A sweet potato peel-specific strong promoter IbGRPpro and its application

Abstract

The present invention belongs to the fields of genetic engineering and molecular biology, and specifically relates to a strong sweet potato peel-specific promoter IbGRPpro and its application. The strong sweet potato peel-specific promoter IbGRPpro is selected from any of the following nucleotide sequences: (a) the sequence shown in SEQ ID NO:1; (b) a DNA molecule sequence that hybridizes with the sequence shown in SEQ ID NO:1; (c) a DNA molecule sequence that has greater than 90% similarity to the sequence shown in SEQ ID NO:1 and can regulate the specific expression of a target gene in sweet potato peel tissue; (d) a sequence that is partially truncated or assembled based on the sequence shown in SEQ ID NO:1, and the truncated or assembled sequence can still function as a promoter. The strong sweet potato peel-specific promoter IbGRPpro of the present invention can direct gene expression in a peel-specific manner and has great application prospects in the fields of plant genetic transformation and genetic engineering, providing a basis for specifically improving the potato peel cell barrier and the expression of antibacterial genes.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and molecular biology, and particularly relates to a sweet potato peel-specific strong promoter IbGRPpro and an application thereof. Background Art

[0002] Sweet potato is an important crop used as food, feed and industrial raw material, and as a new energy plant in the world today, its status is particularly important. my country is the world's largest sweet potato producer, with an annual planting area of ​​3.482 million hectares / hm2. 2 , accounting for 43.0% of the world's total cultivated area and an annual output of 73.361 million tons, accounting for 71.1% of the world's total production. When sweet potatoes have high moisture content, they are highly susceptible to soft rot during storage. If left untreated, this can lead to widespread rot. Therefore, screening and identifying potato peel-specific promoters can facilitate the subsequent expression of specific disease-resistance genes in the peel, improving the peel's properties and preventing pathogen infection without compromising the quality of the flesh, thereby extending the storage and shelf life of sweet potatoes. This is of great significance to the industry.

[0003] Sweet potato is a vegetatively propagated crop. Interspecific and intraspecific hybrid incompatibility severely limits resource utilization and parental assortment in sweet potato breeding. Breeding practices have shown that the availability of sweet potato germplasm resources is scarce, severely hampering conventional breeding. Therefore, genetic engineering to improve sweet potato disease resistance is a viable approach.

[0004] Promoters, when used in genetic transformation to transfer exogenous genes into plant explants for expression, play a crucial role in the expression level and specificity of the introduced transgene. Tissue-specific promoters have been extensively studied and used due to their safety, but the search for promoters in sweet potato peel tissue remains inadequate. There is an urgent need to identify and isolate strong, highly specific, and efficient promoters specifically for peel tissue, construct vectors for the expression of exogenous genes in sweet potato tubers, and control the specific and high expression of marker genes, Cas nuclease-encoding genes, and other functional exogenous genes in peel tissue. This approach has significant application value in improving transgenic efficiency and plant safety, while also saving experimental time and costs. It holds great promise for application in plant genetic transformation and genetic engineering, providing a foundation for subsequent specific improvements in the peel cell barrier and the expression of antimicrobial genes, thereby reducing common sweet potato pests and diseases such as soft rot, root rot, and insect infestations, extending storage life, improving sweet potato quality, and increasing its economic value.

[0005] In view of this, the technical problem to be solved by the present invention is to explore a sweet potato peel-specific promoter, which can be expressed in large quantities in the potato peel, which is of great significance for subsequently driving the specific expression of specific disease-resistant functional genes in the potato peel, improving the properties of the potato peel, avoiding pathogen infection, without affecting the quality of the potato flesh, and extending the storage time and shelf life of the sweet potato. Summary of the Invention

[0006] The present invention aims to provide a sweet potato peel-specific strong promoter IbGRPpro and its application, and to enable the promoter to be specifically and highly expressed in potato peel tissue by constructing an expression vector.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0008] The first aspect of the present invention provides a sweet potato peel-specific strong promoter IbGRPpro, wherein the sweet potato peel-specific strong promoter IbGRPpro is selected from any one of the following nucleotide sequences:

[0009] (a) the sequence shown in SEQ ID NO: 1;

[0010] (b) a DNA molecule sequence that hybridizes with the sequence shown in SEQ ID NO: 1;

[0011] (c) a DNA sequence having a similarity of 90% or more to the sequence shown in SEQ ID NO: 1 and capable of regulating the specific expression of the target gene in potato peel tissue;

[0012] (d) A sequence that is partially truncated or assembled based on the sequence shown in SEQ ID NO: 1, and the truncated or assembled sequence can still function as a promoter.

