Application of BnaPE1 protein or its encoding gene in regulating photosynthesis and / or leaf shape in Brassica napus

Abstract

This invention discloses a BnaPE1 protein and its encoding gene's application in regulating photosynthesis and / or leaf shape in Brassica napus. The amino acid sequence of the BnaPE1 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the coding region of the BnaPE1 gene is shown in SEQ ID NO.1. This invention isolates and applies a DNA fragment containing the BnaPE1 gene, which endows Brassica napus with enhanced photosynthesis and increased and deeper leaf lobes, and has important application value for cultivating new Brassica napus varieties with improved photosynthetic efficiency and yield.

Description

Technical Field

[0001] This invention relates to the application of a BnaPE1 protein or its encoding gene in regulating photosynthesis and / or leaf shape in Brassica napus, and belongs to the field of genetic engineering. Background Technology

[0002] Rapeseed is one of the world's most important oilseed crops, and rapeseed oil is the world's third largest source of edible vegetable oil. my country is a major producer and consumer of rapeseed oil. Rapeseed is the most widely planted oilseed crop in my country and the second largest source of vegetable oil after soybeans. Among the three types of rapeseed—Chinese cabbage type, Brassica napus type, and Chinese mustard type—Brassica napus type is the most widely planted in my country, and increasing yield is one of the core objectives of Brassica napus breeding. Photosynthesis is the basic pathway for plants to absorb light energy and synthesize organic matter, and it is a crucial factor determining rapeseed yield and quality. 90%-95% of rapeseed yield comes from photosynthetic products. Currently, research on the photosynthetic characteristics of Brassica napus type is still limited. Photosynthetic rate is an important manifestation of photosynthesis; improving the photosynthetic efficiency of rapeseed is beneficial for further tapping its yield potential and is of great significance for breeding high-efficiency rapeseed varieties. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide an application of the BnaPE1 protein, its encoding gene, and expression cassettes containing the BnaPE1 gene, recombinant vectors, recombinant microorganisms, or transgenic cell lines in controlling photosynthesis and / or leaf shape in Brassica napus.

[0004] Technical solution: This invention provides the application of BnaPE1 protein in regulating photosynthesis and / or leaf shape in Brassica napus, wherein the amino acid sequence of BnaPE1 protein is shown in SEQ ID NO:2, and the number of amino acids is 545.

[0005] SEQ ID NO:2:MGLTSSLRRNNKSFLRIFMILVLSCIPGRTNLCSNHSVTTPIDLPP DIRSSLDSLDLEGYISFNDVHNASKDFGNRYQLPPLAILHPRSVSDISTMVRHIVHLGSTSNLTVAARGHGHSLQGQSLAHQGVVINMESLRSPDIKIHKGKQPYVDVSGGESWINILRETLKYG LSPKSWTDYLHLTVGGTLSNAGISGQAFKYGPQINNVYQLEIITGKGEVMTCSEKQNSELFYSVLGGLGQFGIITRARIALGPAPHMVKWIRVLYSDFSAFSRDQEHLITKKNGFDYVEGFVTVN RTDLLDNWRSSFSPNDSIGASQFKSEGKTLYCLEVVKYFKLEEANSTNLEVEKLLSELSYIPSTLFSSEVTYIEFLDRVHIAEIKLRAKGLWEVPHPWLNLLIPKSSIFEFATEVFNNILTSNNN GPILIYPVNQSKWNKHLSLITPDEDIFYLVAFLPSAVPNPNSGTSNLEYLLRQNQRVLNFCAAANINVKQYLPHYETQREWRSHFGNRWETFAKRKHTYDPLAILAPGHRIFQKATQLSPIQLL.

[0006] The present invention also provides the application of the BnaPE1 gene encoding the above-mentioned BnaPE1 protein in regulating photosynthesis and / or leaf shape in Brassica napus, wherein the nucleotide sequence of the BnaPE1 gene is shown in SEQ ID NO:1 and the sequence length is 1638bp.

[0007] The present invention also provides the application of expression cassettes containing the BnaPE1 gene, recombinant vectors, recombinant microorganisms or transgenic cell lines in regulating photosynthesis and / or leaf shape in Brassica napus, wherein the nucleotide sequence of the BnaPE1 gene is shown in SEQ ID NO:1.

[0008] Furthermore, the leaf shape includes the number and depth of leaf lobes.

