EsPLS1 gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an EsPLS1 gene for regulating and controlling the size of siberian wildrye leaves and application of the EsPLS1 gene. The nucleotide sequence of the EsPLS1 gene is as shown in SEQ ID NO.1, and the size of a plant leaf can be positively regulated and controlled by overexpressing the gene in a plant. Therefore, the EsPLS1 gene can be used for increasing the leaf size of the siberian wildrye, and a theoretical basis is provided for the thought of increasing the leaf size by using the EsPLS1 gene so as to increase the biomass.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the regulation of leaf size in monocotyledonous plants, particularly wheat or rice. EsPLS1 Genes and their applications. Background Technology

[0002] Old Mango ( Siberian elympus L . As a high-quality gramineous forage grass with strong resilience and wide adaptability, it has broad application prospects in the future sustainable development of agriculture and ecological security construction in my country. Its prospects are mainly reflected in two directions: diversified development and ecological-economic synergy. On the one hand, through variety selection and optimized cultivation management, *Triticum aestivum* will continue to serve as a backbone forage grass for livestock production in arid and high-altitude regions, providing reliable high-quality roughage for livestock. On the other hand, its strong soil and water conservation and slope protection capabilities make it an irreplaceable pioneer plant and ecological guardian in ecological projects such as the ecological restoration of degraded grasslands, soil erosion control, and the reclamation of mining sites.

[0003] With the development of molecular breeding technology, breeding new varieties of wheat with higher yield, better quality, and stronger resistance will become the core driving force for the upgrading of the industry. From an ecological perspective, the scientific planting and utilization of forage grasses such as wheat can effectively curb grassland degradation and restore fragile ecosystems. From an industrial perspective, a stable and efficient supply of forage grass is the foundation for reducing costs, increasing efficiency, and enhancing competitiveness in animal husbandry, and is directly related to the livelihoods of farmers and herdsmen and the prosperity of the regional economy.

[0004] Leaf size (i.e., leaf area) is one of the key traits determining aboveground biomass accumulation in plants, and there is a close but complex relationship between the two. The core of this relationship lies in the function of the leaf as the primary organ for photosynthesis: theoretically, a larger leaf area means a stronger light-capturing capacity, thus providing more photosynthetic products for plant growth and directly promoting an increase in aboveground biomass. Under sufficient light and unrestricted conditions (such as water and CO2), a larger light-receiving area can directly intercept and absorb more light quanta, thereby driving stronger photosynthesis and synthesizing more carbohydrates (such as sugars and starch). These carbohydrates are the basic building blocks of plant bodies (stems, leaves, branches, etc.) and are directly converted into aboveground biomass. In the breeding and cultivation practices of many crops (such as rice, wheat, corn, and soybeans), cultivating and maintaining a canopy with a large leaf area has always been a core objective for increasing economic yield (usually a portion of aboveground biomass). Therefore, increasing leaf area can enhance the plant's photosynthetic capacity and resource capture efficiency, thus having a significant positive impact on aboveground biomass accumulation. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a EsPLS1 Genes that participate in regulating leaf area in monocotyledonous plants, especially in regulating the leaf area of ​​*Triticum aestivum*. This invention is the first to discover, through regulation... EsPLS1 Gene expression can significantly increase rice leaf area, which may effectively optimize the leaf type of wheat, thereby increasing the yield of wheat per plant and achieving efficient accumulation of aboveground biomass. This provides an innovative molecular breeding technology solution for solving the problem of insufficient forage supply and reducing dependence on foreign sources.

[0006] To achieve the technical objective of this invention, the first aspect of this invention provides a... EsPLS1 The gene, characterized by overexpression of the gene... EsPLS1 Genes are used to increase the leaf area of ​​monocotyledonous plants, and their nucleotide sequences are shown in SEQ ID NO.1.

[0007] In particular, the monocotyledonous plant is wheat or rice.