[0013] The second aspect of the present invention provides a gene expression cassette specifically expressed in potato peel tissue, wherein the gene expression cassette comprises the sweet potato peel-specific strong promoter IbGRPpro as described in the first aspect of the present invention.

[0014] In some technical solutions of the present invention, the expression cassette further includes a terminator.

[0015] In some technical solutions of the present invention, the downstream of the nucleic acid sequence further comprises any one of the following three genes or a combination thereof: a screening marker gene, a nuclease encoding gene, and a target functional gene.

[0016] In some technical solutions of the present invention, the selection marker gene is selected from any one of the following:

[0017] 1) Genes that encode enzymes that produce color changes in plants;

[0018] 2) Genes that encode light-emitting compounds in plants;

[0019] 3) Genes that confer resistance to antibiotics, chemical agents, or herbicides.

[0020] In some technical solutions of the present invention, the screening marker gene is GUS Gene, GFP / YFP Fluorescent genes, Luc luciferase gene, NPT II Kanamycin resistance gene, HPT Hygromycin resistance gene or Bar Herbicide resistance genes.

[0021] In some technical solutions of the present invention, the nuclease encoding gene is CRISPR / Cas Nuclease-encoding genes.

[0022] In some technical solutions of the present invention, the CRISPR / Cas The nuclease encoding gene is SpCas9 Genes and their variants encode genes, ScCas9 Gene, SaCas9 Gene, Cas12a Gene or Cas13a Gene.

[0023] In some technical solutions of the present invention, the target functional gene is a gene specifically expressed in potato peel tissue.

[0024] The third aspect of the present invention provides a recombinant vector comprising the gene expression cassette according to the second aspect of the present invention.

[0025] The fourth aspect of the present invention provides a recombinant bacterium, which comprises the recombinant vector according to the third aspect of the present invention.

[0026] The fifth aspect of the present invention provides a use of the sweet potato peel-specific strong promoter IbGRPpro as described in the first aspect of the present invention in screening transgenic sweet potato peel cells.

[0027] The sixth aspect of the present invention provides a use of the sweet potato peel-specific strong promoter IbGRPpro as described in the first aspect of the present invention in gene editing of a sweet potato peel tissue cell line.

[0028] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a sweet potato peel-specific strong promoter IbGRPpro, which can guide gene expression in a potato peel-specific manner, has good application prospects in the fields of plant genetic transformation and genetic engineering, and provides a basis for subsequent specific improvement of potato peel cell barriers and expression of antibacterial genes, thereby reducing common sweet potato diseases and pests such as soft rot, root rot, and insect infestation, extending storage time, and improving sweet potato quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a bar graph showing the expression levels of the IbGRPpro promoter in various sweet potato tissues.

[0030] Figure 2These are the staining results of GUS expression driven by the blank wild type, 35S:GUS, and IbGRPpro:GUS promoters in various tissues of sweet potato tuber fibrous roots.

[0031] Figure 3 This is the activity analysis of GUS of wild type, 35S:GUS, and IbGRPpro:GUS in the flesh, peel, fibrous roots, and fibrous root sections of plants. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] Example 1 Sweet potato ( Ipomoea batatas ) Acquisition of IbGRPpro promoter

[0034] 1. Screening of sweet potato peel tissue-specific expression promoters

[0035] In order to isolate and clone the sweet potato peel tissue-specific expression promoter, Figure 1 Different letters indicate statistically significant differences. Two-way ANOVA followed by T-test indicated significant differences at P < 0.05. Data are expressed as mean ± standard deviation (n = 3). The present invention screened out potato peel-specific genes that are highly expressed in potato peel tissue from sweet potato transcriptome data. IbGRP .

