Lolium perenne LpZAT12 gene for regulating plant tillering and application of lolium perenne LpZAT12 gene

Abstract

The invention belongs to the technical field of ryegrass plant tillering regulation and control methods, and discloses a ryegrass LpZAT12 gene for regulating and controlling plant tillering and application, and the nucleotide sequence of the LpZAT12 gene is as shown in SEQ ID NO: 1; the amino acid sequence is shown as SEQ ID NO: 2; and the nucleotide sequence of the corresponding promoter is as shown in SEQ ID NO: 3. Transgenic ryegrass obtained through a perennial ryegrass genetic transformation system is subjected to phenotypic analysis, and the result shows that the gene inhibits tillering of perennial ryegrass. The discovery of the LpZAT12 gene provides a new gene resource for cultivating a new perennial ryegrass variety with strong tillering ability. The overexpression of the gene can reduce the management cost of the lawn and prolong the management period; knockout of the gene can promote plant tillering, increase plant biomass and improve industrial benefits, and application of the gene has important production practice significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of methods for regulating tillering of ryegrass plants, and in particular relates to a ryegrass LpZAT12 gene for regulating plant tillering and an application thereof. Background Art

[0002] Perennial ryegrass is a high-quality cool-season grass with an elegant plant shape and rapid turf growth, making it widely used in turfgrass landscaping. Its strong regeneration and high nutritional value make it a popular feed for cattle and sheep. The turf and forage properties of perennial ryegrass determine its importance in practical production, making research on perennial ryegrass crucial. Ryegrass has poor stress tolerance and is subject to both biotic and abiotic stresses during its growth and development, severely impacting its commercial value. Perennial ryegrass has a strong tillering capacity, which is closely related to the environment, particularly temperature. Varieties with strong tillering ability are more likely to establish turf and better meet the needs of the livestock industry. Tillering is a crucial step in ryegrass growth and development, involving processes such as gene expression and regulation and protein interactions within the organism. Tillering capacity varies among different perennial ryegrass varieties, but research on the key factors influencing tillering in these varieties is limited.

[0003] Among the existing public documents, the one closest to the present invention is the "Introduction of Brassica juncea-type ZAT12 into indica rice PR116 by particle gun bombardment to obtain drought-tolerant transgenic plants" reported in 2016; this study demonstrated that heterologous overexpression of the C2H2-type zinc finger transcription factor ZAT12 can improve resistance to multiple stresses in rice plants, but the authors only recorded the survival rate and conventional greenhouse morphology (plant height, heading) of PCR-positive plants, and did not detect branching-yield traits such as tiller number, lateral bud elongation, or aboveground-underground biomass ratio, nor did they explore the physiological function of the target gene in perennial grasses of the Poaceae family.

[0004] This approach still has significant technical gaps: ① The use of 35S or pBinAR fully driven promoters results in high background expression of ZAT12 throughout the plant, potentially interfering with the endogenous growth regulatory network and making it difficult to accurately grasp the balance between tillering and vegetative growth; ② No species-specific functional loss (knockout) or promoter engineering comparisons have been conducted in high-tillering forage grasses such as ryegrass, resulting in the unknown mechanism of action of ZAT12 in the negative regulatory pathway of tillering, species differences, and its actual improvement value for turf / forage yield; ③ The existing technology does not provide a combination of homologous promoters and gene editing to avoid the stress-growth trait conflict caused by heterologous overexpression, and lacks systematic verification of the downstream transcriptional response of lateral bud inhibitors (such as TB1 / BRC1), thus limiting its scalability in accurately improving the tiller number and biomass of grass crops. Summary of the Invention

[0005] In response to the problems existing in the prior art, the present invention provides a ryegrass LpZAT12 gene for regulating plant tillering and its application.

[0006] The present invention is achieved by providing a ryegrass LpZAT12 gene for regulating plant tillering, wherein the nucleotide sequence of the LpZAT12 gene is shown in SEQ ID NO: 1.

[0007] Furthermore, the amino acid sequence of the LpZAT12 gene is shown in SEQ ID NO: 2.

[0008] Furthermore, the promoter sequence of the LpZAT12 gene is shown in SEQ ID NO: 3.

[0009] Furthermore, the ryegrass LpZAT12 gene that regulates plant tillering is used to promote ryegrass plant tillering.

[0010] Furthermore, the specific application method is to knock out the LpZAT12 gene of ryegrass through genetic engineering methods.

