Promoter zmmir529-pro specifically expressed during early development of maize tassel primordium and use thereof

Abstract

A promoter ZmMIR529-pro specifically expressed during the early development of a maize tassel primordium and a use thereof. The nucleotide sequence of the promoter ZmMIR529-pro is as shown in SEQ ID NO: 1, or a nucleotide sequence complementary to the sequence as shown in SEQ ID NO: 1, or a nucleotide sequence having 75% or more homology with the sequence as shown in SEQ ID NO: 1 or the complementary sequence of the sequence as shown in SEQ ID NO: 1. The promoter ZmMIR529-pro specifically expressed during the early development of a maize tassel primordium can initiate the specific expression of a target gene in a maize tassel, thereby avoiding the adverse effect caused by continuous expression of the target gene in plant tissue. The promoter has significant value for specific modification of the traits of maize tassel using a transgenic means and for application in maize genetic breeding.

Description

Promoter ZmMIR529-pro specifically expressed in early development of maize tassel primordium and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, in particular to a kind of for plant genetic breeding and trait improvement field, especially a kind of promoter ZmMIR529-pro specifically expressed in early development of maize tassel primordium and application thereof. BACKGROUND

[0002] During the different stages of plant growth and development, and the interaction between plants and the environment, its structure, morphology and function will change dramatically. These temporal and spatial changes are controlled by a series of genes expressed in specific organs, tissues, at specific times, and these precise switches that control the temporal and spatial expression of plant genes are plant tissue-specific promoters. Plant tissue-specific promoters can initiate the expression of target genes in specific tissues of plants, such as flowers, stems, leaves, seeds or roots. This is of great significance to the biological safety and genetic modification of transgenic crops.

[0003] There have been relevant researches on plant tissue-specific promoters in model plants (Arabidopsis, tobacco), food crops (corn, rice, wheat, etc.) and economic crops (cotton, rapeseed, peanut, etc.), and a variety of tissue-specific promoters have been isolated, including leaf-specific promoters, stem-specific promoters, root-specific promoters, flower-specific promoters and seed-specific promoters. Therefore, finding some promoters that can more specifically regulate gene expression during cell, tissue and organ development is of great significance for basic research and crop improvement under certain conditions.

[0004] Corn is a typical monoecious plant, and the development of tassel is synchronized with that of ear, and tassel does not produce seeds, and has a certain competitive effect on seed formation and nutrient collection. Therefore, reducing the tassel in agricultural production is beneficial to reduce the energy competition and shading effect. The development of tassel involves a variety of metabolic activities, and gene expression is active, mainly involving transcription factors, protein metabolism, carbohydrate metabolism, stress, signal transduction, material transport, cell composition and other aspects and pathways. Therefore, it is of great application value to deeply explore the tassel-specific promoter of corn and to improve the tassel traits and yield of corn through genetic engineering. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art. The purpose of the present application is to provide a kind of promoter ZmMIR529-pro specifically expressed in early development of maize tassel primordium and application thereof, to solve the problem of inaccurate control of the development of maize tassel in the prior art.

[0006] The technical scheme adopted by the present application to solve the technical problems is:

[0007] The first aspect of the present application provides a promoter ZmMIR529-pro specifically expressed in the early development of maize tassel primordium, the nucleotide sequence of which is the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence having a complementary sequence to the sequence shown in SEQ ID NO: 1, or a nucleotide sequence having more than 75% homology with the sequence shown in SEQ ID NO: 1 and also having a promoter function; or a nucleotide sequence having more than 75% homology with the complementary sequence of the sequence shown in SEQ ID NO: 1 and also having a promoter function.

[0008] Further, the nucleotide sequence of the promoter ZmMIR529-pro specifically expressed in the early development of maize tassel primordium has more than 90% homology with the sequence shown in SEQ ID NO: 1, or more than 90% homology with the complementary sequence of the sequence shown in SEQ ID NO: 1.

[0009] It should be noted that any DNA molecule artificially modified, having more than 75% identity with the nucleotide sequence shown in SEQ ID NO: 1 and having a promoter function, is derived from the nucleotide sequence of the present application and is equivalent to the nucleotide sequence of the present application.