[0009] Furthermore, the method for constructing the recombinant vector containing the BnaPE1 gene includes the following steps: using the cDNA of Brassica napus as a template, the BnaPE1 gene is amplified by PCR and ligated into the TA / Blunt-Zero vector, transformed into competent Escherichia coli cells, the positive colonies are digested with enzymes, the DNA fragments are recovered and ligated into the enzyme-digested pMDC83 vector to obtain the recombinant vector.

[0010] Furthermore, the primers used for the PCR amplification of the BnaPE1 gene have sequences as shown in SEQ ID NO:9-10.

[0011] Furthermore, the transgenic cell line containing the BnaPE1 gene is obtained by introducing a recombinant vector of the BnaPE1 gene into Agrobacterium.

[0012] The present invention also provides a method for cultivating transgenic plants with high photosynthetic efficiency. The method obtains transgenic plants by increasing the content or activity of BnaPE1 protein in the target plant. The amino acid sequence of the BnaPE1 protein is shown in SEQ ID NO:2.

[0013] Furthermore, the plants include rice, tobacco, soybeans, tomatoes, and wheat.

[0014] The present invention also provides a method for identifying high photosynthetic efficiency varieties of Brassica napus, by extracting genomic RNA from Brassica napus and detecting the expression level of the BnaPE1 gene.

[0015] Furthermore, the primer sequences used for detection are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0016] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: Using Brassica napus as the research material, this invention analyzes the gene expression levels in the tissues and organs of Brassica napus, finding that BnaPE1 is highly expressed in the apical meristem, axillary buds, and siliques of the stem. This invention isolates the BnaPE1 gene from Brassica napus and finds that overexpression of the BnaPE1 gene in rapeseed significantly reduces the content of cytokinins (trans-zeatin (tZT), isopentened adenine (iP), and cis-zeatin (cZT)) while significantly increasing the photosynthetic efficiency of Brassica napus. Furthermore, it increases and deepens leaf lobes and increases chlorophyll content, indicating that the BnaPE1 gene plays an important role in regulating photosynthesis and leaf shape in Brassica napus. Therefore, isolating the BnaPE1 gene from Brassica napus and identifying its biological functions in enhancing photosynthesis and altering leaf shape is of great practical significance for breeding new Brassica napus varieties with enhanced photosynthetic efficiency. Attached Figure Description

[0017] Figure 1 Expression of BnaPE1 in various tissues and organs of Brassica napus.

[0018] Figure 2 Schematic diagram of the construction of the BnaPE1 overexpression vector (pMDC83-BnaPE1).

[0019] Figure 3 The expression of BnaPE1 in pMDC83-BnaPE1 transgenic positive plants, where J9712 is a wild plant; the other numbered plants are pMDC83-BnaPE1 transgenic positive Brassica napus.

[0020] Figure 4 The cytokinin content of BnaPE1-OE transgenic Brassica napus, where J9712 is the control plant; BnaPE1-OE is a BnaPE1 overexpressing transgenic Brassica napus.

[0021] Figure 5 Leaf phenotype of BnaPE1-OE transgenic Brassica napus seedlings, with J9712 as the control plant; BnaPE1-OE is BnaPE1 overexpression transgenic Brassica napus.

[0022] Figure 6 The chlorophyll content of BnaPE1-OE transgenic Brassica napus, with J9712 as the control plant; BnaPE1-OE is a BnaPE1 overexpression transgenic Brassica napus.

[0023] Figure 7 The photosynthetic efficiency of BnaPE1-OE transgenic Brassica napus at the bolting stage, where J9712 is the control plant; BnaPE1-OE is a BnaPE1 overexpressing transgenic Brassica napus. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] The following embodiments define the present invention and describe the methods for cloning DNA fragments containing the BnaPE1 coding region and verifying the function of the BnaPE1 gene. Based on the following description and these embodiments, those skilled in the art can determine the essential features of the invention and can make various changes and modifications to adapt it to different uses and conditions without departing from the spirit and scope of the invention.

[0026] Example 1: qPCR analysis of BnaPE1 expression levels in various tissues and organs of Brassica napus

[0027] The semi-winter pure line J9712 of Brassica napus was planted in the field. During various growth and development stages, the effects of different tissues and organs on the Brassica napus (including the January root, January leaf, axillary bud, shoot apical meristem, stem, adult leaf, bud, ovary, 14-day fertilized seed (14DAP), 14-day fertilized silique wall (14DAP), 24-day fertilized seed (24DAP), 24-day fertilized silique wall (24DAP), 34-day fertilized seed (34DAP), 34-day fertilized silique wall (34DAP), 50-day fertilized seed (50DAP), and 50-day fertilized silique wall (50DAP) were investigated. Samples (50 DAP) were collected and flash-frozen in liquid nitrogen. Total RNA was extracted from various tissues and organs of Brassica napus using the RNAisolater Total RNA Extraction Reagent (Cat. No.: R401-01) from Nanjing Novizan Biotechnology Co., Ltd. After passing the agarose gel electrophoresis and UV spectrophotometry tests, reverse transcription was performed using the RNAHiScript Q RT SuperMix for qPCR (+gDNAwiper) kit (Cat. No.: R223-01) from Nanjing Novizan Biotechnology Co., Ltd. The system is as follows:

[0028] Table 1

[0029]

[0030] After reacting at 42℃ for 2 minutes, add the following reagents.