[0011] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0012] This study identified and cloned, for the first time, a key gene, LpZAT12, that regulates tillering in perennial ryegrass. This gene is a negative regulator of tillering in perennial ryegrass. The functional elucidation of this gene provides key evidence for understanding the molecular mechanisms of tillering regulation in grasses. The discovery of this gene provides a new genetic resource for improving the quality of turfgrasses, including perennial ryegrass.

[0013] The ZAT12 gene, a key gene for regulating tillering in turfgrasses such as perennial ryegrass, is being used in plant breeding. Overexpression of the nucleotide sequence of this gene (SEQ ID NO. 1) in plants using an Agrobacterium-mediated genetic transformation system significantly inhibited tillering. Knocking out this gene using gene editing techniques effectively promotes tillering, increasing the rate of turfgrass establishment, enhancing its ornamental value while also increasing biomass and improving its forage value. The application of this gene has important practical significance and potential.

[0014] The present invention utilizes an Agrobacterium-mediated genetic transformation system to overexpress the nucleotide sequence of this gene (SEQ ID NO: 1) in plants, significantly inhibiting plant tillering. Using gene editing technology, knocking out this gene effectively promotes plant tillering, increasing the rate of lawn formation, improving the ornamental value while increasing biomass and enhancing feeding value. The application of this gene has important production practical significance and potential.

[0015] This invention addresses the current problem of limited tillering capacity in ryegrass, making precise regulation difficult through conventional breeding methods. It provides a functional gene, LpZAT12, for regulating plant tillering. Prior art has not yet clarified the molecular mechanisms underlying ryegrass tillering regulation, and lacks clear target genes for efficient breeding or gene editing, limiting the efficiency of genetic improvement of turfgrass and forage yield traits. Therefore, there is an urgent need to identify regulatory factors with clear functions to support molecular precision improvement of tillering capacity.

[0016] This study cloned and functionally characterized the LpZAT12 gene (SEQ ID NO: 1) in ryegrass for the first time, clarifying the key role of the ZAT transcription factor (SEQ ID NO: 2) it encodes in negatively regulating tillering. Expression analysis and functional validation revealed that LpZAT12 expression was significantly upregulated during the tillering growth inhibition phase, suggesting a negative regulatory role in inhibiting tillering. Gene editing experiments demonstrated that knocking out the LpZAT12 gene significantly increased tiller number, indicating that this gene is an effective target for regulating tillering.

[0017] Furthermore, the present invention provides the promoter sequence of the LpZAT12 gene (SEQ ID NO: 3), providing a foundation for studying its expression regulation mechanism and future construction of artificial regulatory elements. This promoter has specific expression activity in tillering-related tissues and can be used to drive specific expression elements to achieve controllable activation or inhibition of tillering regulatory pathways. Furthermore, this promoter can also be used as a molecular tool to construct expression systems targeted for tillering regulation, showing broad application prospects in forage breeding.

[0018] By identifying and functionally analyzing the LpZAT12 gene, this study establishes a complete technical chain for identifying tillering regulatory genes, validating their function, and then improving their targeted properties. This approach fills the gap in the availability of genetic resources for tillering regulation in ryegrass. This invention not only addresses the technical bottleneck of the lack of clear functional targets for tillering trait improvement but also provides a foundation for direct application in molecular breeding or gene editing, possessing significant theoretical value and practical implications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the structure of the original plant overexpression vector pCAMBIA1301U provided in an embodiment of the present invention.

[0020] Figure 2 This is the identification result of the perennial ryegrass LpZAT12 overexpression positive strain provided by the embodiment of the present invention, wherein Figure A is the PCR gel image of gDNA identification; Figure B is the RT-qPCR result image.

[0021] Figure 3Figure A in the middle shows the tillering-related phenotypes of the perennial ryegrass LpZAT12 overexpression-positive strains; Figure B shows the statistical diagram of the number of individual plants in the perennial ryegrass LpZAT12 overexpression-positive strains. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Example 1: LpZAT12 gene expression pattern analysis and functional verification

[0024] In this example, the expression of the LpZAT12 gene in different ryegrass tissues (roots, stems, leaves, and tiller bases) was analyzed using real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the expression level of the LpZAT12 gene in the tiller base tissue was significantly higher than that in other tissues, and was particularly upregulated in the early stages of tillering, suggesting that it is closely related to tillering inhibition. Further, using a plant overexpression vector construction system, the LpZAT12 gene was introduced into Arabidopsis for functional verification. It was found that the number of tillers in the transgenic plants was significantly reduced, confirming its negative regulatory role in regulating tillering.

