Gene vector, construction method therefor and use thereof

Abstract

Provided are a gene vector, a construction method therefor and the use thereof. The gene vector comprises a sequence as shown in SEQ ID NO. 1, the transcription product of the sequence as shown in SEQ ID NO. 1 comprises a sequence as shown in SEQ ID NO. 2, and the transcription product is not specifically recognized and cleaved by miR529. The transcription product of the gene vector provided by the embodiments is not specifically recognized and cleaved by miR529, which can reduce the degradation of a target gene SPL25 transcript caused by specific recognition and cleavage via miR529, thereby leading to an overexpression of the SPL25 gene in grain crops. The overexpression of the SPL25 gene can increase the number of branches per panicle in grain crops, while having no significant effect on other characters, which is beneficial for the direct use of the gene in genetic improvement and molecular breeding.

Description

Gene vector and its construction method and application Technical Field

[0001] The present application relates to the field of transgenic technology, and in particular to a gene vector and a construction method and application thereof. Background Art

[0002] Grain crops are one of the main sources of food for humans and are essential for maintaining normal life and development. With the continuous growth of the population and the continuous reduction of per capita arable land, there is an urgent need to increase grain crop yields to meet the growing food demand. The number of ears of grain crops is one of the most important factors affecting their yield. Therefore, studying the regulatory mechanisms of ear number (such as the number of branches per ear) is of great significance for cultivating grain crops and improving their yields.

[0003] However, in related technologies, the regulation of the number of panicle branches will be accompanied by changes in other traits (such as plant height), which is not conducive to direct application in genetic improvement. Summary of the Invention

[0004] The present application provides a gene vector and a construction method and application thereof, which can help increase the number of ear branches of grain crops while having no obvious effect on other traits.

[0005] In a first aspect, an embodiment of the present application provides a gene vector comprising a sequence as shown in SEQ ID No. 1, a transcription product of the sequence as shown in SEQ ID No. 1 comprising a sequence as shown in SEQ ID No. 2, and the transcription product is not specifically recognized and cleaved by miR529.

[0006] According to any of the aforementioned embodiments of the first aspect of the present application, it includes the sequence shown as SEQ ID No. 3.

[0007] In a second aspect, the present invention provides a method for constructing a gene vector according to the second aspect of the present invention, comprising:

[0008] By utilizing the degeneracy of codons, the miR529 target site in the SPL25 gene sequence was edited, causing base mutations in the sequence without changing the type of amino acids. This resulted in a gene vector that could not complement the miR529 sequence, and the gene vector prevented the transcription product SPL25 mRNA from being specifically recognized and cleaved by miR529.

[0009] According to any of the foregoing embodiments of the second aspect of the present application, further comprising:

[0010] The mutated gene sequence was obtained by using SnapGene Viewer software, homologous sequence alignment, and codon degeneracy to design base mutations;

[0011] The mutated gene sequence was cloned using a kit to construct an rSPL25 gene vector that was not complementary to the miR529 sequence.

[0012] According to any of the aforementioned embodiments of the second aspect of the present application, the base mutation is to mutate 5'-CGTGCTCTCTCTCTT-3' to 5'-AGGGCGTTGAGCCTG-3'.

[0013] In a third aspect, an embodiment of the present application provides an rSPL25 gene, comprising the sequence shown as SEQ ID No. 4.

[0014] In a fourth aspect, an embodiment of the present application provides an application of the rSPL25 gene according to the third aspect of the present application in regulating the number of ear branches in transgenic corn plants.

[0015] In a fifth aspect, the present invention provides a method for cultivating transgenic corn plants, comprising:

[0016] Introducing the rSPL25 gene vector of the first aspect of the present application into Agrobacterium to obtain a target Agrobacterium containing the rSPL25 gene;

[0017] The target Agrobacterium was used to infect the corn inbred line C01, and transgenic corn plants were obtained after antibiotic selection.

[0018] According to any of the aforementioned embodiments of the fifth aspect of the present application, in the transgenic corn plant, the rSPL25 gene is overexpressed.

[0019] The transcription product of the gene vector provided in the embodiment of the present application is not specifically recognized and cleaved by miR529, which can reduce the occurrence of degradation of the target gene SPL25 mRNA due to specific recognition and cleavage by miR529, and thus the SPL25 gene can be overexpressed in food crops. The overexpression of the SPL25 gene can effectively increase the number of ear branches of food crops while having no obvious effect on other agronomic traits, thereby facilitating the direct application of the gene in genetic improvement and molecular breeding.

