Zjnoot2 gene for regulating formation of zizyphus jujuba mill cv. dama's thorn and application thereof in cultivating thornless zizyphus jujuba mill

Abstract

The application provides a ZjNOOT2 gene for regulating formation of Ziziphus jujuba Mill. thorn and application of the ZjNOOT2 gene in breeding thornless Ziziphus jujuba Mill., and the nucleotide sequence of the gene is shown as SEQ ID NO:1. The application first clones a thorn formation related gene ZjNOOT2 from Ziziphus jujuba Mill., and performs directional knockout on the gene through CRISPR / Cas9 to obtain three types of knockout mutants. Phenotype analysis shows that the Ziziphus jujuba Mill. plants with the ZjNOOT2 gene knocked out exhibit a less-thorn or thornless phenotype, and the trait can be stably inherited. The results show that the ZjNOOT2 gene is involved in key regulation of thorn formation of Ziziphus jujuba Mill. The application provides a functional gene target and a precise breeding strategy for thornless Ziziphus jujuba Mill. variety improvement, and can be used for breeding a new thornless Ziziphus jujuba Mill. variety, and effectively solves production problems such as difficult harvesting and easy damage.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a ZjNOOT2 gene that regulates the formation of thorns in jujube and its application in the cultivation of thornless jujube. Background Technology

[0002] Sour jujube (Ziziphus jujube var. spinosa) is a plant belonging to the genus Ziziphus in the family Rhamnaceae. Its kernel (sour jujube kernel) is a medicinal and edible resource with significant medicinal value. It has remarkable effects in calming the nerves, improving sleep, anti-oxidation, and immune regulation, and market demand has been increasing year by year. However, wild and cultivated sour jujubes generally have well-developed stipular thorns, which are long and hard. These thorns not only seriously hinder manual harvesting and mechanized operations, but also easily pierce the fruit skin during picking and transportation, leading to an increased incidence of post-harvest diseases, a decrease in the rate of marketable fruit, and a significant increase in production costs.

[0003] Although traditional hybridization or mutation breeding holds promise for improving thorny traits, the complex genetic background of jujube, the long breeding cycle, and the lack of stable, genetically derived thornless mutants in existing germplasm resources have prevented the successful development of commercially viable thornless jujube varieties. Thorns, as crucial defense organs for plants against external stresses, are subject to strict genetic regulation during their development. In recent years, gene editing technologies such as CRISPR / Cas9 have enabled precise improvement of target traits in various woody plants, providing an efficient technical pathway for thornless jujube breeding. However, the application of this technology hinges on identifying the key functional genes regulating thorn formation and development in jujube. To date, no research reports on the cloning and functional verification of jujube thorn-related genes have been published domestically or internationally. This lack of key gene information severely restricts the practical application of gene editing technology in the innovation of thornless jujube germplasm.

[0004] In view of this, there is an urgent need in this field to screen key genes that regulate the development of jujube thorns and to create new germplasm with significantly reduced or completely absent thorns using gene editing technology, so as to solve the harvesting problem that the industry has long faced, and at the same time provide a theoretical basis and gene resources for the molecular improvement of thorn traits in jujube plants. Summary of the Invention

[0005] The purpose of this invention is to provide a ZjNOOT2 gene that regulates the formation of thorns in jujube and its application in the cultivation of thornless jujube, so as to solve the industrial problems of difficult harvesting, high production costs, and lack of thornless varieties caused by the well-developed stipular thorns of jujube in the existing technology.

[0006] To achieve the above objectives, the technical solution specifically adopted by the present invention is as follows: The inventors, through transcriptome sequencing and differentially expressed gene screening, identified a significantly highly expressed, unknown functional gene in the young stipular spines of jujube, named ZjNOOT2. This gene encodes a protein containing a conserved domain, and its expression characteristics are highly spatiotemporally synchronized with the development of jujube spines, suggesting its involvement in the initiation and developmental regulation of spine primordia. Functional experiments confirmed a positive correlation between ZjNOOT2 gene expression and jujube spine formation: normal gene expression maintains primordia cell division and differentiation, promoting jujube spine development and maturation; gene editing to knock out or disrupt the function of this gene interferes with or blocks normal primordia development, resulting in jujube exhibiting a stipular spine-turning-leaf or stipular spine-less phenotype. This invention elucidates for the first time the crucial role of the ZjNOOT2 gene in regulating jujube spine formation, providing a core functional gene target for molecular breeding of spineless jujube.

