Lpbpm5.6 gene for regulating plant type of perennial ryegrass, low expression vector, construction method and application thereof
By cloning and constructing a low-expression vector for the LpBPM5.6 gene, the plant architecture of perennial ryegrass was regulated, filling the gap in the existing technology for regulating ryegrass plant architecture. This resulted in an ideal plant architecture characterized by short stature, high tillering, and few fibrous roots, thereby improving the agronomic traits of turfgrass.
Patent Information
- Application Number
- CN202411476149.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-22
- Publication Date
- 2026-02-03
- Estimated Expiration
- 2044-10-22
AI Technical Summary
The lack of genes in existing technologies to regulate the plant type of perennial ryegrass prevents it from exhibiting the ideal plant type of short stature, multiple tillers, and few fibrous roots, thus affecting the improvement of turfgrass.
The LpBPM5.6 gene of perennial ryegrass was cloned, and a low-expression vector was constructed. Its expression level was reduced by genetic engineering. Perennial ryegrass callus tissue was infected with Agrobacterium tumefaciens to obtain perennial ryegrass plants with low expression of LpBPM5.6.
This method achieves the ideal plant shape of perennial ryegrass, characterized by short stature, high tillering, and few fibrous roots, significantly improving the yield and quality of turfgrass.
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Figure CN119082136B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant molecular breeding, and particularly relates to a LpBPM5.6 gene for regulating the plant type of perennial ryegrass, a low-expression vector, a construction method and application thereof. BACKGROUND
[0002] Plant type is an important agronomic trait, and genes for regulating tillering, leaf angle and roots can significantly improve the yield of crops such as rice, maize and wheat by improving plant type. Unlike cereal crops with less tillering and upright stems and leaves, dwarf height, more tillering and creeping stems and leaves are the "ideal plant type" of turf grass.
[0003] Lolium perenne L. is a perennial grass of the Poaceae family, with a developed root system and more fibrous roots, which are distributed in a network in the 15-20 cm soil layer, which helps it better absorb water and nutrients in the soil. The stem is upright, hollow, clump-shaped, soft, 80-100 cm high, with internodes and more tillers, which enables the ryegrass to quickly form a dense grass, with strong tillering ability, fast establishment and long green period, and is often used for ornamental greening and golf course planting.
[0004] In the prior art, genes for regulating the plant type of Poaceae plants are mainly involved in plant hormone pathways, such as dwarf1, dwarf3, dwarf8 and dwarf11 related to gibberellin biosynthesis pathway, brd1, nana plant1 and nana plant2 related to brassinosteroid biosynthesis pathway, and brevies plant1 and pin1 related to auxin biosynthesis pathway. These genes are mainly found in corn, rice and wheat, and are involved in the morphological development of plant type. However, there is no report on genes for regulating the plant type of perennial ryegrass.
[0005] In summary, there is an urgent need to provide a gene for regulating the plant type of perennial ryegrass to make the perennial ryegrass exhibit an "ideal plant type". SUMMARY
[0006] The application aims to provide a LpBPM5.6 gene for regulating the plant type of perennial ryegrass, a low-expression vector, a construction method and application thereof. The low expression level of LpBPM5.6 makes the perennial ryegrass exhibit a plant type of dwarf height, more tillers and fewer fibrous roots, which provides a good application for improving the plant type of turf grass.
[0007] The LpBPM5.6 gene for regulating plant type of perennial ryegrass has a nucleotide sequence as shown in SEQ ID No. 1.
[0008] The application provides a low-expression vector constructed by using the LpBPM5.6 gene of perennial ryegrass, and the low-expression vector comprises the following steps:
[0009] RNA of perennial ryegrass leaves is extracted, and cDNA is obtained by reverse transcription; a pair of primers is synthesized, and cDNA is used as a template to perform PCR amplification, so that an attB-PCR product is obtained, and the primer sequences are shown in SEQ ID NO. 4 and SEQ ID NO. 5; the attB-PCR product is mixed with a donor vector, and BP recombination mixed enzyme is added for incubation, termination, transformation into competent cells, and thus a gateway system entry vector is obtained; the entry vector and an expression vector are mixed, and LR recombination cloning enzyme is added for incubation, termination, transformation into competent cells, and thus the low-expression vector is obtained.