[0036] Example 2 Amplification of the IbGRPpro Promoter and Construction of the Promoter Expression Vector

[0037] An 888 bp fragment (nucleotide sequence shown in SEQ ID NO: 1, named fragment 1) was cloned from the sweet potato variety Xushu 18. IbGRP The promoter sequence of the gene was amplified by designing primers. The upstream and downstream primers are shown in Table 1:

[0038] Table 1 IbGRP Primer combination for amplifying gene promoter sequences

[0039]

[0040] An 888 bp IbGRPpro fragment to be detected was obtained. The target band was ligated to the pMD19-T cloning vector and transformed into competent Escherichia coli DH5α. The cells were cultured at 37°C for 20 h, and single-clone bacterial liquid PCR analysis was performed. The positive clone bacterial liquid was sequenced and verified. The bacterial liquid with the correct sequencing results was selected for expansion and plasmid extraction.

[0041] The vector pMDC162 was digested with Pac I and Kpn I to recover the large vector fragment. At the same time, primers were designed to amplify the obtained plasmid. The upstream and downstream primers are shown in Table 2:

[0042] Table 2 Plasmid amplification primers

[0043]

[0044] The recovered large vector fragment was ligated with the target fragment amplified from the plasmid using T4 DNA Ligase from Treasure Biotechnology (Dalian) Co., Ltd. for homologous recombination. The reaction product was transformed into competent E. coli DH5α cells, and the screening antibiotic was Kan (50 μg / mL). Single E. coli clones were isolated and identified by PCR using Kan (50 μg / mL). After screening, positive bacterial cultures were sent to Qingke for sequencing. Successful sequencing results were compared with the target sequence, and plasmids were extracted using a Rapid Plasmid DNA Miniprep Kit (Beijing Zhuangmeng International Biological Co., Ltd.) based on the comparison results and set aside.

[0045] Example 3 Functional Verification of the Potato Peel Tissue-Specific Strong Promoter IbGRPpro

[0046] 1. Transformation of Agrobacterium with promoter expression vector

[0047] (1) Take out 200 μL of EHA105 competent cells (purchased from Beijing Biode Biotech Co., Ltd.) from a -80°C freezer, thaw on ice, add 1 μg of the IbGRPpro promoter expression vector obtained in Example 2, and mix well.

[0048] (2) Freeze in liquid nitrogen for 1 min and incubate at 37°C for 5 min.

[0049] (3) Add 800 μL of LB liquid medium and incubate at 28°C for 2-6 h.

[0050] (4) Take 100 μL of the bacterial solution and spread it evenly onto LB solid medium (containing 100 μg / mL rifampicin (Rif) and 25 μg / mL kanamycin (Kan)). Seal the culture dish and incubate the dish at 28°C for 2 days.

[0051] (5) A single colony that was positive for PCR was inoculated into LB liquid medium containing 100 μg / mL Rif and 25 μg / mL Kan. The culture was cultured at 28°C for 30 h until the logarithmic growth phase. An appropriate amount of Agrobacterium was diluted 30-fold with liquid MS medium for later use. This is the Agrobacterium culture solution containing the IbGRPpro promoter expression vector.

[0052] 2. Agrobacterium transformation of sweet potato skin, flesh and sweet potato fiber roots

[0053] Activate the transformed Agrobacterium tumefaciens and shake overnight until the OD600 is between 0.8 and 1.0. Collect the cells by centrifugation at 5500 rpm for 7 min at room temperature, remove the supernatant, and resuspend the cells in infection solution. The OD600 should be between 1.0 and 2.0. The specific configuration of the infection solution is shown in Table 3:

[0054] Table 3 Preparation method of infection solution

[0055]

[0056] (2) Let the bacterial solution stand for 2-3 hours in a dark place at 28°C.

[0057] (3) Cut the potato peels and 0.5 cm thick potato slices, as well as the sweet potato fiber roots, soak them in the pretreated bacterial solution, and vacuum them with a circulating water vacuum pump for 10 min.