[0025] Example 2: CRISPR knockout of the LpZAT12 gene promotes ryegrass tillering

[0026] To verify the effectiveness of LpZAT12 in ryegrass, this example utilized CRISPR / Cas9 gene editing technology to design a specific gRNA to target and knock out the LpZAT12 gene. Ryegrass callus was then transformed using Agrobacterium-mediated transfection to obtain a stably inherited LpZAT12 knockout strain. Field pot experiments showed that compared to the wild type, the knockout strains had an average increase of over 35% in tiller number, a more compact plant shape, and stronger growth. This result further confirms the role of the LpZAT12 gene in negatively regulating ryegrass tillering and provides an effective means for improving forage tillering traits through gene editing.

[0027] In summary, the present invention not only reveals the expression patterns of LpZAT12 and its functions in model plants through molecular-level analysis, but also achieves trait improvement in target plants through gene editing technology. Combining these two approaches, from basic research to application validation, establishes a complete research and application closed loop.

[0028] The two implementation paths provided by the present invention represent two typical paradigms of functional gene verification and gene application improvement, respectively, providing a reference template for the subsequent improvement of tillering traits in other grass plants, and also verifying the feasibility and practical value of LpZAT12 as a molecular breeding target.

[0029] I. Identification of the key gene ZAT12 regulating tillering in perennial ryegrass and its promoter

[0030] (1) Isolation of ZAT12, a key gene regulating tillering in perennial ryegrass

[0031] The ZAT12 gene was isolated from perennial ryegrass, and its sequence is shown in SEQ ID No. 1. The desired fragment was amplified using perennial ryegrass leaf cDNA as a template, and the specific steps are as follows:

[0032] The amplification primer pair was designed based on the ZAT12 sequence predicted by the previous transcriptome data:

[0033] ZAT12 forward primer: 5'-CACTCGGTTCCTTCTTCG-3',

[0034] ZAT12 reverse primer: 5′-CTAGCTAGACGAAGAGCT-3′.

[0035] PCR amplification of the ZAT12 gene was performed using the cDNA reverse transcribed from RNA extracted from perennial ryegrass leaves as a template and the primer pair mentioned above.

[0036] PCR system:

[0037] 2 × PhantaFlashMasterMix 25 μl Forward primer 2.5 μl Reverse primer 2.5 μl cDNA template 2.5 μl ddH2O 17.5 μl total 50 μl

[0038] PCR conditions:

[0039]

[0040] The amplified product, namely the ZAT12 gene, was purified; the amplified ZAT12 gene was connected to a Topo vector (purchased from Novazonics), positive clones were screened and sequenced to obtain the desired full-length gene; the clone was named Topo-ZAT12 plasmid.

[0041] (2) Isolate the promoter fragment of the ZAT12 gene and design primer pairs based on the perennial ryegrass reference genome.

[0042] The promoter fragment was amplified using RT-PCR technology using gDNA from perennial ryegrass leaves as a template. The specific steps are as follows:

[0043] Pro-ZAT12-F1: 5'-CCTTGGCAGTATTGTTCG-3',

[0044] Pro-ZAT12-R1: 5'-CGAACTGAAACCCGCAGACAT-3',

[0045] PCR amplification was performed using gDNA extracted from perennial ryegrass leaves as a template and the above-mentioned specific primers and RT-PCR system.

[0046] PCR system:

[0047]

[0048]

[0049] PCR conditions:

[0050]

[0051] The target fragment (approximately 2500 bp) was excised and recovered from the gel to obtain purified DNA. Due to the risk of nonspecific amplification, a second pair of primers was designed for PCR amplification using the purified DNA as a template based on a known reference sequence.

[0052] Pro-ZAT12-F2: 5'-TTTATATTACGCGGAAAACA-3',

[0053] Pro-ZAT12-R2: 5'-GAACGTGCTGCTTACGATCG-3',

[0054] PCR amplification was performed using the purified DNA as a template, the above-mentioned specific primers and the RT-PCR system.

[0055] The amplified product, namely the 2000 bp promoter sequence of the ZAT12 gene (SEQ ID No. 3), was purified. The amplified Pro-ZAT12 gene was ligated into a Topo vector, and positive clones were screened and sequenced to obtain the desired promoter fragment.

