LpBPM5.6 gene for regulating perennial ryegrass plant type, low expression vector, construction method and application thereof

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to an LpBPM5.6 gene for regulating and controlling a perennial ryegrass plant type, a low expression vector, a construction method and application of the LpBPM5.6 gene. The invention discloses a perennial ryegrass LpBPM5.6 gene for regulating and controlling a plant type. The nucleotide sequence of the perennial ryegrass LpBPM5.6 gene is shown as SEQ ID No. 1. According to the invention, a gene full-length sequence, namely LpBPM5.6, for regulating and controlling the perennial ryegrass plant type is cloned for the first time. The LpBPM5.6, as a gene for regulating and controlling the plant type of perennial ryegrass, can enable the perennial ryegrass to present an ideal plant type. The inventor constructs an LpBPM5.6 low expression vector to obtain a ryegrass plant with low expression of the LpBPM5.6 gene. Results show that compared with wild perennial ryegrass, after low expression of the gene, the tiller number of the perennial ryegrass can be increased by more than 20%, the plant height and fresh weight can be reduced by more than half, fibrous roots are obviously reduced, and obvious changes of the characters provide support for plant type improvement of turfgrass such as the perennial ryegrass.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular breeding, and specifically relates to an LpBPM5.6 gene for regulating the plant type of perennial ryegrass, a low expression vector, a construction method and applications thereof. Background Art

[0002] Plant type is an important agronomic trait of plants. Genes that regulate tillering, leaf angle, and roots can significantly increase the yield of crops such as rice Oryza sativa, corn Zea mays, and wheat Triticum aestivum by improving plant type. The "ideal plant type" of lawn grass is obviously different from the "ideal plant type" of crops such as cereals with few tillers and upright stems and leaves. Short plant height, many tillers, and creeping stems and leaves are the "ideal plant type" of lawn grass.

[0003] Lolium perenne L., a perennial grass plant, has a well-developed root system and many fibrous roots, which are distributed in a network in the 15-20 cm soil layer, which helps it better absorb water and nutrients from the soil. The stems are upright, hollow, clumping, soft in texture, 80-100 cm high, with internodes and many tillers, which allows ryegrass to quickly form dense grass. It also has the characteristics of strong tillering ability, fast turf formation speed, and long green period, and is often used for ornamental greening and golf course fairway construction.

[0004] In the prior art, genes that regulate the plant type of Gramineae plants are mainly involved in plant hormone pathways, such as dwarf1, dwarf3, dwarf8 and dwarf11 related to the gibberellin biosynthesis pathway; brd1, nana plant1 and nana plant2 related to the brassinolide biosynthesis pathway; breviesplant1 and pin1 related to the auxin biosynthesis pathway. These genes are mostly found in corn, rice and wheat, and are involved in the morphological construction of plant type; however, there are no reports on genes that regulate the plant type of perennial ryegrass.

[0005] In summary, there is an urgent need to provide a gene that regulates the plant type of perennial ryegrass so that perennial ryegrass can present an "ideal plant type". Summary of the invention

[0006] The purpose of the present invention is to provide a LpBPM5.6 gene, a low expression vector, a construction method and an application thereof for regulating the plant type of perennial ryegrass. The low expression level of LpBPM5.6 makes perennial ryegrass present a plant type with short plant height, many tillers and few fibrous roots, which provides a good application for improving the plant type of lawn grass.

[0007] The technical solution adopted by the present invention is a perennial ryegrass LpBPM5.6 gene for regulating plant type, and its nucleotide sequence is shown in SEQ ID No.1.

[0008] The present invention provides a low expression vector constructed using the perennial ryegrass LpBPM5.6 gene, comprising the following steps:

[0009] Extract RNA from perennial ryegrass leaves, reverse transcribe to obtain cDNA; synthesize a pair of primers, use cDNA as a template, perform PCR amplification to obtain attB-PCR products, the primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5; mix the attB-PCR product with a donor vector, add BP recombination enzyme mix to incubate, terminate, transform into competent cells, and obtain an entry vector of the Gateway system; mix the entry vector and an expression vector, add LR recombination cloning enzyme to incubate, terminate, transform into competent cells, and obtain a low expression vector.

[0010] Preferably, the donor vector is pDONA207, and the expression vector is PC336.

[0011] The present invention also provides an application of the perennial ryegrass LpBPM5.6 gene or the low expression vector in regulating the plant type of perennial ryegrass.