[0008] This invention has found that, compared to the wild type, after overexpression... EsPLS1 The leaf area of ​​the rice variety with the overexpression gene is much larger than that of wild-type rice, a result that confirms the effectiveness of overexpression. EsPLS1 Genes can regulate leaf size. Therefore, it can be concluded that… EsPLS1 Genes can be used to increase the leaf area of ​​old wheat, providing a basis for utilization. EsPLS1 Genetic breeding provides a theoretical basis for high-yield old wheat varieties.

[0009] In particular, quantitative amplification of the above EsPLS1 The nucleotide sequences of the quantitative PCR primers for the gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] In particular, cloning the above EsPLS1 The nucleotide sequences of the PCR primers for the gene are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0011] The amplification program is as follows: 97℃ for 3 min, 97℃ for 15 s, 58℃ for 30 s, 72℃ for 1 min 30 s, 35 cycles, 72℃ for 5 min.

[0012] To achieve the technical objective of this invention, a second aspect of this invention provides a protein encoding the above-mentioned gene, the protein sequence of which is shown in SEQ ID NO.2.

[0013] To achieve the technical objective of this invention, a third aspect of this invention provides a recombinant expression vector comprising the above-described... EsPLS1Genes that regulate the leaf size of monocotyledonous plants, especially the leaf size of old wheat or rice plants.

[0014] In particular, the recombinant expression vector is used to express the... EsPLS1 Gene insertion into the P1300-35S-C-Myc overexpression vector plasmid Xba I and CPN Obtained between I sites.

[0015] Among them, the EsPLS1 The gene was amplified and inserted using CDS primer sets such as SEQ ID NO.7 and SEQ ID NO.8.

[0016] To achieve the technical objective of this invention, a fourth aspect of this invention provides a recombinant engineered bacterium comprising the above-described recombinant expression vector.

[0017] In particular, the recombinant engineered bacteria are obtained by transferring the recombinant expression vector into competent Escherichia coli cells.

[0018] To achieve the technical objective of this invention, the fifth party of this invention provides the above-mentioned... EsPLS1 The application of genes in regulating leaf area in monocotyledonous plants, through overexpression in said plants. EsPLS1 Genetic implementation.

[0019] To achieve the technical objective of this invention, a sixth aspect of this invention provides the application of the above-mentioned recombinant expression vector in regulating leaf area of ​​monocotyledonous plants, which is achieved by overexpressing the vector in the plant. EsPLS1 Genetic implementation.

[0020] To achieve the technical objective of this invention, a seventh aspect of this invention provides the application of recombinant engineered bacteria in regulating leaf size in monocotyledonous plants, which is achieved by overexpressing [a specific type of bacteria] in the plant. EsPLS1 Genetic implementation.

[0021] The monocotyledonous plant is either wheatgrass or rice.

[0022] Beneficial effects The present invention provides EsPLS1 Genes can be used to regulate the size of leaves in monocotyledonous plants, especially *Triticum aestivum*, which is beneficial for increasing the yield of *Triticum aestivum* forage and can be used to improve *Triticum aestivum* forage varieties. Attached Figure Description

[0023] Figure 1 for EsPLS1 The relative expression levels of genes in different tissues at different stages of *Oryza sativa* (a type of wheat); Figure 2 For old wheat EsPLS1 Image showing the results of gene cloning; Figure 3 for EsPLS1 Gene overexpression vector structure diagram; Figure 4 for EsPLS1 Image showing the results of RNA level identification in gene-overexpressing plants; Figure 5 for EsPLS1 Phenotypic results of transgenic rice plants with overexpressed genes. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The bioinformatics software and products used in the present invention are all commercially available. Various processes and methods not described in detail are all conventional methods known to those skilled in the art. The source of materials used, trade names, and components that need to be listed are indicated when they first appear. Unless otherwise specified, the same reagents used thereafter are the same as those initially indicated.