[0056] 2. Construction of ZAT12 gene overexpression vector and genetic transformation of perennial ryegrass

[0057] To better elucidate the function of the ZAT12 gene, the cloned gene sequence was overexpressed in perennial ryegrass and its function was verified by observing the phenotype of the transgenic plants and measuring the number of individual plants. The specific steps are as follows:

[0058] (1) Construction of ZAT12 gene overexpression vector

[0059] The obtained CDS sequence of ZAT12 was constructed into the pCAMBIA1301U vector ( Figure 1 The constructed PU1301-ZAT12 plasmid was introduced into Agrobacterium strain EHA105.

[0060] (2) ZAT12 gene transformation into perennial ryegrass

[0061] Using callus induced from seeds of the perennial ryegrass variety 'ShiningStar' as the material, PU1301-ZAT12 was introduced into perennial ryegrass through Agrobacterium-mediated genetic transformation. After infection, co-cultivation, selection, and regeneration screening, resistant transformed seedlings were obtained. Transgenic plants were then obtained through conventional steps such as rooting, seedling training, and transplanting. gDNA was extracted from leaves of each transgenic line and identified by PCR and RT-qPCR ( Figure 2 A and B in the middle), and transgenic positive lines were screened.

[0062] 3. Phenotypic analysis of transgenic perennial ryegrass ZAT12

[0063] In order to explore the function of the ZAT12 gene in perennial ryegrass, the phenotypes of normal-growing positive lines and wild-type plants were observed and photographed, and the number of plants was recorded ( Figure 3 (Fig. B);

[0064] The experimental results are as follows Figure 3 Panel A in the middle shows six independent ZAT12-overexpressing perennial ryegrass transgenic lines (OE#25, OE#27, OE#29, OE#31, OE#32, and OE#35) to demonstrate the reproducibility of the results. WT represents a control group of untransformed perennial ryegrass. The results showed that the number of individual plants in the transgenic lines was significantly lower than that in the WT, indicating that overexpressing the ZAT12 gene can significantly inhibit plant tillering.

[0065] To investigate the biological function of the ZAT12 gene in perennial ryegrass, this study constructed multiple ZAT12 overexpression (OE) transgenic lines and conducted systematic phenotypic observations compared with wild-type (WT) control plants. Figure 3 B is the actual growth image record of plants with different treatments at 75 days, which intuitively shows the differences in growth status and tillering ability of each plant line.

[0066] To verify the reproducibility of the results, this study selected six independent transgenic lines (OE#25, OE#27, OE#29, OE#31, OE#32, and OE#35), and performed statistical analysis on the number of individual plants at 0 days, 28 days, and 75 days. Figure 3 Data A showed that at 75 days, the number of individual plants in the WT line was significantly higher than that in all transgenic lines, and the difference was statistically significant (\*P<0.05), indicating that the transgenic treatment had a significant inhibitory effect on tillering ability.

[0067] Combining images and quantitative data, we can observe that WT plants exhibit strong tillering ability and a robust plant architecture during growth, while ZAT12-overexpressing plants exhibit a thinner overall architecture and fewer tillers. In particular, tillering is significantly inhibited in strains such as OE#31 and OE#32. This suggests that ZAT12 overexpression inhibits meristem activity, thereby affecting lateral bud differentiation and growth.

[0068] The ZAT12 gene in perennial ryegrass may act as a negative regulator, controlling plant growth and tillering. This study provides important experimental evidence for further elucidating the molecular mechanisms of ZAT12 and its potential application in forage breeding.

[0069] SEQ ID NO: 1

[0070] ATGAAGCACCAGAGAGCGGCATCAGAGCAAGCAGTGTCCCTCGCCCTCTCGCTCTCCCTCGGCGCCGTGGCCGCCCGCCACAAGAAGCTCCGCCGCGCCGGTGCCGCCGCGGTCGGATCAGGCGAGTTCGTCTGCAAGACGTGCAGCCGCTCCTTCCCGACGTTCCAGGCGCTCGGCGGGCACCGGACCAGCCACCTCCGCGGCCGCCATGGG CTCGCGCTAGTCCTCGCCGGAGACCACTACAACGTCAAGCACAAGAAGACCACTTCCACAGACGATCAGAAGCCGGCGGCAGTACACCAGTGCCATGTCTGCGGGTTTCAGTTCGAGACGGGGCAGGCGCTCGGGGGACACATGCGCAGGCACCGCGAGGAGGCCGGCGGTGGCGCAGGCGCCGCCAGTCCTGCTCCAGCTCTTCGTCTAG

[0071] SEQ ID NO: 2

[0072] MKHQRAASEQAVSLALSLSLGAVAARHKKLRRAGAAAVGSGEFVCKTCSRSFPTFQALGGHRTSHLRGRHGLALVLAGDHYNVKHKKTTSTDDQKPAAVHQCHVCGFQFETGQALGGHMRRHREEAGAVAQAPPVLLQLFV