[0012] The low expression vector is used to prepare perennial ryegrass plants with low expression of LpBPM5.6, and the preparation process includes the following steps:

[0013] The low expression vector is transformed into Agrobacterium tumefaciens competent cells to obtain positive recombinant Agrobacterium tumefaciens; the positive recombinant Agrobacterium tumefaciens infects perennial ryegrass callus and co-cultivates in the dark on a co-culture medium; the product of the dark co-culture is transferred to a resistance culture medium to screen positive perennial ryegrass callus; the positive perennial ryegrass callus is transferred to a differentiation culture medium and a seedling strengthening culture medium in sequence to obtain perennial ryegrass plants with low expression of LpBPM5.6.

[0014] Preferably, the Agrobacterium tumefaciens competent cell is any one of EHA105, GV3101 and LB4404.

[0015] Preferably, the infection process is: at 1.0×10 5 Pa~1.5×10 5 Pa soaked the perennial ryegrass callus in the positive recombinant Agrobacterium tumefaciens suspension for 20 minutes, and then incubated at 80rpm ~ 80rpm for 20min ~ 25min.

[0016] Preferably, the differentiation medium has a formula of: adding 0.5 mg to 0.6 mg of benzylaminopurine, 30 g to 32 g of maltose, 3 g to 4 g of plant gel and 150 mg to 152 mg of timentin to each liter of MS medium.

[0017] Preferably, the formula of the seedling-strengthening culture medium is as follows: 30 g to 32 g of maltose, 3 g to 4 g of plant gel and 150 mg to 152 mg of timentin are added to each liter of MS culture medium.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention clones for the first time a full-length gene sequence regulating the plant type of perennial ryegrass, namely LpBPM5.6. As a gene regulating the plant type of perennial ryegrass, LpBPM5.6 can make perennial ryegrass present an "ideal plant type".

[0019] The inventors constructed a low-expression vector of LpBPM5.6 and obtained ryegrass plants with low expression of the LpBPM5.6 gene. The results showed that compared with wild perennial ryegrass, low expression of the gene increased the number of tillers of perennial ryegrass by more than 20%, reduced the plant height and fresh weight by more than half, and significantly reduced the number of fibrous roots. These significant changes in traits provide support for the improvement of plant type of lawn grasses such as perennial ryegrass. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2 shows the plant type results of LpBPM5.6 low-expression ryegrass plants. A: The expression level of LpBPM5.6 in various organs; B: The expression level of LpBPM5.6 in LpBPM5.6 RNAi materials; C-E: GFP fluorescence images of empty vector, DAPI nuclear staining images, and the results of merging GFP fluorescence images and DAPI nuclear staining images; F-H: GFP fluorescence images of LpBPM5.6 vector, DAPI nuclear staining images, and the results of merging GFP fluorescence images and DAPI nuclear staining images; I-L: Plant height, tiller number, fresh weight and maximum root length of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics; M-O: Representative photos of the roots of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics; P: Heat map of root morphology indicators of WT and LpBPM5.6 RNAi; Q: Plant height, tiller number, fresh weight and maximum root length of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics. Representative pictures of the aboveground parts of RNAi plants; R: Representative pictures of the underground parts of WT and LpBPM5.6RNAi plants after 21 days of hydroponics.

[0021] Figure 2Plant type results of LpBPM5.6 overexpressing ryegrass plants, A: expression level of LpBPM5.6 in LpBPM5.6 overexpressing materials; B: representative pictures of aboveground parts of WT and LpBPM5.6 overexpressing plants after 21 days of hydroponics; C-F: plant height, tiller number, fresh weight and maximum root length of WT and LpBPM5.6 overexpressing plants after 21 days of hydroponics; G: representative pictures of roots of WT and LpBPM5.6 overexpressing plants after 21 days of hydroponics, from left to right: WT, LpBPM5.6 overexpressing ryegrass plant OE#1, LpBPM5.6 overexpressing ryegrass plant OE#2; H: heat map of root morphology indicators; I: representative pictures of underground parts of WT and LpBPM5.6 overexpressing plants after 21 days of hydroponics. DETAILED DESCRIPTION

[0022] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0023] The inventive concept of the present invention is as follows: At present, the genes regulating the plant type of Gramineae plants are mainly involved in the biosynthesis pathway of plant hormones, such as the biosynthesis pathway of gibberellins, the biosynthesis pathway of brassinolide and the biosynthesis pathway of auxins. These genes are mostly found in corn, rice and wheat and participate in the morphological construction of their plant types; however, there are no reports on the genes regulating the plant type of perennial ryegrass.