[0010] Preferably, the donor vector is pDONA207, and the expression vector is PC336.
[0011] The application further provides application of the LpBPM5.6 gene of perennial ryegrass or the low-expression vector in regulating plant type of perennial ryegrass.
[0012] The low-expression vector is used for preparing perennial ryegrass plants with low expression of LpBPM5.6, and the preparation process comprises the following steps:
[0013] The low-expression vector is transformed into Agrobacterium tumefaciens competent cells, and positive recombinant Agrobacterium tumefaciens is obtained; the positive recombinant Agrobacterium tumefaciens infects perennial ryegrass callus, and the perennial ryegrass callus is co-cultured in dark; the product of dark co-culture is transferred to a resistant culture medium, and positive perennial ryegrass callus is screened; the positive perennial ryegrass callus is sequentially transferred to a differentiation culture medium and a strong seedling culture medium, and thus the perennial ryegrass plants with low expression of LpBPM5.6 are obtained.
[0014] Preferably, the Agrobacterium tumefaciens competent cells are any one of EHA105, GV3101 and LB4404.
[0015] Preferably, the infection process is as follows: the perennial ryegrass callus is soaked in the positive recombinant Agrobacterium tumefaciens suspension at 1.0×10 5 Pa~1.5×10 5 Pa The perennial ryegrass callus is soaked in the positive recombinant Agrobacterium tumefaciens suspension for 20 min, and then incubated at 80 rpm~80 rpm for 20 min~25 min.
[0016] Preferably, the formula of the differentiation culture medium is: 0.5mg-0.6mg benzylaminopurine, 30g-32g maltose, 3g-4g plant gel and 150mg-152mg thidiazuron are added to each liter of MS medium.
[0017] Preferably, the formula of the seedling culture medium is: 30g-32g maltose, 3g-4g plant gel and 150mg-152mg thidiazuron are added to each liter of MS medium.
[0018] Compared with the prior art, the beneficial effects of the present application are: the present application first clones a full-length sequence of a gene regulating the plant type of perennial ryegrass, i.e. 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 obtain perennial ryegrass plants with low expression of LpBPM5.6 gene by constructing a low expression vector of LpBPM5.6. The results show that, compared with wild-type perennial ryegrass, after low expression of the gene, the tiller number of perennial ryegrass increases by more than 20%, the plant height and fresh weight decrease by more than 50%, and the fibrous roots are significantly reduced. The significant changes in these traits provide support for the plant type improvement of perennial ryegrass and other turfgrasses. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 For the plant type results of LpBPM5.6 low expression perennial ryegrass plants, A: expression level of LpBPM5.6 in each organ; B: expression level of LpBPM5.6 in LpBPM5.6 RNAi material; C-E: results of GFP fluorescence image, DAPI nuclear staining image, and combined results of GFP fluorescence image and DAPI nuclear staining image of empty vector; F-H: GFP fluorescence image, DAPI nuclear staining image, and combined results of GFP fluorescence image and DAPI nuclear staining image of LpBPM5.6 vector; 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 pictures of root systems of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics; P: heat map of root system morphological indexes of WT and LpBPM5.6 RNAi; Q: representative pictures of aboveground parts of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics; R: representative pictures of underground parts of WT and LpBPM5.6 RNAi plants after 21 days of hydroponics.
[0021] Figure 2For the plant type results of LpBPM5.6 overexpression ryegrass plants, A: expression level of LpBPM5.6 in LpBPM5.6 overexpression materials; B: representative pictures of the aerial parts of WT and LpBPM5.6 overexpression plants after 21 days of hydroponics; C-F: plant height, tiller number, fresh weight and maximum root length of WT and LpBPM5.6 overexpression plants after 21 days of hydroponics; G: representative pictures of the root systems of WT and LpBPM5.6 overexpression plants after 21 days of hydroponics, from left to right are WT, LpBPM5.6 overexpression ryegrass plant OE#1, LpBPM5.6 overexpression ryegrass plant OE#2; H: heat map of root system morphology index; I: representative pictures of the underground parts of WT and LpBPM5.6 overexpression plants after 21 days of hydroponics. DETAILED DESCRIPTION
[0022] The present application will be further described in the following specific examples, but the scope of the present application is not limited thereto. The details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and these modifications or replacements all fall within the protection scope of the present application.