[0020] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiment below. The accompanying drawings are only for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present application. In the accompanying drawings:

[0022] FIG1 shows a schematic diagram of miR529 recognizing SPL25 / rSPL25 gene transcript target sites provided in some embodiments of the present application;

[0023] FIG2 shows a schematic diagram of the rSPL25 vector map provided in some embodiments of the present application;

[0024] FIG3 shows a schematic diagram of the SPL25 gene sequence alignment between wild-type (WT) and rSPL25 transgenic plants provided in some embodiments of the present application;

[0025] FIG4 shows the tassel images of wild type (WT) and rSPL25 transgenic plants;

[0026] FIG5 shows statistical analysis graphs of tassel traits of wild-type (WT) and rSPL25 transgenic plants, wherein (A) is a statistical analysis graph of the number of tassel branches, (B) is a statistical analysis graph of the number of spikelet pairs, and (C) is a statistical analysis graph of the dry weight of the tassel.

[0027] The above drawings illustrate specific embodiments of the present application, which will be described in more detail below. These drawings and the textual description are not intended to limit the scope of the present application in any way, but rather to illustrate the concepts of the present application to those skilled in the art by reference to specific embodiments. DETAILED DESCRIPTION

[0028] The following embodiments of the technical solution of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only examples and are not intended to limit the scope of protection of the present application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the term "comprising" in the specification and claims of this application and the above-mentioned figure descriptions is intended to cover non-exclusive inclusions.

[0030] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be understood to indicate or imply relative importance or implicitly specify the quantity, specific order, or primary and secondary relationship of the technical features indicated. In the description of the embodiments of this application, the meaning of "plurality" is more than two, unless otherwise specifically defined.

[0031] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0032] In the description of the embodiments of this application, the term "and / or" is simply a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist simultaneously, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship.

[0033] As described in the above background technology, in the relevant technology, the genes that regulate the ear development of grain crops are mainly quantitative trait loci (QTL), and only a few single genes are involved in regulating this trait. Moreover, most of the single genes discovered so far, in addition to regulating the ear trait, also affect other traits such as plant height, which is not conducive to direct application in genetic improvement.

[0034] In view of this, the embodiments of the present application provide a gene vector and a construction method and application thereof, which can help increase the number of ear branches of grain crops while having no obvious effect on other traits.

[0035] In a first aspect, an embodiment of the present application provides a gene vector comprising a sequence as shown in SEQ ID No. 1, a transcription product of the sequence as shown in SEQ ID No. 1 comprising a sequence as shown in SEQ ID No. 2, and the transcription product is not specifically recognized and cleaved by miR529.

[0036] The transcription product of the gene vector provided in the embodiments of the present application is not specifically recognized and cleaved by miR529, which can reduce the occurrence of degradation of the transcription product (i.e., SPL25 mRNA) due to specific recognition and cleavage by miR529. As a result, the SPL25 gene can be overexpressed in food crops. The overexpression of the SPL25 gene can effectively increase the number of ear branches in food crops while having no significant effect on other agronomic traits, thereby facilitating the direct application of the gene in genetic improvement and molecular breeding.

[0037] In this application, SPL (SQUAMOSA promoter-binding protein-like) protein is a type of transcription factor unique to plants and is widely present in green plants. SPL plays an important regulatory role in multiple physiological and biochemical processes such as plant growth regulation, developmental stage transition, morphogenesis, fruit development, and response to adverse stress. MicroRNA (miRNA) is a type of non-coding small molecule RNA with a length of approximately 21 nucleotides that is ubiquitous in organisms. It plays an important role in regulating plant organ development, signal transduction, and response to adverse stress. miRNA is mainly involved in the regulation of gene post-transcriptional levels. In plants, it can cause mRNA degradation or translation inhibition by specific base pairing with target mRNA.

[0038] In the following example of the present application, the gene vector includes the sequence shown as SEQ ID No.3.

[0039] In the second aspect, based on the same inventive concept, the embodiments of the present application provide a method for constructing a gene vector according to the second aspect of the present application, comprising:

[0040] S100. Utilizing the degeneracy of codons, the miR529 target site in the SPL25 gene sequence (such as the sequence shown in SEQ ID No. 5) was edited to cause base mutations in the sequence without changing the type of amino acids, thereby constructing a gene vector that cannot complement the miR529 sequence. Furthermore, the gene vector prevents the transcription product SPL25 mRNA from being specifically recognized and cleaved by miR529.

[0041] In this application, codon degeneracy refers to the situation in which, within one or more triplet genetic codes encoding the same amino acid, the first and second bases are mostly identical, with only the third base being different. For example, ACU, ACC, ACA, and ACG are all codons for threonine, while UGU, UGC, UGA, and UGG are all codons for valine. Thus, a point mutation at the third base of a codon does not affect the type of amino acid translated or the final protein product.

[0042] Please refer to Figure 1 , which shows the target sites recognized by miR529 in SPL25 and rSPL25 transcripts and their encoded amino acids. Red fonts indicate rSPL25 mutant bases. Represents a codon reading frame. A, adenine; G, guanine; C, cytosine; U, uracil; Ser, serine; Cys, cysteine; Ala, alanine; Leu, leucine.

[0043] Furthermore, in some embodiments of the present application, it also includes:

[0044] S110. Using SnapGene Viewer software, homologous sequence alignment, and codon degeneracy, base mutations in the SPL25 gene were designed to obtain the mutated gene sequence.