[0024] BPM proteins are adaptor proteins for Cullin3-based E3 ubiquitin ligases in animals and plants. In Arabidopsis, they form a small family of six members, which are involved in the regulation of physiological processes such as plant growth, fertility, stomatal movement, fatty acid metabolism, and ABA signaling. The Arabidopsis genome encodes only six BPM genes, corresponding to AtBPM 1-6. As substrate-specific binding proteins for CUL3-based E3 ligases, BPM proteins bind to different transcription factor family members and are therefore considered to be important regulators of various developmental processes and stress responses. Overexpression of BPM3 or BPM5 enhances the sensitivity of plants to abscisic acid, inhibits seed germination, seedling morphology, and root growth. At the same time, plants overexpressing BPM3 or BPM5 have reduced water loss, increased stomatal closure, and improved drought resistance.

[0025] BPM proteins not only affect the transduction of hormone signals in plants, but have also been shown to be related to plant flowering. In Arabidopsis, bpm mutants exhibit late flowering, while AtBPM1 overexpression plants exhibit early flowering, so it is predicted that BPM positively regulates the flowering time of plants. In addition, transcription factors AtMYB56 and AtMYB106 bind to the promoter of AtFT, a key regulator of the vegetative-to-reproductive transition, thereby inhibiting its transcription, while CULBPM mediates the ubiquitination and degradation of transcription factors AtMYB56 and AtMYB106, thereby increasing AtFT transcription and promoting flowering. Therefore, BPM proteins play an important role in plant flowering.

[0026] These findings demonstrate that BPMs are involved in hormone-mediated stress responses and flowering regulation and are protein post-translational regulators of plant physiological responses. It is noteworthy that although all six Arabidopsis BPM proteins have been shown to interact with proteins such as AtHB6, WRI1, MYB56, and DREB2A, only specific members have been shown to interact with PP2C, and different BPMs have similar functions in binding to the three MYC proteins. In addition, only BPM1, BPM2, and BPM4 interact with MYB106, and only BPM1 and BPM3 interact with RRTF1. This means that there is both functional redundancy and individual different functions in this highly conserved protein family, some of which may only be manifested under specific environmental conditions. Protein families containing BPM domains have been found in almost all eukaryotic genomes. It regulates the ubiquitination and proteasomal degradation of specific substrates. BPMs are widely involved in plant growth and development and the regulation of tolerance to biotic or abiotic stresses. In recent years, BPMs have also been shown to be used to improve key agronomic traits of some crops, but the function of BPMs in perennial turfgrasses is still unclear.

[0027] Before the invention, the inventors had never imagined that the LpBPM5. gene was related to the morphological construction of ryegrass plant type. Based on this, the present invention provides a ryegrass LpBPM5.6 gene for regulating plant type, and its nucleotide sequence is shown in SEQ ID No.1.

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is further described below in conjunction with specific embodiments and drawings. In the description of the present invention, unless otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art.

[0029] Example 1: Construction method of LpBPM5.6 gene low expression vector

[0030] RNA was extracted from leaves of the perennial ryegrass variety Peak III and reverse transcribed to obtain cDNA. The RNA extraction kit was purchased from Promege, and the reverse transcription kit was purchased from Vazyme. Based on the sequence information of LpBPM5.6 obtained by transcriptome sequencing, a pair of primers was synthesized for amplification of LpBPM5.6, the sequence of the forward primer LpBPM5.6-F is shown in SEQ ID NO.2, and the sequence of the reverse primer LpBPM5.6-R is shown in SEQ ID NO.3.

[0031] SEQ ID NO. 2: LpBPM5.6-F, 5'-ATGGGCGTGGGCAAGCACATC-3'.

[0032] SEQ ID NO. 3: LpBPM5.6-R, 5'-TCAGACCCTCGGTCTCACTCT-3'.

[0033] The LpBPM5.6 gene was cloned by PCR using the cDNA of the perennial ryegrass variety Peak III as a template, and its nucleotide sequence is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1:

[0035]

[0036] The Gateway kit purchased from Thermo was used to recombinant the LpBPM5.6 specific sequence of 200 bp to 300 bp into the Gramineae specific RNAi vector PC336 to construct the LpBPM5.6-RNAi vector that low-expresses LpBPM5.6. The steps for constructing the LpBPM5.6-RNAi vector are as follows:

[0037] Step 1: extract RNA from leaves of perennial ryegrass variety Summit III and obtain cDNA by reverse transcription.

