is not affected by high temperature.

[0028] Embodiment 4: PcWRKY1 Regulation of high temperature tolerance of protoplasts of Polygonatum cyrtonema For research PcWRKY1 In this case, PEG-mediated transient genetic transformation in protoplasts of Polygonatum cyrtonema was used to transform PcWRKY1 The overexpression was transferred into the protoplasts of Polygonatum cyrtonema, and then the cells were cultured in the dark for 16 h and then subjected to high temperature stress at 38°C for 1 h. The protoplasts not subjected to high temperature treatment were used as the control group, and their MDA content and CAT enzyme activity were measured. Figure 4 As shown, transient overexpression in protoplasts of Polygonatum cyrtonema PcWRKY1 The increase of MDA content induced by high temperature was inhibited and the increase of CAT enzyme activity was promoted. These results indicate that PcWRKY1 It can improve the tolerance of Polygonatum cyrtonema protoplasts under high temperature stress.

[0029] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

Claims

1. A Polygonatum multiflorum transcription factor gene PcWRKY1 , whose DNA sequence is shown in SEQ ID NO.

1.

2. The polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 The encoded protein has an amino acid sequence as shown in SEQ ID NO.

2.

3. The polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Or the use of the encoded protein according to claim 2 in regulating the high temperature resistance of Polygonatum cyrtonema.

4. The polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Or the use of the encoded protein according to claim 2 in the preparation of a reagent for regulating the high temperature resistance of Polygonatum cyrtonema.

5. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Or the use of the encoded protein according to claim 2 in the preparation of a reagent for regulating the MDA content and / or CAT enzyme activity of Polygonatum cyrtonema.

6. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Or the use of the encoded protein according to claim 2 in regulating the MDA content and / or CAT enzyme activity of Polygonatum cyrtonema.

7. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Application in breeding / identifying varieties / lines of Polygonatum cyrtonema with high temperature stress resistance.

8. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Application in the preparation of reagents for breeding / identification of high temperature stress resistance traits of Polygonatum cyrtonema.

9. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Application in improving the high temperature tolerance of Polygonatum cyrtonema protoplasts.

10. The Polygonatum cyrtonema transcription factor gene according to claim 1 PcWRKY1 Application in the preparation of reagents for improving the high temperature tolerance of Polygonatum cyrtonema protoplasts.

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