Application of HYR gene in regulating plant height and tillering in tall fescue

Abstract

The present invention relates to RENT The application of genes in regulating plant height and tillering of tall fescue belongs to the field of plant genetic engineering. The present invention first constructs a light-induced expression promoter pRbcS5 drive RENT The plant recombinant expression vector of the gene was then transformed into tall fescue using Agrobacterium-mediated method. RENT Analysis of overexpressed transgenic tall fescue RENT Biological functions of genes in tall fescue; overexpression provided by the present invention RENT The genetically modified tall fescue plants can provide more theoretical basis for regulating the plant height and tillering of tall fescue and other grass plants, and can be used to cultivate transgenic turf grass varieties with reduced plant height and increased tillering, which has practical application value in optimizing the plant type of turf grass.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, mainly involving high-yield rice genes RENT And its application in regulating tall fescue plant height and tillering. Background Art

[0002] Tall Fescue ( Fescue arundinacea Schreb . Tall fescue is a perennial cool-season turfgrass of the genus Festuca in the Poaceae family. It is a commonly used turfgrass in my country and is widely distributed in Guizhou, Sichuan, Guangxi, and Xinjiang. Tall fescue boasts a long green period, attractive ornamental value, a soft texture, resistance to trampling, a well-developed root system, and strong stress resistance. Therefore, it has a wide range of uses, including river protection, road slope protection, and lawns in sports venues. Tall fescue is also nutritious and juicy, making it suitable for forage and fodder. Tall fescue is a clumping plant with a well-developed root system, strong environmental adaptability, and strong regeneration capabilities, making it highly suitable for widespread use.

[0003] Plant height and tillering are two important phenotypic characteristics of turfgrass. Tillering ability directly affects the turfgrass's resistance to trampling and its turf value. More tillers help turfgrass effectively utilize soil nutrients and improve fertilizer utilization efficiency. More tillers can also expand the turfgrass' coverage area and inhibit weed growth. Furthermore, more tillers are crucial for recovery and regeneration after abiotic stresses such as drought and salinity. Shorter plants and more tillers allow turfgrass to establish quickly, which, in turn, reduces mowing and reduces maintenance costs. Therefore, reducing turfgrass plant height and increasing its tiller number are of great practical significance.

[0004] Many factors can affect plant tillering and plant height, such as genetics, hormones, development, and environment. Among them, the role of plant hormones in plant tillering has been studied in depth. For example, the content and distribution of auxin in plants are closely related to plant tillering and plant height. The auxin export carrier protein PIN-FORMED (PIN) is a type of carrier protein that plays a key role in the polar transport of auxin. OsPIN1b Gene mutations lead to reduced plant height and increased tillering; while overexpression OsPIN9 Cytokinin also plays an important role in plant tillering. For example, mutation of cytokinin activating enzyme gene LOG ( LONELY GUY) leads to reduced tillering and reduced yield. Gibberellic acid can inhibit the growth of tillering buds and thus inhibit tillering. Gibberellic acid loss-of-function mutants often show increased branching. Gibberellic acid oxidase is the main catalytic enzyme in the gibberellin metabolic pathway that inactivates gibberellins. Overexpression of gibberellin oxidase in rice generally leads to phenotypes such as dwarf plants and increased tillering. Strigolactone is a newly discovered plant hormone in the 21st century. Most of the genes involved in the synthesis and signal transduction pathways of strigolactone are related to tillering and plant height, such as the genes in the synthesis pathway. D17 ( DWARF17 ) and D27 ( DWARF27 ), signal transduction pathways D14 ( DWARF14 ) and D3 ( DWARF ) mutations will result in dwarf plants and increased tillering.

[0005] HYR (HIGHER YIELD RICE) is a new type of transcription factor discovered in recent years. Although some studies have shown that 35S Promoter drive RENT Overexpression can improve rice yield and stress resistance, but RENT However, their role in regulating plant height and tillering, particularly in tall fescue, has not been reported. Light-induced expression promoters are a type of light-regulated promoter that can regulate gene expression in response to changes in light intensity and circadian rhythms. Using light-induced expression promoters can overcome the drawbacks of constitutive promoters, which consume excessive energy and substances through continuous expression, and achieve the goal of dynamically regulating the expression of target genes. The Rubisco small subunit promoter is a typical light-induced expression promoter. Using the Rubisco small subunit promoter to initiate expression of target genes can achieve the goal of dynamically regulating target gene expression. Summary of the Invention

[0006] To solve the above problems, the present invention utilizes overexpression technology to express Rubisco small subunit 5 promoter. pRbcS5 drive RENT Gene expression, by studying its biological function in the growth and development of tall fescue, the present invention provides RENT The new use of genes in regulating tall fescue plant height and tillering provides a basis for breeding dwarf tall fescue varieties with high tillering suitable for turf grass.