[0023] The inventive concept of the present application is as follows: At present, the genes for regulating the plant type of Poaceae plants are mainly involved in the biosynthetic pathways of plant hormones, such as the gibberellin biosynthetic pathway, the brassinosteroid biosynthetic pathway and the auxin biosynthetic pathway. These genes are mostly found in corn, rice and wheat for participating in the morphological establishment of their plant types; and there is no report on the genes for regulating the plant type of perennial ryegrass.
[0024] BPM protein is an adapter protein of Cullin3-based E3 ubiquitin ligase in animals and plants, forms a small family consisting of six members in Arabidopsis, and is involved in the regulation of physiological processes such as plant growth, fertility, stomatal movement, fatty acid metabolism and ABA signal transduction. The Arabidopsis genome only encodes 6 BPM genes, corresponding to AtBPM 1-6. BPM protein, as a substrate-specific binding protein of CUL3-based E3 ligase, binds to different transcription factor family members, and is therefore considered to be an important regulator of various developmental processes and stress responses. Overexpression of BPM3 or BPM5 enhances the sensitivity of plants to abscisic acid, inhibits seed germination, seedling morphological establishment 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 hormone signaling in plants but have also been shown to be involved in flowering. In Arabidopsis thaliana, bpm mutants exhibit late flowering, while plants overexpressing AtBPM1 exhibit early flowering, thus predicting that BPM positively regulates flowering time. Furthermore, transcription factors AtMYB56 and AtMYB106 bind to the promoter of AtFT, a key regulator of the trophic-to-reproductive transition, thereby inhibiting its transcription. CULBPM mediates the ubiquitination and degradation of 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, serving as post-translational regulators of plant physiological responses. Notably, while 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 exhibit similar functions in binding to the three MYC proteins. Furthermore, only BPM1, BPM2, and BPM4 interact with MYB106, and only BPM1 and BPM3 interact with RRTF1. This suggests that within this highly conserved protein family, there is both functional redundancy and distinct individual functions, some of which may only be expressed under specific environmental conditions. Protein families containing BPM domains have been found in almost all eukaryotic genomes. They regulate ubiquitination and proteasome degradation of specific substrates, and BPMs are widely involved in the regulation of plant growth, development, and tolerance to biotic and abiotic stresses. In recent years, BPMs have also been shown to improve key agronomic traits in some crops; however, the function of BPMs in perennial turfgrass remains unclear.
[0027] Prior to this invention, the inventors had not anticipated that the LpBPM5. gene was related to the morphogenesis of ryegrass plant architecture. Based on this, the present invention provides a ryegrass LpBPM5.6 gene that regulates plant architecture, the nucleotide sequence of which is shown in SEQ ID No. 1.
[0028] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are 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 cultivar Peak III, and cDNA was obtained by reverse transcription. The RNA extraction kit was purchased from Promege, and the reverse transcription kit from Vazyme. Based on the LpBPM5.6 sequence obtained from 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'-ATGGGCTGGGCAAGCACATC-3'.
[0032] SEQ ID NO. 3: LpBPM5.6-R, 5'-TCAGACCCTCGGTCTCACTCT-3'.
[0033] Using cDNA from the perennial ryegrass cultivar Peak III as a template, the LpBPM5.6 gene was cloned by PCR, and its nucleotide sequence is shown in SEQ ID NO.1.
[0034] SEQ ID NO.1:
[0035]
[0036] Using the Gateway kit purchased from Thermo, a 200bp-300bp LpBPM5.6-specific sequence was recombined into the Gramineae-specific RNAi vector PC336 to construct the LpBPM5.6-RNAi vector, which expresses LpBPM5.6 at low levels. The construction steps of the LpBPM5.6-RNAi vector are as follows:
[0037] Step 1: RNA was extracted from the leaves of the perennial ryegrass variety Peak III and cDNA was obtained by reverse transcription.