[0025] This invention has discovered a new gene. EsPLS1 By analyzing leaf size data collected from a population of wild *Eriocaulon buergerianum*, two extreme leaf size materials, YS-92 (small leaves) and YS-137 (large leaves), were identified. Transcriptome analysis was performed on the flag leaf of these two materials at the heading stage. After data quality control analysis, DEGs and DEPs screening, GO enrichment, and KEGG pathway analysis, 12 genes showed significant expression differences, which were significantly associated with growth, development, cell elongation, and cell division. Finally, the most significantly different gene expression was selected. EsPLS1 Gene.

[0026] The EsPLS1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0027] EsPLS1 The encoded protein sequence is shown in SEQ ID NO.2: MANPLAGLQDHLKHARDYAFEGLYDTSIIFFDGAIAQINKHLANLDDTFIRTKWMNCKKAISEEVEIVRQLDAQLKSFKEAPGTMRSSSPPIRSNNKSFLFQPLDEYPTSSPTTFDDPDVWAPPRDTSTP SRRSARGQPSARKSSQDGAWARGPSKTGTPSRGAKPSGSKASSAVRSSTASSTGGRKGKSSSKADSASSDAEEGKSKKAQYEGPDGDLAAMLERDVLDSTPGVRWDDVAGLSEAKRLLEEAVVLPLWMPE YFQGIRRPWKGVLMFGPPGTGKTLLAKAVATECGTTFFNVSSATLASKWRGESERMVRCLFELARAYAPSTIFIDEIDSLCTSRGASGEHESSRRVKSELLVQIDGVNNSSTNEDGQPKIVMVLAATNFP WDIDEALRRRLEKRIYIPLPSFESRKSLISINLRTVEVATDVNIDEVARRTEGYSGDDLTNVCRDASMNGMRRKIAGKTRDEIKNMSKDDISKDPVAMCDFEEALVKVQKSVSPSDIERHEKWMAEFGSA.

[0028] The inventors selected the expression differences that were most significant. EsPLS1 The genes have undergone extensive analysis, experiments, and research. The following are representative examples that clearly demonstrate their effectiveness. EsPLS1 Part of the research experiment on the key role of old wheat in the growth process.

[0029] Example 1: Spatiotemporal expression pattern analysis of *Ophiopogon japonicus* genes 1. Obtaining cDNA from different tissue samples of *Eriocheir sinensis* Seeds of variety "YS-137" were sown in seedling trays and cultured in a greenhouse. Multiple tissue samples were collected from *Eriobotrya stenoptera* from the seedling stage to the milk stage, including the seedling stage, early tillering stage, mid- and late tillering stage, early and late jointing stage, booting stage, heading stage, flowering stage, and milk stage. These tissue samples included roots, stems, leaves, and ears. All collected tissue samples were flash-frozen in liquid nitrogen and stored at -80℃ for later use. RNA was extracted from these tissues using the AFT Spin Universal Plant Fast RNA Extraction Kit (Wuhan Aibotek Biotechnology Co., Ltd.). RNA quality was assessed by agarose gel electrophoresis, and RNA quantity was determined by Nanodrop. cDNA was then synthesized using the EX RT kit reverse transcription kit (Wuhan Aibotek Biotechnology Co., Ltd.). The reverse transcription cDNA system is shown in Table 1. The reverse transcription conditions were 45℃ for 2 min, 50℃ for 15 min, and 85℃ for 2 min, inactivating the reverse transcriptase. This cDNA was diluted 2-fold to serve as a template, yielding cDNA samples from each tissue sample at the two aforementioned stages.