[0007] The specific scheme adopted in the present invention is:

[0008] The first aspect of the present invention is to provide a gene RENT Application in regulating tall fescue plant height and tillering.

[0009] The second aspect of the present invention is to provide a gene expression cassette for regulating plant height and tillering of tall fescue, the gene expression cassette comprising a gene RENT and activate the gene RENT Rubisco small subunit promoter pRbcS5 The gene RENT The nucleotide sequence of the coding region is shown in SEQ ID NO. 1, and the promoter pRbcS5 The nucleotide sequence is shown in SEQ ID NO.2.

[0010] The third aspect of the present invention is to provide a recombinant expression vector comprising the above-mentioned gene expression cassette.

[0011] The fourth aspect of the present invention is to provide a transformant obtained by transforming the above-mentioned recombinant expression vector into a host cell.

[0012] The fifth aspect of the present invention is to provide an expression system for light-induced target gene expression, wherein the expression system utilizes the above-mentioned gene expression cassette and is driven by a light-induced expression promoter. pRbcS5 Regulating rice RENT Gene expression.

[0013] A sixth aspect of the present invention is to provide the use of the above-mentioned gene expression cassette, recombinant expression vector or transformant in any of the following:

[0014] (1) Regulating plant height and / or tillering of grass plants;

[0015] (2) Cultivate new varieties of genetically modified plants.

[0016] Preferably, the plant is tall fescue.

[0017] The sixth aspect of the present invention is to provide a method for dwarfing plants and increasing tillering, wherein the method is to overexpress RENT gene, and then obtain positive plants with dwarfed plants and increased tillering.

[0018] Furthermore, using the Rubisco small subunit promoter pRbcS5 start up RENT Gene, the steps are: pRbcS5 and RENT The gene was connected to the vector PcactF through two rounds of ligation to construct a recombinant expression vector; then the recombinant expression vector was transformed into the recipient plant material through the Agrobacterium-mediated method to obtain transgenic plants with dwarfed plants and increased tillering.

[0019] The method specifically comprises the following steps:

[0020] (1) According to the multiple cloning site sequence of PcactF, pRbcS5 Sequence and RENT Design primers based on gene sequence to amplify the rice Rubisco small subunit promoter pRbcS5 as well as RENT Genes, after two rounds of connection pRbcS5 and RENT Genes are linked into PcactF; the first round of ligation will pRbcS5 Lianrujing Kpn and Monkey Double enzyme digestion of PcactF yielded PcactF-pRbcS5; the second round of ligation RENT Lianrujing Hall The enzyme-digested PcactF-pRbcS5 was finally constructed. RENT Overexpression vector PcactF-pRbcS5-HYR;

[0021] (2) The PcactF-pRbcS5-HYR obtained in step (1) was transformed into Agrobacterium EHA105 by freeze-thaw method;

[0022] (3) Agrobacterium-mediated RENT The overexpression vector PcactF-pRbcS5-HYR was transferred into tall fescue callus tissue, and T0 generation resistant plants were obtained by tissue culture. Positive transgenic plants were identified by PCR amplification, and the expression level of the target gene was determined by qRT-PCR.

[0023] Beneficial effect: The present invention constructs a light-induced expression promoter through overexpression technology pRbcS5 drive RENT The plant recombinant expression vector of the gene was transformed into tall fescue by Agrobacterium-mediated method. RENT Overexpressed transgenic tall fescue can be used for analysis RENT Biological functions of genes in tall fescue; overexpression provided by the present invention RENT The genetically modified tall fescue plants can provide more theoretical basis for regulating the plant height and tillering of tall fescue and other grass plants, and can be used to cultivate transgenic turf grass varieties with reduced plant height and increased tillering, which has practical application value in optimizing the plant type of turf grass. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the recombinant plant expression vector PcactF-pRbcS5-HYR for regulating the plant type of tall fescue using overexpression technology.

[0025] Figure 2The figures are the phenotypes of transgenic tall fescue and the statistical analysis of plant height and tillering; (A) is the phenotype of wild-type plants and transgenic plants, and (B) is the statistical analysis of plant height and tiller number. DETAILED DESCRIPTION

[0026] A gene that regulates the height and tillering of tall fescue plants RENT The nucleotide sequence of its coding region is shown in SEQ ID NO: 01.

[0027] A light-induced expression promoter for regulating gene expression pRbcS5 , whose nucleotide sequence is shown in SEQ ID NO: 02.