[0007] Based on the above findings, the present invention provides the following technical solution: At the gene level, this invention provides a ZjNOOT2 gene that regulates the formation of jujube thorns, the nucleotide sequence of which is shown in SEQ ID NO:1. This gene is a natural functional gene isolated and cloned from jujube, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2. Obtaining this gene sequence provides a molecular basis for the design and functional verification of subsequent gene editing targets.

[0008] At the application level, this invention provides the application of the aforementioned ZjNOOT2 gene in the cultivation of thornless jujube plants. The core technical idea of ​​this application is to block or interfere with the genetic program of jujube thorn development by inhibiting the expression of the endogenous ZjNOOT2 gene or reducing the biological activity of its encoded protein, thereby achieving the technical effect of reducing or completely inhibiting jujube thorn formation. The method of inhibiting gene expression can flexibly employ any combination of one or more of the following: CRISPR / Cas9 gene knockout, RNA interference (RNAi), and antisense nucleic acid technology. These methods can all achieve varying degrees of functional loss of the ZjNOOT2 gene.

[0009] At the tool level, this invention provides a CRISPR / Cas9 gene editing and recombinant vector for jujube based on the ZjNOOT2 gene. The design logic of this vector is as follows: utilizing the efficient site-specific editing capability of the CRISPR / Cas9 system, site-specific DNA double-strand breaks are introduced into the coding region of the ZjNOOT2 gene, triggering the cell's endogenous non-homologous end joining (NHEJ) repair pathway, thereby generating random insertion or deletion (indel) mutations at the target site, causing frameshifting of the gene coding frame, and ultimately achieving functional knockout of the ZjNOOT2 gene. Specifically, the recombinant vector contains two core functional units: a Cas9 expression cassette and an sgRNA expression cassette. The sgRNA expression cassette contains a specific sgRNA fragment targeting the coding region of the ZjNOOT2 gene, and the nucleotide sequence of the sgRNA is cagatgcatcctggcggca (the target site is adjacent to the PAM motif NGG). The recombinant vector backbone can be a pYLCRISPRCas9 vector, and can also contain plant selection marker genes (such as hygromycin resistance genes or GFP fluorescent marker genes) to facilitate the screening and identification of transformants, as well as to facilitate subsequent hybridization breeding to screen for offspring without foreign gene insertion.

[0010] At the methodological level, this invention provides a breeding method for thornless jujube. The overall technical idea of ​​this method is: using the ZjNOOT2 gene as the target site, by inhibiting the expression of this gene in jujube plants, the thorn development program of jujube is interfered with or blocked, thereby obtaining thornless jujube plants with improved thorn traits. As a preferred implementation method, this method uses the above-mentioned CRISPR / Cas9 gene editing recombinant vector to knock out the endogenous ZjNOOT2 gene of jujube. The specific implementation path includes the following steps: (1) Recombinant vector construction: select the knockout target site in the coding region of ZjNOOT2 gene, synthesize sgRNA with the sequence cagatgcatcctggcggca, amplify and purify the sgRNA-ZjNOOT2 fragment by PCR, and construct the pYLCRISPRCas9-ZjNOOT2 recombinant plasmid after enzyme digestion and ligation; (2) Jujube genetic transformation: use Agrobacterium rhizogenes-mediated genetic transformation method to infect jujube. Materials, the recombinant vector was introduced into jujube cells to obtain ZjNOOT2 gene-edited transformation seedlings; (3) Screening of positive edited plants: Design specific amplification primers targeting the ZjNOOT2 gene (forward primer F: gctggactatctgaacctgctg; reverse primer R: tatggacacttgcccactgtac), PCR amplification of the genomic DNA of the transformation seedlings, the amplification products were identified by Sanger sequencing, and the indel mutation status of the target region was analyzed by comparing with the wild type sequence, and ZjNOOT2 gene knockout positive edited plants were screened to obtain thornless jujube germplasm with improved thorn traits.