[0058] After drying the potato peel surface and the bacterial liquid in the root hairs, the potato peel was placed in 2,4-D solid culture medium with 200 μL / 100 mL of acetosyringyl AS and cultured in the dark for 2 days.

[0059] 3. GUS staining and result analysis

[0060] (1) The above-mentioned Agrobacterium-transformed sweet potato skin, sweet potato flesh and sweet potato fiber roots were used as the experimental group, the wild-type sweet potato was used as the blank group, and the sweet potato infected with the 35S promoter was used as the control group. The above three groups of experimental materials were stained.

[0061] (2) Take a photo of the discolored material. The result is as follows: Figure 2 shown.

[0062] (3) Image J software was used to quantitatively analyze the images of the transiently transformed sweet potatoes, so that the degree of color change of the materials could be accurately and effectively evaluated. The data obtained from Image J software was input into Excel software, and the results were as follows: Figure 3 shown.

[0063] Figure 2 In the figure, A is the flesh, skin, fibrous roots and cross section of the blank wild-type sweet potato test tube plantlet; B is the flesh, skin, fibrous roots and cross section of the 35S:GUS sweet potato test tube plantlet; C is the flesh, skin, fibrous roots and cross section of the 1bGRPpro:GUS sweet potato test tube plantlet. Figure 2 As shown in A, the sweet potato flesh driven by IbGRPpro is transparent milky white after bleaching, with only a few small blue spots; Figure 2As shown in B, the sweet potato peel infected with IbGRPpro and 35S strong promoter turned dark blue after bleaching; Figure 2 As shown in C, the cross section of the sweet potato fibrous root shows that the inner part of the root is transparent milky white after bleaching, and the epidermis of the root is dark blue. Figure 3 Different letters indicate statistically significant differences (P < 0.05) as determined by two-way ANOVA followed by a Student's T-test. Data are presented as mean ± standard deviation (n = 3). Both IbGRPpro and the negative blank control showed low GUS activity in potato flesh, while both IbGRPpro and the strong 35S promoter showed high GUS activity in potato peel.

[0064] These results clearly show that IbGRPpro is abundantly expressed in potato peel but barely expressed in other tissues, consistent with the FPKM values. Overall, the results strongly suggest that this promoter is a suitable candidate to direct gene expression in a potato peel-specific manner.

[0065] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional 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 invention.

[0066] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A sweet potato peel-specific strong promoter IbGRPpro, characterized in that The nucleotide sequence of the sweet potato peel-specific strong promoter IbGRPpro is shown in SEQ ID NO:

1.

2. A gene expression cassette specifically expressed in potato peel tissue, characterized in that: The gene expression cassette comprises the sweet potato peel-specific strong promoter IbGRPpro according to claim 1.

3. The gene expression cassette according to claim 2, wherein The gene expression cassette further comprises a terminator; and the downstream of the sweet potato peel-specific strong promoter IbGRPpro further comprises any one or a combination of the following three genes: a screening marker gene, a nuclease encoding gene, and a target functional gene.

4. The gene expression cassette according to claim 3, wherein The screening marker gene is selected from any one of the following: 1) Genes that encode enzymes that produce color changes in plants; 2) Genes that encode light-emitting compounds in plants; 3) Genes that confer resistance to antibiotics, chemical agents, or herbicides.

5. The gene expression cassette according to claim 3, wherein The nuclease encoding gene is CRISPR / Cas Nuclease-encoding genes.

6. The gene expression cassette according to claim 3, wherein The target functional gene is a gene specifically expressed in sweet potato peel tissue.

7. A recombinant vector, characterized in that The recombinant vector comprises the gene expression cassette according to claim 2.

8. A recombinant bacterium, characterized in that The recombinant bacteria comprises the recombinant vector according to claim 7.

9. Use of the sweet potato peel-specific strong promoter IbGRPpro according to claim 1 in screening transgenic sweet potato peel cells.

10. Use of the sweet potato peel-specific strong promoter IbGRPpro according to claim 1 in gene editing of a sweet potato peel tissue cell line.

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