[0028] The present invention provides an expression system for light-induced target gene expression, wherein the target gene RENT Expression is regulated by light. Studies have found that RENT The positive plants obtained after overexpression showed dwarfing and increased tillering phenotypes, indicating that RENT The gene has important application value in improving the plant height and tillering of tall fescue.

[0029] Therefore, the present invention provides a nucleotide sequence as shown in SEQ ID NO: 01 and SEQ ID NO: 02 using a light-induced expression promoter to regulate rice RENT Application of genes in controlling plant height and tillering in tall fescue.

[0030] Specifically, the overexpression technology was used to increase the RENT The expression level of the gene is reduced, thereby obtaining tall fescue with dwarf plants and increased tillering.

[0031] The present invention provides a method for dwarfing plants and increasing tillering, comprising: utilizing an overexpression technique using the Rubisco small subunit promoter sequence shown in SEQ ID NO. 2 pRbcS5 Start SEQ ID NO. 1 shown RENT Gene.

[0032] Specifically, the rice Rubisco small subunit promoter pRbcS5 and RENT The gene was linked into the expression vector PcactF to construct a recombinant expression vector; the recombinant expression vector was transformed into recipient plant materials through Agrobacterium-mediated method to obtain transgenic plants with dwarfed plants and increased tillering.

[0033] The method comprises the following steps:

[0034] (1) According to the multiple cloning site sequence of PcactF, pRbcS5 Sequence and RENTDesign primers based on gene sequence to amplify the rice Rubisco small subunit promoter pRbcS5 as well as RENT Genes, after two rounds of connection pRbcS5 and RENT The first round of ligation will pRbcS5 Lianrujing Kpn and Monkey Double enzyme digestion of PcactF yielded PcactF-pRbcS5; the second round of ligation RENT Lianrujing Hall The enzyme-digested PcactF-pRbcS5 was finally constructed. RENT Overexpression vector PcactF-pRbcS5-HYR.

[0035] (2) The recombinant expression vector was transferred into Agrobacterium EHA105 by freeze-thaw method.

[0036] (3) Agrobacterium-mediated RENT The overexpression vector PcactF-pRbcS5-HYR was transferred into tall fescue callus tissue, and T0 generation resistant plants were obtained by tissue culture. Positive transgenic plants were identified by PCR amplification, and the expression level of the target gene was determined by qRT-PCR.

[0037] The PCR primers used in step (1) are:

[0038] Amplification of the rice Rubisco small subunit promoter pRbcS5 Primers (sequences shown in SEQ ID NO: 03 and SEQ ID NO: 04, respectively):

[0039] PrbcS5-Kpn-F: 5'-tacgaattcgagctcggtaccTTTACGGTACTTTCCTTCATTCTGAT-3'

[0040] PrbcS5-Apa-R: 5'-caggtcgactctagagggcccTGCTAGCTCTCTAGCTTTCTGCTTG-3'.

[0041] Expanded rice RENT Gene primers (sequences shown in SEQ ID NO: 05 and SEQ ID NO: 06, respectively):

[0042] HYR-Sal-F: 5'-gcagggccctctagagtcgacATGAACGTTTTTTTGTGCATGTTGTTTACTG-3'

[0043] HYR-Sal-R: 5'-agggcatgcctgcaggtcgacTTAATGATGATGATGATGATGTCTCGCATAC-3'.

[0044] The PCR primers used in step (3) for screening positive plants are (sequences are shown in SEQ ID NO: 07 and SEQ ID NO: 08, respectively):

[0045] HPT-F: 5'-CTGAACTCACCGCGACGTCTGTC-3'

[0046] HPT-R: 5'-TAGCGCGTCTGCTGCTCCATACA-3'.

[0047] The following is further described with reference to specific embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0048] In the following examples, the experimental methods without specific experimental conditions are generally based on conventional experimental conditions.

[0049] Unless otherwise specified, all materials and reagents used were obtained from commercial sources.

[0050] The primers used in the examples were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. Hound No. 5 tall fescue was used as the recipient material for genetic transformation.

[0051] Example 1: Rice Rubisco small subunit promoter pRbcS5 Sequence analysis.

[0052] Sequence source: The sequence containing Rubisco small subunit 5 ( RbcS5 ) genome sequence (AC134236.4), RbcS5 2000 bp upstream of the start codon is pRbcS5 Sequence, the nucleotide sequence of which is shown in Table SEQ ID NO:02.

[0053] Example 2: Rice RENT Sequence analysis.

[0054] Gene source: obtained from the National Center for Bioinformatics (NCBI) RENT The gene (Os03g02650) sequence, the nucleotide sequence of its coding region is shown in the sequence listing SEQ ID NO: 01, and the sequence length is 804 bp.