[0010] In combination with the embodiments, the nucleotide sequence of the promoter ZmMIR529-pro has more than 99% homology with the sequence shown in SEQ ID NO: 1; or more than 99% homology with the complementary sequence of the sequence shown in SEQ ID NO: 1.

[0011] The promoter ZmMIR529-pro specifically expressed in the early development of maize tassel primordium provided by the present application can initiate the specific expression of a target gene in the tassel of maize, thereby avoiding the adverse effects of the continuous expression of the target gene in other tissues of the plant, which is of great value for the specific improvement of the tassel traits of maize using transgenic means and the application in the genetic breeding of maize.

[0012] The second aspect of the present application provides a recombinant vector, which comprises the promoter ZmMIR529-pro of the first aspect and other regulatory elements, the promoter ZmMIR529-pro being operably linked to the other regulatory elements, and the promoter ZmMIR529-pro controlling the initiation of the operable unit.

[0013] According to an embodiment of the present application, the other regulatory elements include, but are not limited to, a replication origin, an enhancer, a screening marker, a multiple cloning site, a transcription termination signal, a fluorescent group, a protein tag, etc.

[0014] According to an embodiment of the present application, the recombinant vector is obtained by introducing the promoter ZmMIR529-pro into a plasmid.

[0015] According to an embodiment of the present application, the recombinant vector is pCAMBIA3301-ZmMIR529pro::GUS.

[0016] The third aspect of the present application provides a transgenic plant comprising the promoter ZmMIR529-pro of the first aspect.

[0017] According to an embodiment of the present application, the transgenic plant is prepared by introducing the recombinant vector of the second aspect into a plant.

[0018] The present application also provides an application of the above-mentioned promoter ZmMIR529-pro in breeding of monocotyledonous plant tassel early development tissue-specific expression. The tassel tissue is an important reproductive tissue of monocotyledonous crops, and the provided promoter ZmMIR529-pro can be used to regulate the specific expression of a target gene in the monocotyledonous plant tassel early development tissue, thereby being applied in breeding. The monocotyledonous plants mentioned include, but are not limited to, corn, wheat, asparagus, bamboo shoots, onions, garlic, leeks, yams, rice, sorghum, lilies, etc. According to a preferred embodiment, the monocotyledonous plant mentioned can be a tassel tissue of corn.

[0019] The present application also provides an application of the above-mentioned promoter ZmMIR529-pro in breeding of monocotyledonous plant tassel tissue-specific expression. The promoter ZmMIR529-pro can initiate the specific expression of a target gene in the tassel tissue, thereby being applied in breeding of monocotyledonous plant tassel tissue-specific expression.

[0020] The present application has the following beneficial effects:

[0021] The promoter ZmMIR529-pro of the present application, which is specifically expressed in the early development of the tassel primordium of corn, can initiate the specific expression of a plant gene in the tassel part, thereby avoiding the adverse effects caused by the continuous expression of a target gene in other tissues of a plant, and having important significance and wide application value for targeted trait improvement of corn through genetic engineering. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a comparison chart of expression values of the ZmMIR529-pro driven MIR529 gene in different tissues by qRT-PCR, wherein V6 is the sixth leaf fully expanded stage, and R1 is the silk stage of the female ear.

[0023] Figure 2 is an expression chart of the ZmMIR529-pro driven MIR529 gene in the maize male ear meristem, wherein A is an expression chart of the ZmMIR529-pro promoter gene, which mainly occurs in the flower ear primordia, branch primordia and spikelet pair primordia, and B is a negative control material.