[0011] Regarding species applicability, the core species of this invention is jujube (jujube). Ziziphus jujuba (Rhamnaceae), but the application scope of the technical solution is not limited to this. Considering the conservation of functional homologous genes among different species, the technical concept of this invention is also applicable to any plant containing the ZjNOOT2 homologous gene or its functional homologous gene, including but not limited to hairy-leaved jujube of the Rhamnaceae family, other species of the genus Ziziphus, and other thorny economic plants such as black locust of the Fabaceae family and barberry of the Berberidaceae family. Therefore, those skilled in the art will understand that technical solutions that use the suppression of the ZjNOOT2 homologous gene as a strategy to improve the thorny trait of plants are all extensions of the technical concept of this invention.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) It fills the gap in the key gene resources for the formation of jujube thorns.

[0013] Currently, there are no research reports on the cloning and functional verification of genes related to thorns in jujube, either domestically or internationally. This invention is the first to clone and identify the ZjNOOT2 gene from jujube, clarifying its key positive regulatory function in thorn formation. This provides the first clear functional gene target for molecular improvement of thorn traits in jujube plants and even other thorny plants, filling a gap in core gene resources in this field.

[0014] (2) Successfully created thornless jujube germplasm with stable phenotype and industrial application value.

[0015] This invention utilizes CRISPR / Cas9 gene editing technology to directionally knock out the ZjNOOT2 gene, obtaining three mutant systems with different editing types. Among them, zjnoot2-3 exhibits complete thornlessness and lacks leaf-like stipule structures, and its phenotype is stably inherited after propagation, subculture, and transplantation. This is the first time, both domestically and internationally, that genetically stable thornless jujube edited materials have been obtained, directly solving the long-standing problems of harvesting difficulties and fruit damage in the industry.

[0016] (3) It significantly reduced the labor costs and economic losses in jujube production.

[0017] The introduction of thornless traits eliminates the need for special thorn-proofing measures in the cultivation, harvesting, and transportation of jujubes, enabling routine and mechanized operations and significantly reducing labor costs. At the same time, it avoids post-harvest diseases and quality decline caused by thorns and scratches on the fruit skin, significantly improving the marketable fruit rate and providing direct economic benefits.

[0018] (4) It provides a technical paradigm that can be referenced for the trait improvement of other thorny economic plants.

[0019] The technical route of "key gene identification → CRISPR / Cas9 targeted knockout → thornless germplasm creation" established in this invention is not only applicable to jujube, but can also be extended to other thorny economic plants such as hairy jujube, black locust, and Sichuan pepper, and has broad methodological value and application prospects. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 Electrophoresis diagram of the PCR amplification products of the ZjNOOT2 gene; Figure 2 A schematic diagram of the ZjNOOT2 gene knockout site and information on mutant editing; Figure 3 Different phenotypes of wild-type and ZjNOOT2 mutant jujube; In the figure: (a) is WT; (b) is Zjnoot2-1; (c) is Zjnoot2-2; (d) is Zjnoot2-3.

[0021] Figure 4 Potted seedlings of the ZjNOOT2 mutant of jujube; In the figure: (a) is Zjnoot2-1; (b) is Zjnoot2-2; (c) is Zjnoot2-3. Detailed Implementation

[0022] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0023] I. Source of Materials In the following examples, the pYLsgRNA-AtU3d and pYLCrisprCas9p35s-H-35sEGFP plasmids were constructed and preserved in our laboratory; Agrobacterium rhizogenes K599 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. The RNAprep Pure plant total RNA extraction kit was purchased from TIANGEN; the PrimerScript™ II 1st Strand cDNA Synthesis Kit was purchased from Takara; the KOD ONE high-fidelity enzyme was purchased from TOYOBO; and the Ultra-Universal TOPO Cloning Kit was purchased from Vazyme. Escherichia coli DH5α competent cells were preserved in our laboratory. All other reagents, unless otherwise specified, were of analytical grade and purchased from common reagent suppliers.

[0024] II. Primer Information The primer sequences used in this invention are shown in Table 1.

[0025] Table 1. Primer sequence information used in this invention

[0026] III. Examples Example 1: Cloning and Sequence Analysis of the ZjNOOT2 Gene Extraction of total RNA from jujube and synthesis of cDNA Using young jujube tissue as material, total RNA was extracted using the RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN), following the kit's instructions. Subsequently, first-strand cDNA was synthesized using the PrimerScript™ II 1st Strand cDNA Synthesis Kit (Takara) according to the kit's instructions.