[0045] S120. The mutated gene sequence is cloned using a kit to construct a gene vector that does not complement the miR529 sequence.

[0046] In the above embodiments, the kit may be any kit well known in the art, such as an In-Fusion PCR Cloning Kit.

[0047] In some embodiments of the present application, the base mutation is to mutate 5'-CGTGCTCTCTCTCTT-3' to 5'-AGGGCGTTGAGCCTG-3'.

[0048] In a third aspect, based on the same inventive concept, the embodiments of the present application provide an rSPL25 gene, comprising the sequence shown as SEQ ID No. 4.

[0049] In the fourth aspect, based on the same inventive concept, an embodiment of the present application provides an application of the rSPL25 gene in the third aspect of the present application in regulating the number of male spike branches in transgenic corn plants.

[0050] In a fifth aspect, based on the same inventive concept, the present application provides a method for cultivating transgenic corn plants, comprising:

[0051] Introducing a gene vector comprising the sequence shown in SEQ ID No. 1 or SEQ ID No. 3 into Agrobacterium to obtain a target Agrobacterium containing the rSPL25 gene;

[0052] The target Agrobacterium was used to infect the corn inbred line C01, and transgenic corn plants were obtained after antibiotic selection.

[0053] According to any of the aforementioned embodiments of the fifth aspect of the present application, in the transgenic corn plant, the rSPL25 gene is overexpressed.

[0054] In the above examples, according to the map shown in FIG2 , the maize inbred line C01 was transformed by Agrobacterium-mediated method, and transgenic maize plants were obtained after antibiotic screening.

[0055] In the examples of the present application, the rSPL25 gene of the rSPL25 transgenic plant was amplified by PCR, and the PCR product was sequenced and compared with the sequence of the wild type (WT). The results are shown in Figure 3. In Figure 3, the SPL25 gene sequences of the wild type (WT) and the rSPL25 transgenic plant are compared, wherein MT represents the rSPL25 base mutant plant, / / represents the gene sequence with consistent alignment, and red font represents the rSPL25 mutant base.

[0056] FIG4 shows the tassel images of wild-type (WT) and rSPL25 transgenic plants. As can be seen from FIG4 , the transgenic corn plants obtained using the gene vector provided in the examples of the present application have significantly increased tassel branch numbers.

[0057] Figure 5 shows statistical analysis of tassel traits for wild-type (WT) and rSPL25 transgenic plants. (A) shows the number of tassel branches, (B) shows the number of spikelet pairs, and (C) shows the dry weight of the tassel. rSPL25-1 and rSPL25-5 represent two independent transgenic events. As can be seen in Figure 5, transgenic maize plants derived using the gene vectors provided in the Examples of this application exhibit higher tassel branch numbers, higher spikelet pairs, and higher tassel dry weight than wild-type maize plants.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application, and they should all be included in the scope of the claims and specification of the present application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall within the scope of the claims.

Claims

1. A gene vector, characterized in that: It includes the sequence shown as SEQ ID No.1, the transcription product of the sequence shown as SEQ ID No.1 includes the sequence shown as SEQ ID No.2, and the transcription product is not specifically recognized and cut by miR529.

2. The SPL25 gene vector according to claim 1, characterized in that Including the sequence shown as SEQ ID No.

3.

3. A method for constructing a gene vector as claimed in claim 1 or 2, characterized in that: include: By utilizing the degeneracy of codons, the miR529 target site in the SPL25 gene sequence is edited so that the sequence undergoes base mutations without changing the type of amino acids, thereby constructing a gene vector that cannot complement the miR529 sequence, and the gene vector prevents the transcription product SPL25 mRNA from being specifically recognized and cleaved by miR529.

4. The construction method according to claim 3, characterized in that: Also includes: The mutated gene sequence was obtained by using SnapGene Viewer software, homologous sequence alignment, and codon degeneracy to design base mutations; The mutated gene sequence is cloned by a kit to construct a gene vector that is not complementary to the miR529 sequence.

5. The construction method according to claim 4, characterized in that: The base mutation is to mutate 5'-CGTGCTCTCTCTCTT-3' to 5'-AGGGCGTTGAGCCTG-3'.

6. A rSPL25 gene, characterized in that: Including the sequence shown as SEQ ID No.

4.

7. Use of the rSPL25 gene as claimed in claim 6 in regulating the number of ear branches of transgenic corn plants.

8. A method for cultivating transgenic corn plants, characterized in that: include: Introducing the gene vector described in claim 1 or 2 into Agrobacterium to obtain a target Agrobacterium containing the rSPL25 gene; The target Agrobacterium was used to infect the corn inbred line C01, and transgenic corn plants were obtained after antibiotic selection.

9. The cultivation method according to claim 8, characterized in that: In the transgenic corn plants, the rSPL25 gene is overexpressed.

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