[0024] Figure 3 is a map of the recombinant vector pCAMBIA3301-ZmMIR529pro::GUS containing the above-mentioned promoter ZmMIR529-pro. Embodiment of the present application

[0025] The embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, and the embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0026] The present application collects samples of 6 different tissues of maize at V6 and R1 stages (V6 is the sixth leaf fully expanded stage; R1 is the silk stage of the female ear), sets up 3 biological replicates for each group, and performs RNA extraction. The specificity of the ZmMIR529-pro promoter in the male ear site is analyzed by using qRT-PCR technology. In addition, the specificity of the ZmMIR529-pro promoter driven gene product in the male ear is analyzed by using in situ hybridization technology, and the results show that the promoter ZmMIR529-pro can specifically express the exogenous gene in the maize male ear, thereby avoiding the adverse effects of continuous expression of the exogenous gene in other tissues of the plant, which has important value for using transgenic means to specifically improve the male ear traits of maize and applying to maize genetic breeding.

[0027] According to the specific embodiment, the nucleotide sequence of the maize male ear primordium early development specific expression promoter ZmMIR529-pro provided is shown in SEQ ID NO: 1.

[0028] SEQ ID NO: 1 is shown as follows:

[0029]

[0030] Due to the genetic diversity of the corn inbred, the nucleotide sequence of the promoter ZmMIR529-pro can also be a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1; or a nucleotide sequence having more than 75% homology with the nucleotide sequence shown in SEQ ID NO: 1 and having a promoter function; or a nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1 having more than 75% homology and having a promoter function. According to the specific embodiments, the homology mentioned is more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%.

[0031] The promoter includes various regulatory elements, such as cis-regulatory elements, cis-regulatory element abscisic acid response, cis-regulatory element light response element, cis-regulatory element meristem regulatory element, MYBHv1 binding region element, light response element, gibberellin response element, and the like, as shown in Table 1 below.

[0032] Table 1 Analysis of regulatory elements in the ZmMIR529-pro promoter sequence

[0033] Element name Sequence Function A-box CCGTCC Cis-regulatory element ABRE ACGTG Cis-regulatory element abscisic acid response element ACEGACACGTATG Cis-regulatory element light response element CAT-box GCCACT Cis-regulatory element meristem regulatory element CCAAT-box CAACGG MYBHv1 binding region element G-box CACGTT Cis-regulatory element light response GA-motif ATAGATAA Light response element GARE-motif TCTGTTG Gibberellin response element I-box gGATAAGGTG Light response element LTR CCGAAA Cis-regulatory element low temperature response MYB CAACAGTATA-box TATA core transcription regulatory start element TGA-element AACGAC Auxin response element

[0034] The present application also provides a recombinant vector comprising the above-mentioned promoter and further comprising other regulatory elements, wherein the promoter and the other regulatory elements are operably linked. The other regulatory elements mentioned include, but are not limited to, at least one of the following: replication origin, enhancer, selectable marker, multiple cloning site, transcription termination signal, fluorescent group, protein tag. The DNA replication origin ori is responsible for monitoring the origin of plasmid replication, and prokaryotic DNA molecules only have a single replication origin; eukaryotic DNA molecules have multiple replication origins. The selectable marker includes various antibiotic resistance genes, which are beneficial for subsequent positive clone selection. The commonly used markers for prokaryotic cell screening include ampicillin / Amp, chloramphenicol Cam, kanamycin Kan and tetracycline Tet, etc.; and the markers used for eukaryotic cell screening include puromycin Puro, neomycin neo / G418, hygromycin Hyg or Hygro, etc. The multiple cloning site (MCS) is usually located downstream of the promoter, and has multiple enzyme cutting sites of endonucleases. Foreign DNA can be inserted into the plasmid by enzyme cutting / ligation. Transcription termination signal: after transcription to AAUAAA (AATAAA), mRNA will be connected with polyA and transcription will be terminated. The common transcription termination signal site is SV40 polyA, abbreviated as SV40 pA. Fluorescent group: proteins that produce fluorescence by laser excitation, usually in the form of independent expression or fusion expression with target genes, are ingeniously designed on the plasmid, and play an important role in tracking, positive detection and imaging. Such as red fluorescent protein (RFP), cherry red fluorescent protein (mCherry), green fluorescent protein (GFP) and enhanced green fluorescent protein (EGFP) and the like. Protein tags are mostly short peptides, which are usually fused to the 3' end or 5' end of the target gene, such as flag, HA, myc and his, etc. With the tag, the target protein can be easily detected when there is no antibody for the target protein.