[0031] Table 2

[0032]

[0033] After reacting at 50℃ for 15 min, the cDNA was obtained by treating at 85℃ for 5 s. An appropriate amount of cDNA was diluted 20-fold as a template, and the quality of the cDNA obtained from reverse transcription was detected by PCR. The primers used were the transintron primers for the BnaActin gene: BnaActinF1 (5'-TCTTCCTCACGCTATCCTCCG-3') (SEQ ID NO:3) and BnaActinR2 (5'-AACAATGGATGGACCTGACT-3') (SEQ ID NO:4). The PCR products were analyzed by agarose gel electrophoresis. If the result showed a specific band of approximately 500 bp, it indicated that the cDNA template was of good quality and not contaminated with genomic DNA.

[0034] The relative expression levels of BnaPE1 in various tissues and organs of Brassica napus were analyzed using real-time quantitative PCR (qPCR). qPCR was performed using the ABI StepOnePlus real-time PCR system and the AceQ Universal SYBR qPCR Master Mix (Cat.No.: P112-03) from Nanjing Novizan Biotechnology Co., Ltd. cDNA from various tissues and organs of Brassica napus at different growth and development stages was used as template. The specific primers for BnaPE1 were BnaPE1-QF (5'-GACCCCAAATCAACAACGTTTA-3') (SEQ ID NO:5) and BnaPE1-QR (5'-CTATCCTTGCCCTGGTGATTAT-3') (SEQ ID NO:6), and the internal control gene primers were BnaActin-QF (5'-TCTTCCTCACGCTATCCTCCG-3') (SEQ ID NO:7) and BnaActin-QR (5'-AGCCGTCTCCAGCTCTTGC-3') (SEQ ID NO:8). The reaction conditions were as follows:

[0035] Table 3

[0036]

[0037]

[0038] Exploit 2 ˉ△△Ct Figure 1 The background expression of BnaPE1 is relatively low, while its expression level is relatively high in tissues and organs such as the apical meristem, axillary buds, and siliques.

[0039] Example 2: Molecular cloning of the BnaPE1 gene in Brassica napus

[0040] Samplings were taken from three-leaf-one-heart stage seedlings of the Brassica napus variety “Darmor-bzh”. Leaf, stem, and root samples were flash-frozen in liquid nitrogen and then stored at -70°C for RNA extraction. Total RNA was extracted from the seedlings using the method described in Example 1 and reverse transcribed into cDNA, which served as a template for subsequent PCR. Gene-specific primers BnaPE1-F (5'-GACCTCGACTCTAGAACTAGTATGGGATTGACCTCA-3') (SEQ ID NO:9) and BnaPE1-R (5'-CGGGCCCCCCCTCGAGGCGCGCCAGAGAAGTTGGA TGG-3') (SEQ ID NO:10) were designed to amplify the BnaPE1 gene in Brassica napus using PCR. The PCR products were analyzed by agarose gel electrophoresis, and the target fragment band of the expected size was recovered by gel excision. The BnaPE1-T amplified fragment was ligated into the TA / Blunt-Zero vector using the 5min TA / Blunt-ZeroCloning Kit (Cat.No.: C601-02) from Nanjing Novizan Biotechnology Co., Ltd., and then transformed into Trans1-T1 Escherichia coli competent cells. White single colonies were picked for colony PCR positive identification. Positive clones were selected and sent to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing verification. Positive clones with correct sequences were named BnaPE1-T.

[0041] Example 3: Construction of the BnaPE1 gene overexpression vector pMDC83-BnaPE1

[0042] To investigate the function of the BnaPE1 gene, the applicant overexpressed the gene in Brassica napus and analyzed its function by observing the phenotype of transgenic plants under salt stress. The overexpression vector was constructed as follows: the BnaPE1-T intermediate vector plasmid and the pMDC83 backbone vector were double-digested with Spe I + AscI. Then, the target DNA and vector fragments were recovered using the Gel DNA Extraction Mini Kit (Lot: 23122923) from Yangzhou Wanhe Biotechnology Co., Ltd. The target fragment and vector fragment were ligated to construct the BnaPE1 gene overexpression vector (construction method follows Jian Li et al. (2021)), named pMDC83-BnaPE1. Figure 2 ).