[0030] Table 1. Reverse transcription cDNA system

[0031] 2. Old Mango Wheat EsPLS1 Gene expression pattern analysis Based on the old mangoes already obtained EsPLS1 Gene sequence information was used to design quantitative real-time primers EsPLS1-RT and EsPLS1-RT-R using AlleleID 6.0. Using cDNA from different tissues of *Evodia rutaecarpa* obtained in step 1 as templates, qRT-PCR amplification was performed using 2×HQ SYBR qPCR Mix (LoW ROX) reagents and an ABI Q7 quantitative real-time PCR instrument. The quantitative real-time PCR amplification program was: 95℃, 30s, 40 cycles, each cycle including 95℃, 10s, 60℃, 30s, and 72℃, 30s. Each experiment contained 3 biological replicates. Two... -ΔΔCt The relative expression levels of genes are calculated using a method called 2. -ΔΔCt Method calculation EsPLS1 The relative expression levels of genes in different tissues of *Leymus chinensis* are analyzed as follows: Figure 1 As shown in Table 2, the quantitative fluorescence system is as follows. The sequence of EsPLS1-RT is ATCATGGAGGTTCTCGGGGA, and the sequence of EsPLS1-RT-R is CTCGAGGGTCTTCATGTCGG.

[0032] according to Figure 1 The analysis results show that EsPLS1It exhibits significant tissue-specific expression at different developmental stages, from seedling stage to flowering stage during growth and development. EsPLS1 Gene expression in leaf tissues was significantly higher than in other tissues. EsPLS1 The expression level in leaf tissues gradually increased from the seedling stage to the jointing stage, while it gradually decreased after the plant began to develop spikes. This indicates that... EsPLS1 It may play a key regulatory role in the development of leaves of old wheat. Leaves are an important component of aboveground biomass. During the plant's growth and development, leaves produce a large amount of carbohydrates (such as sucrose) through photosynthesis. They are the key "source" organs, assimilating mineral nutrients such as nitrogen and phosphorus into more complex organic matter and transporting them directionally to developing organs. This close relationship determines how the plant's total biomass accumulates and is distributed during vegetative growth.

[0033] Table 2. Quantitative Fluorescence System

[0034] Example 2, Old Mango Wheat Yes PLS1 Cloning of genes According to the old mangoes obtained EsPLS1 RNA was extracted from the *Brassica juncea* material “YS-137” using CDS-F and CDS-R primers designed based on gene sequence information. PCR amplification was performed using reverse-transcribed cDNA as a template. The PCR reaction system is shown in Table 3. After amplification, agarose gel electrophoresis was performed, and the amplification products were recovered and sequenced. EsPLS1 The genome sequence and cDNA sequence of the gene. The CDS-F sequence is ATGGTGCAGCCGGTGTTCGA, and the CDS-R sequence is TCATGTCCTGGCGGCGGGCT.

[0035] PCR amplification program: 97℃, 3 min; 97℃, 15 s, 58℃, 30 s, 72℃, 1 min 30 s, 35 cycles; extension at 72℃ for 5 min.

[0036] PCR products were subjected to 1% agarose gel electrophoresis at 180V for 10 min. This was based on whole-genome data from *Leymus chinensis*. EsPLS1 Gene sequence was obtained, and gene-specific primers were designed. Using *Ophiopogon japonicus* cDNA as a template, PCR amplification was performed using a high-fidelity enzyme. After TA cloning and colony PCR detection, two colony PCR bands were found to be consistent with the expected size. Figure 2 As shown. Sequencing of the above positive colonies revealed that... EsPLS1 The CDS region is 1080 bp in length, and the sequence is correct, indicating that the correct sequence was obtained. EsPLS1 TA plasmid.

[0037] Table 3 PCR reaction system

[0038] Example 3 EsPLS1 Overexpression of transgenic rice to obtain 1. Overexpression EsPLS1 Construction of transgenic vectors.