[0038] Step 2: Using cDNA as a template, perform PCR amplification to prepare attB-PCR products, namely, LpBPM5.6-specific sequences.

[0039] A pair of primers were synthesized for amplifying attB-PCR products, and the primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0040] SEQ ID NO.4:

[0041] ggggacaagtttgtacaaaaaagcaggcttaGCAGGGGGCAAGCACAAGG.

[0042] SEQ ID NO.5:

[0043] ggggaccactttgtacaagaaagctgggtcAGCCTATGTCAGAATCCGG.

[0044] Using the cDNA of the leaves of the perennial ryegrass variety Peak III as a template, primers SEQ ID NO.4 and SEQ ID NO.5 were used to amplify the LpBPM5.6 specific sequence, and a DNA gel recovery kit was used to recover the LpBPM5.6 specific sequence.

[0045] Step 3, BP reaction: Mix the attB-PCR product with the donor vector, add BP recombination mixed enzyme for incubation, terminate, and transform into competent cells to obtain the entry vector of the Gateway system.

[0046] 1. Add the following components to a centrifuge tube and mix at room temperature: 150 ng of attB-PCR product, 150 ng of pDONA207 vector, TE buffer pH 8.0 to 8 μL, and 2 μL of BP Clonase TM II enzyme mix.

[0047] 2. Incubate the reaction at 25°C for 1 hour.

[0048] 3. Add 1 μL of proteinase K solution to the centrifuge tube to terminate the reaction and incubate at 37°C for 10 minutes.

[0049] 4. Transform the above mixture into TOP10 competent cells, select single clones for sequencing, compare sequencing results, and screen positive clones, i.e., entry vectors.

[0050] Step 4, LR recombination reaction: Mix the entry vector and the expression vector, add LR recombination cloning enzyme for incubation, terminate, and transform into competent cells to obtain a low expression vector.

[0051] 1. Add the following components to a centrifuge tube and mix at room temperature: 300 ng of entry vector, 150 ng of PC336 vector, 4 μL of 5X LR clonase TM Reaction buffer: add TE buffer pH 8.0 to 16 μL, and then add 4 μL of LR clonase.

[0052] 2. Incubate the reaction at 25°C for 1 hour.

[0053] 3. Add 2 μL of proteinase K solution to the centrifuge tube to terminate the reaction and incubate at 37°C for 10 minutes.

[0054] 4. Transform the sample into TOP10 competent state, select single clones to extract plasmids, and obtain LpBPM5.6-RNAi vector, i.e., low expression vector.

[0055] Example 2: Application of LpBPM5.6 gene low expression vector

[0056] 1. Experimental methods

[0057] 1.1 Construction of LpBPM5.6 low-expression ryegrass strains, the steps are as follows:

[0058] The LpBPM5.6-RNAi vector described in Example 1 was transformed into Agrobacterium tumefaciens competent cells EHA105 by chemical transformation to obtain positive recombinant Agrobacterium tumefaciens. Three-month-old perennial ryegrass peak III callus was selected and immersed in a suspension of positive recombinant Agrobacterium tumefaciens under vacuum conditions of 1.0×10 5Pa, infect for 20 minutes, incubate on a shaker at 80 rpm for 20 minutes, spread the callus evenly on a culture dish containing moistened filter paper, and use a co-culture medium to co-culture in the dark for 3 days. The co-culture medium formula is: add 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthylacetic acid, and 8 g of agar per liter of MS medium. After the co-culture is completed, transfer to the resistance medium to screen out the positive perennial ryegrass callus. Induce seedlings on the differentiation medium, and after the seedlings are formed, transfer the seedlings to the seedling medium to obtain transgenic seedlings. The formula of the differentiation medium is: add 0.5 mg of benzylaminopurine, 30 g of maltose, 3 g of plant gel, and 150 mg of timentin per liter of MS medium. The formula of the seedling medium is: add 30 g of maltose, 3 g of plant gel, and 150 mg of timentin per liter of MS medium. The transgenic ryegrass materials obtained by identification and screening were used to obtain perennial ryegrass plants with low expression of LpBPM5.6. The positive transgenic materials were rapidly expanded and cultured through tillering to obtain sufficient research materials.