[0043] Example 4: Genetic transformation of pMDC83-BnaPE1 in Brassica napus

[0044] The pMDC83-BnaPE1 overexpression vector was transformed into Agrobacterium competent cells using the following electroporation method: A tube of GV3101 Agrobacterium competent cells was thawed on ice. 1 μL of the target plasmid was added to the competent cells and gently mixed. The mixture of plasmid and Agrobacterium competent cells was added to the bottom of a pre-chilled sterile electroporation cuvette. After electroporation at 1800V, 500 μL of LB liquid medium was quickly added to the cuvette to resuspend the cells. The mixture was then transferred to a new sterile 1.5 mL centrifuge tube and incubated at 28°C and 200 rpm for 30 min. The cells were centrifuged at 6000 rpm for 5 min at room temperature, 450 μL of supernatant was discarded, and the cells were resuspended in the remaining medium. An appropriate amount of the bacterial suspension was spread onto LA medium plates containing 50 mg / L Kan and 50 mg / L Rif and incubated upside down at 28°C for 2 days.

[0045] Using the hypocotyl of sterile rapeseed seedling J9712 as explant, the exogenous fragment was introduced into rapeseed for genetic transformation by Agrobacterium infection. The specific steps are described in patent application 202410912937X. The transgenic plant obtained was named BnaPE1-OE.

[0046] Example 5: Molecular identification of transgenic Brassica napus plants

[0047] Leaves from BnaPE1-OE transgenic plants were collected, and genomic DNA was extracted using the following rapid extraction method: Approximately 0.2g of young leaves from each transgenic plant were placed in a 2mL centrifuge tube, two 4.5mm diameter steel balls were added, and 300μL of DNA buffer (DNA buffer formula: 500mM Tris-HCl (pH=7.5), 300mM NaCl, 300mM Sucrose) was added. The sample was placed in a sampler and sampled at 50Hz for 3min to thoroughly break up the leaf sample; the sample was then incubated in a 95℃ water bath for 10min, and after cooling to room temperature, it was centrifuged at 10000rpm for 5min; 150μL of the supernatant was transferred to a new 1.5mL centrifuge tube and stored at -20℃ for later use.

[0048] The DNA samples were diluted 10-fold, and 1 μL of DNA from each sample was used as a PCR template. Amplification was performed using primer pairs 35S-F (5'-CAAGACCCTTCCTCTAT-3') (SEQ ID NO:11) and BnaPE1-R (5'-CGGGCC CCCCCTCGAGGCGCGCCAGAGAAGTTGGATGG-3') (SEQ ID NO:10), and primer pairs BnaPE1-F (5'-GACCTCGACTCTAGAACTAGTATGGGATTGACCTCA-3') (SEQ ID NO:9) and GFP-R (5'-CATCACCTTCACCCTC-3') (SEQ ID NO:12). The program was as follows: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 7 min. The PCR products were detected by agarose gel electrophoresis. If a specific target fragment was obtained, it proved that the corresponding transgenic plant was a positive plant, indicating that the pMDC83-BnaPE1 vector had been integrated into the rapeseed genome. The PCR system is as follows:

[0049] Table 4

[0050]

[0051] Subsequently, qPCR technology was used to analyze the expression level of the BnaPE1 gene in negative controls and some positive transgenic Brassica napus plants. Positive transgenic plants and control plants with consistent growth stages were selected, and leaves of the same size and location were rapidly frozen in liquid nitrogen. Total RNA extraction, reverse transcription, and qPCR were performed on the leaves according to the method described in Example 1. Figure 3 As shown, compared with the control (J9712) plants, the expression level of BnaPE1 was increased to varying degrees in most transgenic positive plants, with the most significant increases observed in BnaPE1-OE-1, BnaPE1-OE-5, BnaPE1-OE-8, BnaPE1-OE-16, BnaPE1-OE-24, and BnaPE1-OE-30. Subsequently, we measured the cytokinin content in BnaPE1-OE and J9712 plants (sent to Maiwei Metabolism for analysis). The results showed that compared with the control J9712, the contents of trans-zeatin (tZT), isopentened adenine (iP), and cis-zeatin (cZT) in the BnaPE1-OE transgenic plants were significantly decreased. Figure 4 ).