[0039] Use restriction endonucleases Xba I and CPN The plant overexpression vector P1300-35S-C-Myc was double-digested with enzymes, and the vector obtained in Example 2 was amplified using primers P1300-35S-C-Myc-F and P1300-35S-C-Myc-R. EsPLS1- TA plasmids, to obtain those containing vector arms EsPLS1 The CDS region of the gene was extracted, and the vector and fragment were recombined using the SE Seamless Cloning and Assembly Kit. The recombinant product was transformed into DH5α competent E. coli cells, and overexpression was confirmed by sequencing. EsPLS1 The transgenic vector P1300-35S-C-Myc::EsPLS1 has the following structure: Figure 3 As shown.

[0040] The sequence of P1300-35S-C-Myc-F is gagaacacgggggatctagaATGGTGCAGCCGGTGTTCGA, and the sequence of P1300-35S-C-Myc-R is acaggcctttcgaaggtaccTGTCCTGGCGGCGGGCTGCT.

[0041] 2. Overexpression EsPLS1 Construction of transgenic lines The above-mentioned carrier P1300-35S::EsPLS1-C-MYC was electroporated to convert EHA105 competent states, and then coated onto a substrate containing 25 Incubate on LB medium containing 1 mg / L Rifampicin and 50 mg / L Kanamycin at 28°C for 2 days; colonies positive by colony PCR are stored at -80°C with glycerol. Mature rice seeds (Nipponbare) Rice L. spp. japonicaAfter dehulling, the callus was soaked in 70% ethanol for 1 min, then in 30% NaClO + 2 drops of Tween-20 for 20 min, washed with sterile water, and inoculated onto N6 medium (Beijing Cooler Master Technology Co., Ltd.) containing 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D). It was incubated in the dark at 28°C for 21 days. Naturally dividing callus tissue was transferred to fresh induction medium and subcultured for 14 days before transformation. Agrobacterium was resuspended in AAM liquid medium (Beijing Cooler Master Technology Co., Ltd.) containing 100 μM acetosyringone (AS), and the OD600 was adjusted to 0.08-0.1. After soaking the callus tissue in the bacterial suspension for 15 min, the bacterial suspension was discarded, and the callus tissue was placed on sterile filter paper to absorb excess bacterial suspension. It was then transferred to N6D-2 solid co-medium containing 100 μM AS and lined with sterile filter paper, and incubated in the dark at 25°C for 3 days. After 3 days of co-culturing, the callus tissue was washed more than 5 times with clean water, then rinsed once with sterile water containing 400 mg / L carbenicillin. After the callus was dried, it was transferred to N6D-2 medium containing 50 mg / L Hyg(Hygromycin) and 200 mg / L Timentin, and cultured at 28°C under light (16h / 8h photocycle, light intensity 2000 lx) for 4-5 weeks, changing the medium every two weeks. Resistant callus was then selected and placed in a medium containing 0.5 mg / L NAA (… α- Naphthaleneacetic acid, On N6 differentiation medium containing α-naphthaleneacetic acid (NAA), the cells were cultured at 28°C under light (16h / 8h photoperiod, 2000lx light intensity) for 2-3 weeks until green shoots appeared. Then, they were transferred to 1 / 2 MS medium containing 10 mg / L Hyg and cultured until rooting (7-10 days). After 3 days of hardening off, the seedlings were transplanted to a greenhouse for further culture to obtain overexpressed NAA. EsPLS1 Transgenic strains.

[0042] 3. Genetically modified rice EsPLS1 Expression level analysis.

[0043] Leaf tissues were collected from seedlings of the transgenic rice plants obtained in step 2, and extracted using the AFT Spin Universal Plant Fast RNA Extraction Kit. EsPLS1RNA from overexpressing plants was analyzed by agarose gel electrophoresis to determine RNA quality and by Nanodrop ultraviolet spectrophotometer (Thermo Fisher Scientific, MA, USA) to determine RNA quantity. cDNA was then synthesized using the EX RT kit reverse transcription assay. This cDNA was diluted two-fold and used as a template. qRT-PCR amplification was performed using primers EsPLS1-RT-F and EsPLS1-RT-R, the Hieff UNICON qPCRSYBR GREEN Master Mix quantitative reagent, and an ABI Q7 real-time PCR instrument. The RNA was then detected in positive plants. EsPLS1 The expression level, using 2 -ΔΔCt The relative expression level of the target gene was calculated using a method. The sequence of EsPLS1-RT-F is ATCATGGAGGTTCTCGGGGA, and the sequence of EsPLS1-RT-R is CTCGAGGGTCTTC ATGTCGG.