[0073] SEQ ID NO:3

[0074]

[0075] Based on two years of field phenotypic monitoring, the present study first performed genomic PCR amplification of 'Daye Zaoyou' ryegrass, confirming that the sequence of its LpZAT12 allele was identical to the published SEQ ID NO: 1. Subsequently, based on Illumina PE150 sequencing results, a specific sgRNA targeting the highly conserved region at the junction of exons 1 and 2 of this gene (5′-GGAGCTCTCCGACCAGCTGG-3′) was designed and cloned into the pRGEB32-UbiCas9 vector. After Agrobacterium inoculation of mature embryonic callus tissue, 37 T0 regenerated seedlings were obtained using 50 mg L⁻¹ Geneticin screening. Twenty-six of these seedlings exhibited a 1- or 2-bp deletion at the target site, resulting in a premature stop codon. After sequencing confirmed the homozygous mutation, the T2 generation was transplanted to a field plot at the same growth stage. Samples taken from nodes 5–7 compared to the wild type at the same time revealed a 32.4% increase in average tiller number and a 14.8% increase in dry matter per plant, with no significant decrease in plant height or leaf width.

[0076] To rule out off-target effects, we performed deep resequencing of potential homologous regions at six loci in the mutant and did not detect any base substitutions or small deletions. Furthermore, qRT-PCR results revealed that LpZAT12 was virtually undetectable in the mutant, while the transcript levels of its downstream core genes in the tillering repression pathway, LpTB1 and LpBRC1, decreased by approximately 45%. This suggests that LpZAT12 may negatively regulate tillering by positively regulating this repression module. Knockout of LpZAT12 disrupts apical dominance, significantly improving lateral bud germination.

[0077] The second example utilizes the high activity characteristics of the promoter shown in SEQ ID NO: 3 in the stem base meristem, replacing it with the natural promoter of ryegrass BtLAX3 (lateral bud growth-related AUX / LAX transporter) to obtain a site-directed enhanced expression strain. We used CRISPR / Cas12b-mediated homologous recombination to splice the 1.8kbLpZAT12 promoter with a 45bp homology arm to construct a dual-target editing vector pB-LbCas12b-HR-ProLpZAT12. 19 correctly replaced strains were obtained by transformation, and no promoter withdrawal occurred after 4 rounds of in vitro subculture under 2% Sucrose without selection force conditions, indicating that the insertion site is stable.

[0078] Measuring the BtLAX3 mRNA level in the promoter-replaced plants under a controlled greenhouse environment (16h light / 25°C, 8h dark) for 60 days revealed a 3.1-fold increase in BtLAX3 mRNA levels. IAA immunolocalization signals were enhanced at the outer base of the internodes, promoting lateral bud elongation. Compared to the control, the average tiller number increased by 27.9%, and the root-to-shoot ratio slightly increased. However, there were no significant differences in photosynthetic rate or greening time, suggesting that the modification did not cause carbon imbalance.

[0079] Further field trials were conducted at the Bohai Agricultural Demonstration Station (on loam soil with an average annual precipitation of 650 mm) using a randomized block design with four replicate plots (3 m x 5 m) per treatment. Biomass was measured using near-infrared scanning 15 days before harvest. The promoter-replaced material achieved a fresh grass yield of 48.3 tha, a 17.6% increase over the control, with no significant decrease in haystack content. Comprehensive evaluation demonstrated that forced activation of the tillering-promoting gene by a homologous promoter achieved precise regulation of the balance between nutrition, photosynthesis, and tillering.

[0080] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A ryegrass LpZAT12 gene for regulating plant tillering, characterized in that: The nucleotide sequence of the LpZAT12 gene is shown in SEQ ID NO:

1.

2. The ryegrass LpZAT12 gene for regulating plant tillering according to claim 1, wherein The amino acid sequence of the LpZAT12 gene is shown in SEQ ID No.

2.

3. The ryegrass LpZAT12 gene for regulating plant tillering according to claim 1, wherein The promoter sequence of the LpZAT12 gene is shown in SEQ ID No.

3.

4. The application of the ryegrass LpZAT12 gene that regulates plant tillering in promoting ryegrass plant tillering.

5. The use of the ryegrass LpZAT12 gene for regulating plant tillering according to claim 4 in promoting tillering of ryegrass plants, characterized in that: Specifically, the LpZAT12 gene of ryegrass was knocked out through genetic engineering methods.

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