[0035] The recombinant vector can be obtained by introducing the promoter gene into a plasmid. The available plasmids include, but are not limited to, pCAMBIA3301, pCAMBIA1300, pCUNm, etc.

[0036] The technical solutions of the present application will be described below through specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as a limitation on the scope of protection of the present application. In the absence of any description, the reagents or materials used in the examples can be obtained by commercial purchase.

[0037] Example 1

[0038] The ZmMIR529-pro is specifically expressed in the tassel of maize by RNA extraction and qRT-PCR analysis. The maize used is B73 (Zea maysssp. mays L.) B73 plants, which are cultivated in a 26℃, 14-hour photoperiod artificial plant culture room. The cultivation method is a common method in the prior art, and outdoor natural light cultivation is also possible.

[0039] The specific method is as follows:

[0040] 1. RNA extraction

[0041] The corn seeds are soaked and germinated, then planted in nutrient soil, and cultivated to the V6 stage (the sixth leaf fully expanded stage) and the R1 stage (the female ear silking stage). The leaves, stems and roots of the V6 stage, and the leaves, female ears and tassels of the R1 stage are taken, and the TRIzol method is used for RNA extraction. The above different tissue samples are set up in three biological replicates, and qRT-PCR experiments are performed.

[0042] The results are shown in Figure 1, and the sequencing result analysis shows that the ZmMIR529-pro gene expression in the tassel of the R1 stage is the highest.

[0043] 2. Promoter information analysis

[0044] The cis-acting elements in the promoter are analyzed by online analysis of the promoter software PlantCARE (Bioinformatics.psb.ugent.be / webtools / plantcare / html / ) and MEME SUITE (https: / / meme-suite.org / meme / info / status?service=M). Most of the cis-acting elements are responsive to light, temperature and plant hormones, which indicates that external light and temperature conditions play an important role in regulating tassel development, and the development of tassel meristem primordia is regulated by internal hormones.

[0045] 3. The specific expression of the ZmMIR529-pro promoter driven gene product in the tassel is analyzed by in situ hybridization technology, and the results are shown in Figure 2. In which, A is the expression diagram of the ZmMIR529-pro promoter gene, which mainly appears in the flower spike primordium, branch primordium and spike pair primordium (A in the figure, each abbreviation is IM flower spike primordium, BM branch primordium and SPM spike pair primordium), B is the negative control material.

[0046] Example 2

[0047] The recombinant vector pCAMBIA3301-ZmMIR529pro::GUS was prepared according to Example 2, and a map thereof is shown in FIG. 3. The recombinant vector was prepared by the following method:

[0048] 1. The ZmMIR529pro promoter was amplified using the maize B73 material as a template and the pCAMBIA3301-GUS plasmid as a template, and the amplification system (50 μL) was prepared. After PCR, electrophoresis analysis was performed, and after the target band was cut and recovered.

[0049] 2. The pCAMBIA3301-GUS plasmid was digested with HindIII and BamHI, and the enzyme digestion system (20 μL) included 3.0 μL of pCAMBIA3301-GUS, 2.0 μL of CutSmart, 0.2 μL of HindIII, 0.2 μL of BamHI, and 14.6 μL of ddH2O. The reaction conditions were 37 ℃, and the reaction time was 60 min.

[0050] 3. The recovered ZmMIR529pro promoter fragment was connected to the linearized pCAMBIA3301-GUS vector digested with HindIII and BamHI by homologous recombination technology, and the connection assembly reaction system was: 50 ℃ reaction for 1 h, and the DH5α competent cells were transformed by heat shock method. The homologous recombination system included 0.4 μL of pCAMBIA3301-GUS, 5.0 μL of PCR fragment with a concentration of 20 ng·μL -1 of Recombinase 3.0 μL.

[0051] 4. The recombinant product pCAMBIA3301-ZmMIR529pro::GUS was transformed into DH5α competent E. coli by heat shock method, and after resistant culture for 12 h, a single colony was selected and cultured on a shaking table for 3-4 h, and bacterial liquid PCR identification was performed. The PCR product was analyzed by 0.5% agarose gel electrophoresis. The PCR reaction conditions were: 94 ℃ for 3 min, 30 cycles (94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 45 s), and 72 ℃ for 2 min.