[0044] The results are as follows Figure 4 As shown, in transgenic plants EsPLS1 The expression level was 1.0 to 2052.74 times that of the control plants, with the highest levels observed in the OE-4, OE-5, and OE-7 transgenic lines. EsPLS1 The expression levels of the [specific compounds] were 2052.74 times, 1546.72 times, and 1335.66 times that of the control, respectively, and the differences were all highly significant. P <0.001. Based on this, the three transgenic lines OE-4, OE-5, and OE-7 were selected as the subjects of subsequent phenotypic analysis.

[0045] Example 4: Transgenic Rice EsPLS1 Phenotypic analysis Transplanted wild-type (WT) and overexpression EsPLS1 Transgenic rice lines with consistent growth stages were selected for phenotypic observation. Results showed that overexpression... EsPLS1 The leaves of the plant were 1.27 to 2.06 times larger than those of the wild type. Figure 5 This result confirms... EsPLS1 Genes play an important role in the yield trait of old awn wheat by positively regulating leaf area.

[0046] In summary, it can be concluded that the [source] comes from the old mango plant. EsPLS1 After the gene was overexpressed in monocotyledonous rice model plants, the leaf area increased significantly. Based on the theory of biological genetics, it can be inferred that the gene provided by this invention... EsPLS1 Overexpression of the gene in *Leymus chinensis* can regulate the size of *Leymus chinensis* leaves, increase the yield of *Leymus chinensis* forage, and achieve efficient accumulation of aboveground biomass.

[0047] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. EsPLS1 Genes, characterized by, It can regulate the leaf area of ​​monocotyledonous plants; Among them, the EsPLS1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The monocotyledonous plant is either wheatgrass or rice.

2. A protein encoded by the gene of claim 1, wherein the protein sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It includes the content described in claim 1. EsPLS1 The gene, the recombinant expression vector, can regulate the leaf size of monocotyledonous plants, the monocotyledonous plants being wheat or rice.

4. The recombinant expression vector according to claim 3, characterized in that, It is the aforementioned EsPLS1 Gene insertion into the P1300-35S-C-Myc overexpression vector plasmid Xba I and Kpn Obtained between I sites.

5. Recombinant engineered bacteria comprising the recombinant expression vector of claim 3.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The recombinant expression vector was transferred into competent Escherichia coli cells to obtain the cells.

7. The claim 1 EsPLS1 The application of genes in regulating leaf area in monocotyledonous plants is characterized by, It is expressed by overexpression in the plant EsPLS1 Genetic realization; The monocotyledonous plant is either wheatgrass or rice.

8. The application of the recombinant expression vector according to claim 3 in regulating leaf area of ​​monocotyledonous plants, characterized in that, It is expressed by overexpression in the plant EsPLS1 Genetic realization; The monocotyledonous plant is either wheatgrass or rice.

9. The application of the recombinant engineered bacteria according to claim 6 in regulating the leaf size of monocotyledonous plants, characterized in that, It is expressed by overexpression in the plant EsPLS1 Genetic realization; The monocotyledonous plant is either wheatgrass or rice.

10. A method for increasing the leaf area or yield of a target plant, characterized in that, The specific steps include: Using cloning methods to replicate the product described in claim 1 EsPLS1 Gene insertion into an expression vector, wherein the expression vector is introduced into a target plant via a transformation method to achieve the effect described in claim 1. EsPLS1 Overexpression of the gene ultimately increases the leaf area or yield of the target plant; the target plant is wheat or rice.

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