[0038] Step 2: Using cDNA as a template, perform PCR amplification to prepare attB-PCR product, namely the LpBPM5.6 specific sequence.
[0039] A pair of primers were synthesized for amplifying the attB-PCR product. 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 cDNA from leaves of the perennial ryegrass cultivar Peak III as a template, the LpBPM5.6 specific sequence was amplified using primers SEQ ID NO.4 and SEQ ID NO.5, and the LpBPM5.6 specific sequence was recovered using a DNA gel recovery kit.
[0045] Step 3, BP reaction: Mix the attB-PCR product with the donor vector, add BP recombinant mixed enzyme for incubation, terminate the reaction, transform into competent cells, and obtain the gateway system entry vector.
[0046] 1. At room temperature, add the following components to a centrifuge tube and mix: 150 ng of attB-PCR product, 150 ng of pDONA207 vector, add TE buffer (pH 8.0) to a final volume of 8 μL, and then add 2 μL of BP Clonase. TM II mixed enzyme.
[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 stop 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 the sequencing results, and screen positive clones, which are the entry vectors.
[0050] Step 4, LR recombination reaction: Mix the entry vector and expression vector, add LR recombinant cloning enzyme for incubation, terminate the reaction, transform into competent cells, and obtain low expression vector.
[0051] 1. Add the following components to a centrifuge tube and mix at room temperature: 300 ng of initiation vector, 150 ng of PC336 vector, and 4 μL of 5X LR cloning enzyme. TM Add TE buffer (pH 8.0) to 16 μL, then add 4 μL of LR cloning enzyme.
[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 cells, select single clones to extract plasmids, and obtain the LpBPM5.6-RNAi vector, i.e., the low expression vector.
[0055] Example 2: Application of LpBPM5.6 gene low expression vector
[0056] 1. Experimental Methods
[0057] 1.1 Constructing a ryegrass line with low LpBPM5.6 expression, the steps are as follows:
[0058] The LpBPM5.6-RNAi vector described in Example 1 was chemically transformed into *Agrobacterium tumefaciens* competent cells EHA105 to obtain positive recombinant *Agrobacterium tumefaciens*. Three-month-old perennial ryegrass peak III callus tissue was selected and immersed in a suspension of positive recombinant *Agrobacterium tumefaciens* under vacuum conditions of 1.0 × 10⁻⁶. 5Pa, incubate for 20 minutes, shake at 80 rpm for 20 minutes, and evenly spread the callus tissue on petri dishes containing moistened filter paper. Co-culture in the dark for 3 days using a co-medium. The co-medium formulation is: 2 mg of 6-benzylaminopurine, 0.15 mg of α-naphthaleneacetic acid, and 8 g of agar per liter of MS medium. After co-culture, transfer to a resistance medium to screen for positive perennial ryegrass callus tissue. Induce seedlings on differentiation medium, and after seedling formation, transfer the seedlings to a seedling strengthening medium to obtain transgenic seedlings. The differentiation medium formulation is: 0.5 mg of benzylaminopurine, 30 g of maltose, 3 g of plant gel, and 150 mg of termethin per liter of MS medium. The seedling strengthening medium formulation is: 30 g of maltose, 3 g of plant gel, and 150 mg of termethin per liter of MS medium. The transgenic ryegrass materials obtained through identification and screening were used to obtain perennial ryegrass plants with low expression of LpBPM5.6. Positive transgenic materials were rapidly expanded 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, constructing the pUbi:LpBPM5.6-GFP overexpression vector. The LpBPM5.6-GFP vector was transformed into Agrobacterium tumefaciens competent cells EHA105. Three-month-old perennial ryegrass peak III callus was soaked in a suspension of Agrobacterium tumefaciens, followed by co-culturing in the dark on a co-medium for 3 days. The callus was then transferred to a resistance medium and cultured in the dark for 2 months. Callus that had not fully browned was then transferred to a differentiation medium and cultured under light for approximately 1 month. After roots and green shoots emerged from the callus, the seedlings were transferred to a seedling strengthening medium to obtain positive transgenic seedlings, i.e., LpBPM5.6 overexpressing ryegrass lines.