[0059] 1.2 Construction of LpBPM5.6 overexpressing ryegrass lines

[0060] The LpBPM5.6 CDS sequence was recombined into the Gramineae-specific overexpression vector CUB-eGFP driven by the maize Ubi promoter to construct the pUbi: LpBPM5.6-GFP overexpression vector. The LpBPM5.6-GFP vector was transferred into the Agrobacterium tumefaciens competent cell EHA105. The 3-month-old perennial ryegrass peak III callus was soaked in a suspension of Agrobacterium tumefaciens, and then the callus was co-cultured in the dark for 3 days using a co-culture medium, and then transferred to a resistance medium. After culturing in the dark for 2 months, the callus that was not completely browned was transferred to a differentiation medium and cultured in light for about 1 month. After the callus grew roots and green shoots, the seedlings were transferred to a strong seedling medium to obtain positive transgenic seedlings, i.e., LpBPM5.6 overexpression ryegrass lines.

[0061] The LpBPM5.6 low-expression transgenic ryegrass lines and over-expression ryegrass lines were grown in a culture room at 22°C, 40% humidity, and a 16-h / 8-h light-dark cycle.

[0062] 1.3 Functional verification of LpBPM5.6 low-expression and over-expression ryegrass strains

[0063] 1.3.1 RNA extraction and quantitative RT-qPCR

[0064] use The total RNA of the strain was extracted using Super Total RNA Extraction Kit, and cDNA was synthesized using reverse transcription reagent. The cDNA and 2×ChamQ SYBR qPCR mixture were then subjected to RT-qPCR reaction on a LightCycler96 quantifier. LpTBP-1 and LpeIF4A were used as internal reference genes. -ΔΔCT Methods The expression level of LpBPM5.6 gene was calculated.

[0065] 1.3.2 Subcellular localization

[0066] The complete coding region of LpBPM5.6 was cloned into the N-terminal region of the pCAMBIA1300-GFP vector. The Pro35S:LpBPM5.6-GFP vector and the Pro35S:GFP vector were respectively transformed into Nicotiana benthamiana leaves using Agrobacterium GV3101. The fluorescence signal was observed using a laser confocal microscope 3 days later.

[0067] 1.4 Phylogenetic analysis

[0068] Protein sequences were aligned using MAFFT software, and phylogenetic relationships were analyzed using IQ-TREE software. Phylogenetic trees were constructed using the maximum likelihood method using default parameters.

[0069] 1.5 R language drawing heat map

[0070] After culturing perennial ryegrass plants with Hoagland solution for 3 weeks, the root characteristics were analyzed using a root scanner purchased from Hangzhou Wansen Technology Testing Company. By using the normalization method, the raw count value of each data was made to be in the same order of magnitude. Then, the different color changes in the heat map reflect the changes in the data in different plants. The data L represents the total root length, PA represents the projected area, AP represents the pixel area, SA represents the surface area, V represents the volume, AD represents the average diameter, LN represents the number of connections, NN represents the number of nodes, TN represents the number of root tips, FN represents the number of bifurcations, CN represents the number of crosses, and FD represents the fractal dimension.

[0071] 2. Experimental results

[0072] A search of the perennial ryegrass genome database revealed that 279 BPM genes could be retrieved from perennial ryegrass. The number of members of this gene family is about 46 times that of Arabidopsis, 26 times that of rapeseed, 9 times that of corn, and 3.6 times that of wheat. The expansion of the number of this gene family in ryegrass suggests that BPM may have unique functions in perennial grasses. Figure 1As shown in the figure, further research found that LpBPM5.6 was highly expressed in ryegrass leaves, roots, stems and other vegetative organs, and it was speculated that it may play a role in the vegetative organs of perennial ryegrass. Observation of its subcellular localization revealed that LpBPM5.6 was evenly distributed in the cytoplasm, nucleus, cell membrane and other parts.

[0073] The inventors successfully constructed LpBPM5.6 low-expression ryegrass strains, namely RNAi#1 and RNAi#2, and studied their functions in the nutritional development stage of perennial ryegrass and found that LpBPM5.6 can affect the plant type of lawn grass. After 21 days of hydroponics of single tillering, the plant height of LpBPM5.6 low-expression ryegrass strains RNAi#1 and RNAi#2 was about half of that of the wild type WT, and the fresh weight was less than half of that of the WT. However, the number of tillers of RNAi#1 and RNAi#2 increased significantly, which was more than 20% higher than that of WT tillers. Observation of the root system found that the maximum root length of RNAi#1 and RNAi#2 strains was not significantly different from that of the wild type, but the root density was significantly lower than that of WT. Statistics of various root parameters showed that the total length, surface area, volume, average diameter, number of root tips, and number of forks of RNAi#1 and RNAi#2 roots were significantly lower than those of WT. These results indicate that lowering the expression level of LpBPM5.6 in ryegrass can present an "ideal plant type" of turf grass with short plant height and many tillers. At the same time, fewer fibrous roots can reduce the metabolic cost of the underground part of the turf grass, allowing more resources to be concentrated on the aboveground part, thereby improving the quality of the turf under good cultivation conditions.