[0052] 5. The positive clone was selected and cultured in 30 μL of liquid LB medium containing 50 mg·mL-1 of kanamycin and 30 mL of liquid LB medium at 37 ℃ on a shaking table overnight. The plasmid was extracted and subjected to plasmid PCR amplification, and the PCR product was identified by 1% concentration agarose gel electrophoresis.

[0053] 6. The plasmid identified by PCR was sent to Shanghai Shengong Bioengineering Co., Ltd. Guangzhou Branch for sequencing. After comparison, pCAMBIA3301-ZmMIR529pro::GUS was consistent with the sequencing results, the sequence corresponding to the sequencing peak graph was normal, indicating that the recombinant plasmid was successfully constructed.

[0054] 7. The recombinant vector pCAMBIA3301-ZmMIR529pro::GUS was introduced into the isolated immature maize embryos by Agrobacterium-mediated method, then the transformed immature embryos were induced to produce callus; then the callus was regenerated and maize positive seedlings were screened, and the expression specificity of ZmMIR529-pro promoter in maize tassel tissue was detected and verified.

[0055] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example.

[0056] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A promoter ZmMIR529-pro specifically expressed in the early stage of tassel primordium development of maize, characterized in that, The nucleotide sequence of the promoter ZmMIR529-pro is selected from at least one of the following: (a) has the nucleotide sequence shown in SEQ ID NO: 1; or (b) has a nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1; or (c) has a nucleotide sequence with more than 75% homology to the sequence shown in SEQ ID NO: 1, and has a promoter function; (d) has a nucleotide sequence with more than 75% homology to the complementary sequence of the sequence shown in SEQ ID NO: 1, and has a promoter function.

2. The promoter ZmMIR529-pro according to claim 1, characterized in that, The nucleotide sequence of the promoter ZmMIR529-pro has more than 90% homology to the sequence shown in SEQ ID NO: 1; or more than 90% homology to the complementary sequence of the sequence shown in SEQ ID NO:

1.

3. The promoter ZmMIR529-pro according to claim 1, characterized in that, The nucleotide sequence of the promoter ZmMIR529-pro has more than 99% homology to the sequence shown in SEQ ID NO: 1; or more than 99% homology to the complementary sequence of the sequence shown in SEQ ID NO:

1.

4. A recombinant vector, characterized in that, The promoter ZmMIR529-pro of any one of claims 1-3, and other regulatory elements, wherein the promoter ZmMIR529-pro is operably linked to the other regulatory elements, and the promoter ZmMIR529-pro controls the initiation of the other regulatory elements.

5. The recombinant vector of claim 4, wherein, The other regulatory elements include at least one of a replication origin, an enhancer, a selectable marker, a multiple cloning site, a transcription termination signal, a fluorescent group, and a protein tag.

6. The recombinant vector of claim 4, wherein, The recombinant vector is obtained by introducing the promoter ZmMIR529-pro into a plasmid; The recombinant vector is pCAMBIA3301-Zm MIR529pro::GUS.

7. A transgenic plant, characterized in that, The promoter ZmMIR529-pro of any one of claims 1-3.

8. The transgenic plant of claim 7, wherein, Prepared by introducing the recombinant vector of any one of claims 4-6 into a plant.

9. Use of the promoter ZmMIR529-pro of any one of claims 1-3 in breeding for specific expression in early development tissues of a monocotyledonous plant tassel; The monocotyledonous plant is at least one of corn, wheat, asparagus, bamboo shoot, onion, garlic, leek, yam, rice, sorghum, and lily.

10. Use of the promoter ZmMIR529-pro of any one of claims 1-3 in breeding for specific expression in a monocotyledonous plant tassel tissue; The monocotyledonous plant is at least one of corn, wheat, asparagus, bamboo shoot, onion, garlic, leek, yam, rice, sorghum, and lily.

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