[0061] LpBPM5.6 low-expression trans-ryegrass lines and overexpression trans-ryegrass lines were grown in a culture room at 22°C, 40% humidity, and a 16-hour / 8-hour light / dark cycle.
[0062] Functional validation of 1.3LpBPM5.6 low-expression and overexpression ryegrass lines
[0063] 1.3.1 RNA extraction and quantification by RT-qPCR
[0064] use Total RNA was extracted from the strain using the Super Total RNA Extraction Kit, and cDNA was synthesized using reverse transcription reagents. The cDNA and a 2×ChamQ SYBR qPCR mixture were then subjected to RT-qPCR using a LightCycler 96 quantitative analyzer. LpTBP-1 and LpeIF4A were used as internal control genes. Through 2... -ΔΔCT The method was used to calculate the expression level of the LpBPM5.6 gene.
[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 and Pro35S:GFP vectors were transformed into *Nicotiana benthamiana* leaves using *Agrobacterium* GV3101. Fluorescence signals were observed using a laser confocal microscope after 3 days.
[0067] 1.4 Phylogenetic Analysis
[0068] Protein sequences were aligned using MAFFT software, and phylogenetic relationships were analyzed using IQ-TREE software. A phylogenetic tree was constructed using maximum likelihood estimation with default parameters.
[0069] 1.5R language for generating heatmaps
[0070] After culturing perennial ryegrass plants in Hoagland solution for 3 weeks, root traits were analyzed using a root scanner purchased from Hangzhou Wansen Technology Testing Co., Ltd. A normalization method was used to ensure that the raw count values for each data point were on the same order of magnitude. Then, the variations in data across different plants were reflected by different color changes in the heatmap. Data is represented as follows: L represents total root length, PA represents projected area, AP represents pixel area, SA represents surface area, V represents volume, AD represents 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 branches, 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 279 BPM genes, approximately 46 times more numerous than in Arabidopsis thaliana, 26 times more than in rapeseed, 9 times more than in maize, and 3.6 times more than in wheat. This amplification of the gene family in ryegrass suggests that BPM may have a unique function in perennial grasses. Figure 1As shown, further research revealed that LpBPM5.6 is highly expressed in the leaves, roots, and stems of ryegrass, suggesting that it may play a functional role in the vegetative organs of perennial ryegrass. Subcellular localization observation showed that LpBPM5.6 is evenly distributed in the cytoplasm, nucleus, and cell membrane.
[0073] The inventors successfully constructed two ryegrass lines, RNAi#1 and RNAi#2, with low expression of LpBPM5.6, and studied their function during the vegetative development stage of perennial ryegrass. The study found that LpBPM5.6 can influence the plant architecture of turfgrass. After 21 days of hydroponic cultivation of individual tillers, the plant height of the LpBPM5.6-low expression ryegrass lines RNAi#1 and RNAi#2 was approximately half that of the wild-type WT, and their fresh weight was less than half that of WT. However, the number of tillers in RNAi#1 and RNAi#2 increased significantly, exceeding 20% compared to WT. Root observation revealed that the maximum root length of RNAi#1 and RNAi#2 was not significantly different from the wild-type, but their root density was significantly lower than WT. Statistical analysis of root parameters showed that the total root length, surface area, volume, average diameter, number of root tips, and number of branches of RNAi#1 and RNAi#2 were all significantly lower than those of WT. These results indicate that reducing the expression level of LpBPM5.6 in ryegrass can produce an "ideal plant type" of turfgrass with short plant height and many tillers. At the same time, fewer fibrous roots can reduce the metabolic cost of the turfgrass's underground parts, allowing more resources to be concentrated in the above-ground parts, thereby improving the quality of the turfgrass under good cultivation conditions.