[0027] Cloning of the ZjNOOT2 gene Using synthesized jujube cDNA as a template, PCR amplification was performed using the high-fidelity enzyme KOD ONE (TOYOBO) and specific primers ZjNOOT2-F / ZjNOOT2-R (sequences shown in Table 1). The amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 68℃ extension for 20 s, for 34 cycles; 68℃ final extension for 5 min; and storage at 4℃. The amplified products were separated by 1% agarose gel electrophoresis (results shown in Table 1). Figure 1 The target fragment was recovered using an agarose gel DNA recovery kit (TIANGEN). The recovered fragment was ligated into a cloning vector provided by the Ultra-Universal TOPO Cloning Kit (Vazyme), transformed into E. coli DH5α competent cells, and single colonies were picked for sequencing to obtain the CDS sequence of the ZjNOOT2 gene and its deduced amino acid sequence.

[0028] Sequence information of the ZjNOOT2 gene The full-length CDS sequence of the ZjNOOT2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2.

[0029] SEQ ID NO:1 (nucleotide sequence): SEQ ID NO:2 (amino acid sequence): MSSLEESLRSLSLDYLNLLINGQAFSDVTFSVEGRLVHAHRCILAARSLLFFRKFFCGPDPPSGLDPVSGSRMTNSGTSTSRPGNTQVIPVNSVGYEVFLLLLQFLYSGQVSIVPQKHEPRPNC GERGCWHTHCTSAVDLALDTLAAARSFGVEQLALLTQKQLASMVEKASIEDVMKVLLASRKQDMHQLWTTCSHLVAKSGLPPEVLAKHLPIDVVAKIEELRLKSSLARRSLMPHHHHHHHHHD LGAAADLEDQKIRRMRRALDSSDVELVKLMVMGEGLNLDEALALHYAVENCSREVVKALLELGAADVNYPAGPAGKTPLHIAAEMVSPDMVAVLLDHHADPNVRTVDGVTPLDVLRTLTSDFL FKGAVPGLTHIEPNKLRLCLELVQSAALVLSREEGNANAPSSTTIYPPMSDDHSSSSNSSNIANLNLDSRLVYLNLGASAASAQMGSRMDGDDDHSSHNSHREAMNRHGSGGCDPTMYHHSHDY Example 2: Construction of CRISPR / Cas9 knockout vector for ZjNOOT2 gene Target design and sgRNA synthesis A specific target site was selected in the coding region of the ZjNOOT2 gene, and its sgRNA sequence is: cagatgcatcctggcggca (this target sequence is immediately followed by the PAM motif CGG). Using the pYLsgRNA-AtU3d vector as a template, PCR amplification was performed using primer pairs NOOT2:gRT1 / gR-R and UF / NOOT2:AtU3dT1 (sequences are shown in Table 1) to obtain the sgRNA fragment.

[0030] Recombinant vector construction The sgRNA fragment was amplified a second time using the primer combination Pps-R / Pgs-L (sequences shown in Table 1) to construct a complete sgRNA expression cassette. Using Golden Gate Cloning technology, the sgRNA expression cassette was ligated into the gene-editing vector pYLCrisprCas9p35s-H-35sEGFP, which had been digested with the appropriate enzymes, to construct a recombinant plasmid named pYLCrisprCas9-ZjNOOT2. The recombinant plasmid was transformed into *E. coli* DH5α, and after single-colony sequencing confirmed its accuracy, it was transformed into *Agrobacterium rhizogenes* K599 competent cells using a freeze-thaw method for subsequent genetic transformation.

[0031] Example 3: Genetic transformation of jujube and obtaining transgenic plants Genetic transformation mediated by *Agrobacterium rhizogenes* K599 was used to infect jujube explants. Specifically, *Agrobacterium rhizogenes* K599 containing the recombinant plasmid pYLCrisprCas9-ZjNOOT2 was inoculated into TY liquid medium containing the appropriate antibiotic and cultured at 28°C with shaking until the logarithmic growth phase. The cells were collected by centrifugation, resuspended in infection buffer (containing 100 μmol / L acetylsyleugenol), and the OD600 was adjusted to 1.0. Sterile jujube seedling leaves were selected as explants and placed in *Agrobacterium* culture for 15–20 min. After infection, excess culture was absorbed with sterile filter paper, and the explants were transferred to co-culture medium and cultured in the dark at 25°C for 2–3 days. After co-culture, the explants were transferred to selection medium containing hygromycin (selection marker) and carbenicillin sodium (antibacterial agent) for further culture. Under a fluorescence microscope, positive callus or adventitious shoots expressing green fluorescent protein (GFP) were selected and induced to root after multiple subcultures to obtain transgenic plants.