[0055] Example 3: Construction RENT Obtaining recombinant expression vector and transgenic plants.

[0056] According to the plant expression vector PcactF and rice RbcS5 The promoter sequence was designed with primers and obtained from rice genomic DNA by PCR amplification. pRbcS5 The primers used were PrbcS5-Kpn-F and PrbcS5-Kpn-R.

[0057] Use PcactF Kpn and Monkey Double enzyme digestion pRbcS5 The sequence was ligated into PcactF using a one-step recombination cloning kit to obtain PcactF-pRbcS5.

[0058] According to PcactF-pRbcS5 and RENT Sequence design primers were amplified from rice cDNA by PCR technology RENT The primers used for the complete coding frame were HYR-Sal-F and HYR-Sal-R.

[0059] PcactF-pRbcS5 was Hall Linearization by enzyme digestion RENT The sequence was connected into PcactF-pRbcS5 using a one-step recombination cloning kit to obtain PcactF-pRbcS5 - HYR (eg Figure 1 shown).

[0060] The correctly sequenced recombinant vector was transformed into Agrobacterium tumefaciens EHA105 via the freeze-thaw method. Calli of tall fescue Hound No. 5 were infected using Agrobacterium-mediated methods. Transgenic plants were obtained after pre-differentiation and differentiation of resistant calli, and rooting and strengthening of seedlings. Genomic DNA from leaves of T0-generation transgenic plants was extracted using the CTAB method, and transgenic plants were screened by PCR using primers HPT-F and HPT-R.

[0061] Example 4: Phenotypic identification of transgenic plants.

[0062] The transgenic plants obtained were phenotypically identified: transgenic and wild-type plants were grown in plastic pots (13 cm in diameter, 11 cm in depth) filled with sand and peat soil (1 / 1, v / v) under controlled conditions of 22 / 18°C, 14 / 10 h light / dark regime, 75% relative humidity, and a light intensity of 360 µmol m −2 s −1Irrigate every five days with Hoagland nutrient solution. After 40 days of cultivation, perform phenotyping, measure plant height with a ruler, and count tiller numbers.

[0063] like Figure 2 As shown, the results showed that the plant height of the transgenic plants was lower than that of the wild-type plants, while the number of tillers was much higher than that of the wild-type plants, showing excellent turfgrass phenotypic characteristics.

[0064] The above embodiment is a specific implementation of the present invention, but the implementation of the present invention is not limited to the above embodiment. Any other changes, modifications, substitutions, combinations, simplifications and other optimization and improvement behaviors that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the claims of the present invention.

Claims

1. HYR Use of genes in any of the following: (1) Obtaining the phenotype of grass plants with dwarfed plants and increased tillering; (2) Cultivate new transgenic plant varieties that are dwarfed and have many tillers; the dwarfing and many tillers are achieved by overexpressing HYR Genetically realized; described HYR The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO. 1; the plant is tall fescue.

2. A method for dwarfing tall fescue and increasing tillering, characterized in that: The method is to overexpress in plants HYR gene, thereby obtaining positive plants with dwarfed plants and increased tillering; HYR The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

1.

3. The method according to claim 2, wherein: Using the Rubisco small subunit promoter pRbcS5 start up HYR Gene, the steps are: the nucleotide sequence as shown in SEQ ID NO. 2 promoter pRbcS5 and HYR The gene was connected into the vector PcactF through two rounds of ligation to construct a recombinant expression vector; the recombinant expression vector was transformed into the recipient plant material through the Agrobacterium-mediated method to obtain transgenic plants with dwarf plants and increased tillering.

4. The method according to claim 3, wherein: The specific steps include: (1) According to the multiple cloning site sequence of PcactF, pRbcS5 Sequence and HYR Design primers based on gene sequence to amplify the rice Rubisco small subunit promoter pRbcS5 as well as HYR Genes, after two rounds of connection pRbcS5 and HYR Genes are linked into PcactF; the first round of ligation will pRbcS5 Lianrujing Kpn and Apa Double enzyme digestion of PcactF yielded PcactF-pRbcS5; the second round of ligation HYR Lianrujing Sal The enzyme-digested PcactF-pRbcS5 was finally constructed. HYR Overexpression vector PcactF-pRbcS5-HYR; (2) The PcactF-pRbcS5-HYR obtained in step (1) was transformed into Agrobacterium EHA105 by freeze-thaw method; (3) Agrobacterium-mediated HYR The overexpression vector PcactF-pRbcS5-HYR was transferred into tall fescue callus tissue, and T0 generation resistant plants were obtained by tissue culture. Positive transgenic plants were identified by PCR amplification, and the expression level of the target gene was determined by qRT-PCR.

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