[0074] Further construction of LpBPM5.6 overexpressing ryegrass lines was carried out, see [link to article]. Figure 2 OE#1 and OE#2 are Leptochloa ryegrass lines overexpressing LpBPM5.6. After 21 days of hydroponic cultivation with individual tillers, plant architecture analysis of WT and LpBPM5.6 overexpressing ryegrass plants revealed that LpBPM5.6 overexpressing ryegrass plants were slightly taller than WT plants; their fresh weight was also greater; however, the number of tillers in LpBPM5.6 overexpressing ryegrass plants was only 70% of that in WT plants. Root observation showed that LpBPM5.6 overexpressing ryegrass plants did not affect the maximum root length, but their root density was significantly higher than that of WT plants. Statistical analysis of root parameters showed that LpBPM5.6 overexpressing ryegrass plants had higher total root length, surface area, volume, average diameter, number of root tips, and number of branches than WT plants.
[0075] BPM protein is an important component of the Cullin3, CUL3 E3, and ubiquitin ligase complex. In this invention, reducing the expression level of LpBPM5.6 resulted in perennial ryegrass exhibiting a phenotype characterized by shorter plant height, increased tillering, and fewer fibrous roots; conversely, overexpression of LpBPM5.6 resulted in perennial ryegrass exhibiting a phenotype characterized by taller plant height, fewer tillers, and more fibrous roots. These results demonstrate that LpBPM5.6 can regulate the ideal plant type of perennial ryegrass, providing support for the improvement of plant type in perennial ryegrass and other turfgrasses.
[0076] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.
Claims
1. Perennial ryegrass that regulates plant architecture LpBPM5.6 The application of genes in regulating the plant architecture of perennial ryegrass is characterized by, The perennial ryegrass LpBPM5.6 The nucleotide sequence of the gene is shown in SEQ ID No. 1; The regulation of perennial ryegrass plant architecture refers to 1) and 2) below. 1) Perennial ryegrass LpBPM5.6 RNAi vectors of genes promote dwarfing, increased tillering, and reduced fibrous roots in perennial ryegrass plants; 2) Perennial ryegrass LpBPM5.6 Gene overexpression vectors promote increased plant height, reduced tillering, and increased fibrous roots in perennial ryegrass plants.
2. The application according to claim 1, characterized in that, The method for constructing the RNAi vector includes the following steps: RNA was extracted from the leaves of perennial ryegrass and cDNA was obtained by reverse transcription. 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. The attB-PCR product was mixed with the donor vector, incubated with BP recombinant mixed enzyme, the incubation was terminated, and the mixture was transformed into competent cells to obtain the gateway system entry vector. The initiation vector and expression vector were mixed, incubated with LR recombinant cloning enzyme, terminated, and transformed into competent cells to obtain the RNAi vector.
3. The application according to claim 2, characterized in that, The donor vector is pDONA207, and the expression vector is PC336.
4. The application according to claim 1, characterized in that, The RNAi vector is used to prepare low-expression RNAi vectors. LpBPM5.6 The preparation process of perennial ryegrass plants includes the following steps: The RNAi vector was transformed into competent Agrobacterium tumefaciens cells to obtain positive recombinant Agrobacterium tumefaciens. Positive recombinant Agrobacterium tumefaciens infected perennial ryegrass callus and co-cultured in the dark on a co-culture medium; The products from dark co-culture were transferred to a resistance medium, and positive perennial ryegrass callus tissues were screened. Positive perennial ryegrass callus tissue was sequentially transferred to differentiation medium and seedling strengthening medium to obtain low expression LpBPM5.6 Perennial ryegrass plants.
5. The application according to claim 4, characterized in that, The Agrobacterium tumefaciens competent cells are any one of EHA105, GV3101 and LB4404.
6. The application according to claim 4, characterized in that, The infection process is as follows: At 1.0×10 5 Pa ~ 1.5 × 10 5 Pa immersed perennial ryegrass callus in a positive recombinant Agrobacterium tumefaciens suspension for 20 min, followed by incubation at 80 rpm for 20 min to 25 min.
7. The application according to claim 4, characterized in that, The formulation of the differentiation medium is as follows: Add 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 termethin to each liter of MS medium; The formula for the seedling growth medium is as follows: Add 30-32g maltose, 3-4g plant gel and 150-152mg termethin to each liter of MS medium.