[0032] Example 4: Molecular detection and phenotypic analysis of transgenic plants Molecular detection Genomic DNA was extracted from wild-type (WT) and transgenic plants using the CTAB method. PCR amplification of the target site region was performed using specific primers CrNoot2-F / CrNoot2-R (sequences shown in Table 1), and the products were sent to a sequencing company for Sanger sequencing. The sequencing results were decoded and analyzed using the DSDecode online website (http: / / skl.scau.edu.cn / dsdecode / ) to determine the editing type of the target site. The results showed that three ZjNOOT2 knockout lines with different editing types were successfully obtained, named zjnoot2-1, zjnoot2-2, and zjnoot2-3 (target site editing information is shown in Table 1). Figure 2 ).

[0033] Phenotypic analysis Wild-type (WT) and ZjNOOT2 knockout lines were propagated and then transplanted into sandy soil. Observations revealed significant phenotypic changes in the knockout lines compared to the wild-type (normally developed stipular spines) (see results). Figure 3 and Figure 4 Among them, zjnoot2-1 and zjnoot2-2 plants were thornless, but had a certain chance of developing leaf-like stipule structures; while zjnoot2-3 plants were completely thornless and lacked leaf-like stipule structures. After transplanting and conventional cultivation, the thornless phenotype was stably maintained. Figure 4 This indicates that the spineless trait obtained after knocking out the ZjNOOT2 gene can be stably inherited.

[0034] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A ZjNOOT2 gene that regulates the formation of jujube thorns, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A protein encoded by the ZjNOOT2 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. The application of the ZjNOOT2 gene as described in claim 1 in the cultivation of thornless jujube plants, characterized in that, The application is to inhibit the expression of the ZjNOOT2 gene in jujube or reduce the biological activity of the ZjNOOT2 protein, so as to reduce or inhibit the formation of jujube thorns.

4. A CRISPR / Cas9 gene editing recombinant vector for jujube based on the ZjNOOT2 gene as described in claim 1, characterized in that, The recombinant vector contains a Cas9 expression cassette and an sgRNA expression cassette. The sgRNA expression cassette contains a specific sgRNA fragment targeting the coding region of the ZjNOOT2 gene. The nucleotide sequence of the sgRNA is cagatgcatcctggcggca. The recombinant vector is a pYLCRISPRCas9 backbone vector.

5. A breeding method for thornless jujube, characterized in that, The breeding method includes inhibiting the expression of the ZjNOOT2 gene as described in claim 1 in jujube plants to obtain thornless jujube plants.

6. The method for breeding thornless jujube according to claim 5, characterized in that, The knockout of the endogenous ZjNOOT2 gene in jujube using the CRISPR / Cas9 gene editing recombinant vector as described in claim 4 specifically includes the following steps: (1) Construction of recombinant vector: Select the knockout target site in the coding region of ZjNOOT2 gene and synthesize sgRNA with the sequence cagatgcatcctggcggca; sgRNA-ZjNOOT2 fragment was recovered by PCR amplification, purification and recovery, and pYLCRISPRCas9-ZjNOOT2 recombinant plasmid was constructed after enzyme digestion and ligation. (2) Genetic transformation of jujube: The jujube material was infected with Agrobacterium rhizogenes-mediated genetic transformation method to obtain ZjNOOT2 gene-edited transformed seedlings; (3) Screening of positive edited plants: Specific amplification primers targeting the ZjNOOT2 gene were designed, and PCR amplification was performed on the genomic DNA of the transformed seedlings. The amplification products were identified by Sanger sequencing, and ZjNOOT2 gene knockout positive edited plants were screened.

7. The thornless jujube breeding method according to claim 6, characterized in that, The specific amplification primer sequences are as follows: Forward primer F: gctggactatctgaacctgctg; Reverse primer R: tatggacacttgcccactgtac.

8. The CRISPR / Cas9 gene editing recombinant vector according to claim 4, characterized in that, The recombinant vector also contains plant selection marker genes.

9. The application according to claim 3, characterized in that, The expression of the ZjNOOT2 gene was suppressed by any combination of RNA interference, CRISPR knockout, and antisense nucleic acid.

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