[0074] Further construction of LpBPM5.6 overexpressing ryegrass lines was performed. Figure 2 , OE#1 and OE#2 are LpBPM5.6 overexpression ryegrass lines. After 21 days of hydroponics of single tillering, the plant types of WT and LpBPM5.6 overexpression ryegrass plants were analyzed and it was found that LpBPM5.6 overexpression ryegrass plants were slightly taller than WT; LpBPM5.6 overexpression ryegrass plants had a heavier fresh weight than WT; and the number of tillers of LpBPM5.6 overexpression ryegrass plants was only 70% of that of WT. Root system observations showed that LpBPM5.6 overexpression ryegrass plants did not affect the maximum root length, but the root density was significantly higher than that of WT. Statistics of various root system parameters showed that the total root length, surface area, volume, average diameter, number of root tips, and number of forks of LpBPM5.6 overexpression ryegrass plants were higher than those of WT.

[0075] BPM protein is an important component of Cullin3, CUL3 E3, and ubiquitin ligase complex. In the present invention, reducing the expression level of LpBPM5.6 makes perennial ryegrass present a plant type with shorter plant height, increased tillering, and reduced fibrous roots; while overexpression of LpBPM5.6 makes perennial ryegrass present a phenotype of increased plant height, reduced tillering, and increased fibrous roots. These results show that LpBPM5.6 can regulate the ideal plant type of perennial ryegrass, which provides support for the improvement of plant type of lawn grasses such as perennial ryegrass.

[0076] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0077] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A perennial ryegrass LpBPM5.6 gene for regulating plant type, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

1.

2. A low expression vector constructed using the perennial ryegrass LpBPM5.6 gene according to claim 1.

3. The method for constructing a low expression vector according to claim 2, characterized in that: The following steps are involved: RNA was extracted from perennial ryegrass leaves and reverse transcribed to obtain cDNA; A pair of primers was synthesized, and PCR amplification was performed using cDNA as a template to obtain attB-PCR products. The primer sequences are shown in SEQ ID NO.4 and SEQ ID NO.5; Mix the attB-PCR product with the donor vector, add BP recombination enzyme mix, incubate, terminate, and transform into competent cells to obtain the entry vector of the Gateway system; The entry vector and expression vector were mixed, LR recombinase was added for incubation, termination, and transformation into competent cells to obtain a low expression vector.

4. The method for constructing a low expression vector according to claim 3, characterized in that: The donor vector is pDONA207, and the expression vector is PC336.

5. Use of the perennial ryegrass LpBPM5.6 gene according to claim 1 or the low expression vector according to claim 2 in regulating the plant type of perennial ryegrass.

6. The use according to claim 5, characterized in that: The low expression vector is used to prepare perennial ryegrass plants with low expression of LpBPM5.6, and the preparation process includes the following steps: Transforming the low expression vector into competent cells of Agrobacterium tumefaciens to obtain positive recombinant Agrobacterium tumefaciens; Positive recombinant Agrobacterium tumefaciens infected perennial ryegrass callus and co-cultivated in the dark on co-culture medium; The products of dark co-cultivation were transferred to resistance medium and positive perennial ryegrass callus was screened; The positive perennial ryegrass callus tissue was transferred to differentiation medium and seedling medium in sequence to obtain perennial ryegrass plants with low expression of LpBPM5.

6.

7. The use according to claim 6, characterized in that: The Agrobacterium tumefaciens competent cell is any one of EHA105, GV3101 and LB4404.

8. The use according to claim 6, characterized in that: The infection process is: In 1.0×10 5 Pa~1.5×10 5 Pa soaked the perennial ryegrass callus in the positive recombinant Agrobacterium tumefaciens suspension for 20 minutes, and then incubated at 80rpm ~ 80rpm for 20min ~ 25min.

9. The use according to claim 6, characterized in that: The formula of the differentiation medium is: Add 0.5mg-0.6mg benzylaminopurine, 30g-32g maltose, 3g-4g phytogels and 150mg-152mg timentin per liter of MS medium; The formula of the seedling-strengthening culture medium is: Add 30g-32g maltose, 3g-4g plant gel and 150mg-152mg timentin per liter of MS culture medium.

Images