[0052] Example 6: BnaPE1-OE transgenic Brassica napus leaves turn green and have increased leaf clefts

[0053] To investigate the effects of BnaPE1 on the growth and development of Brassica napus, we conducted phenotypic observations on BnaPE1-OE transgenic positive plants (BnaPE1-OE-1, BnaPE1-OE-5, BnaPE1-OE-8) and control plants (J9712). We found that compared to the control, BnaPE1-OE transgenic plants had more and deeper leaf lobes, and the leaf color was significantly darker. Figure 5 Further analysis of chlorophyll content in BnaPE1-OE transgenic positive plants and control plants showed that, compared with the control, the chlorophyll content of BnaPE1-OE plants was significantly increased. Figure 6 The above results indicate that BnaPE1 has a regulatory effect on leaf shape and chlorophyll content in Brassica napus.

[0054] Example 7: Enhanced photosynthetic efficiency of BnaPE1-OE transgenic Brassica napus

[0055] Because the leaves of BnaPE1-OE transgenic Brassica napus turned green, we speculated that BnaPE1 might affect the photosynthesis of Brassica napus. To investigate the effect of BnaPE1 on the photosynthesis of Brassica napus, we used a LI6800 portable photosynthesis system to measure the net photosynthetic rate and stomatal conductance of leaves from BnaPE1-OE transgenic plants and wild-type plants. During the bolting stage of the rapeseed, three different transgenic lines (BnaPE1-OE-1, BnaPE1-OE-5, and BnaPE1-OE-8) and a wild-type control (J9712) were selected. Three to five replicates were set for each line. Fresh, flat leaves were selected for measurement. The photosynthesis system parameters were set as follows: temperature 25℃, relative humidity 60%, CO2 concentration 400 μmol / mol, and light intensity 1200 μmol / m². -2 s -1 The results showed that the photosynthetic rate and stomatal conductance of BnaPE1-OE plants were significantly higher than those of the control material, indicating that overexpression of BnaPE1 can improve the photosynthetic capacity of Brassica napus leaves. Figure 7 Therefore, we believe that increased BnaPE1 expression can enhance photosynthesis in rapeseed.

[0056] This invention isolates the BnaPE1 gene from Brassica napus, which is related to photosynthesis and leaf shape. This provides theoretical guidance for studying the molecular mechanisms regulating plant growth and development. The photosynthesis and leaf shape-related genes isolated in this invention originate from the plant itself, thus having minimal environmental impact. Using the isolated gene for molecular breeding to genetically improve rapeseed photosynthetic efficiency is of great significance for cultivating new Brassica napus varieties with relatively improved photosynthetic efficiency.

Claims

1. The application of BnaPE1 protein overexpression in enhancing photosynthesis and / or regulating leaf shape in Brassica napus, characterized in that, The amino acid sequence of the BnaPE1 protein is shown in SEQ ID NO:2, and the regulation of leaf shape is the increase and deepening of leaf lobes.

2. Encoding the BnaPE1 protein of claim 1 BnaPE1 The application of genes in enhancing photosynthesis and / or regulating leaf shape in Brassica napus is characterized by, The BnaPE1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the regulation of leaf shape is the increase and deepening of leaf lobes.

3. Contains BnaPE1 The application of gene expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic cell lines in enhancing photosynthesis and / or regulating leaf shape in Brassica napus is characterized by, The BnaPE1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the regulation of leaf shape is the increase and deepening of leaf lobes.

4. The application according to claim 3, characterized in that, The containing BnaPE1 The method for constructing a recombinant gene vector includes the following steps: using cDNA from Brassica napus as a template, PCR amplification... BnaPE1 The gene was ligated into the TA / Blunt-Zero vector, transformed into competent E. coli cells, and the positive colonies were digested with enzymes. The recovered DNA fragments were then ligated into the digested pMDC83 vector to obtain the recombinant vector.

5. The application according to claim 4, characterized in that, The PCR amplification BnaPE1 The primers used for the gene are shown in SEQ ID NO:9~10.

6. The application according to claim 3, characterized in that, The containing BnaPE1 Recombinant microorganisms are those that, through the process of... BnaPE1 The gene was obtained by introducing a recombinant vector into Agrobacterium.

7. A method for cultivating a transgenic plant with high photosynthetic efficiency, characterized in that, The method obtains transgenic plants by increasing the content or activity of BnaPE1 protein in the target plant. The amino acid sequence of the BnaPE1 protein is shown in SEQ ID NO:

2. The plant is Brassica napus.

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