Application of BnaPE1 protein or coding gene thereof in regulating photosynthesis and / or leaf shape of brassica napus

Abstract

The invention discloses a BnaPE1 protein and application of a coding gene of the BnaPE1 protein in regulating and controlling photosynthesis and / or leaf shape of brassica napus. The amino acid sequence of the BnaPE1 protein is shown as SEQ ID NO.2, and the nucleotide sequence of a coding region of the BnaPE1 gene is shown as SEQ ID NO.1 in a sequence table. According to the invention, a DNA fragment containing the BnaPE1 gene is separated and applied, and the fragment endows the brassica napus with the capabilities of enhancing photosynthesis and increasing and deepening leaf cracks, and has important application value for culturing a new variety of brassica napus with improved photosynthetic efficiency and yield.

Description

Technical Field

[0001] The invention relates to application of a BnaPE1 protein or a coding gene thereof in regulating photosynthesis and / or leaf shape of Brassica napus, and belongs to the field of genetic engineering. Background Art

[0002] Rapeseed is one of the most important oil crops in the world, and rapeseed oil is the third largest edible vegetable oil in the world. my country is a major producer and consumer of rapeseed oil. Rapeseed is the largest oil crop in my country in terms of planting area, and is also the second largest source of vegetable oil after soybeans. However, the current rapeseed production in China is far from meeting the processing needs and is highly dependent on imports. Improving rapeseed production capacity is not only a livelihood issue to ensure the security of national edible oil supply, but also an important mission to comply with national strategic development and ensure national grain and oil security. Among the three types of rapeseed, cabbage rapeseed and mustard rapeseed, cabbage rapeseed is the most widely planted rapeseed type in my country, and increasing yield is one of the core goals of rapeseed breeding. Photosynthesis is the basic way for plants to absorb light energy to synthesize organic matter, and is an important factor in determining rapeseed yield and quality. 90%-95% of rapeseed yield comes from photosynthetic products. At present, there are not many studies on the photosynthetic characteristics of rapeseed. The photosynthetic rate is an important manifestation of photosynthesis. Improving the photosynthetic efficiency of rapeseed is conducive to further tapping the yield potential of rapeseed and is of great significance for breeding high-light-efficiency rapeseed varieties. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a BnaPE1 protein, its encoding gene, and an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the BnaPE1 gene for use in controlling photosynthesis and / or leaf shape of Brassica napus.

[0004] Technical solution: The present invention provides the use of BnaPE1 protein in regulating photosynthesis and / or leaf shape of Brassica napus. The amino acid sequence of the 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 use of the BnaPE1 gene encoding the BnaPE1 protein in regulating photosynthesis and / or leaf shape of Brassica napus. The nucleotide sequence of the BnaPE1 gene is shown in SEQ ID NO: 1, and the sequence length is 1638 bp.

[0007] The present invention also provides the use of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the BnaPE1 gene in regulating photosynthesis and / or leaf shape of Brassica napus. 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 fissures.

[0009] Furthermore, the method for constructing a recombinant vector containing the BnaPE1 gene includes the following steps: using the cDNA of Brassica napus as a template, PCR amplifying the BnaPE1 gene and connecting it with a TA / Blunt-Zero vector, transforming Escherichia coli competent cells, digesting the positive colonies, recovering the DNA fragments and connecting them with the pMDC83 vector after digestion to obtain a recombinant vector.

[0010] Furthermore, the primers used for PCR amplification of the BnaPE1 gene have sequences as shown in SEQ ID NOs: 9-10.

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

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

[0013] Furthermore, the plants include rice, tobacco, soybean, tomato and wheat.

[0014] The present invention also provides a method for identifying Brassica napus varieties with high photosynthetic efficiency, which comprises extracting Brassica napus genomic RNA 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 the prior art, the present invention has the following significant advantages: The present invention uses Brassica napus as research material, and by analyzing the gene expression level of Brassica napus in tissues and organs, it is found that BnaPE1 is highly expressed in the apical meristem, axillary buds and siliques of Brassica napus stems. The present invention isolates the BnaPE1 gene from Brassica napus, and finds that overexpressing the BnaPE1 gene in rapeseed can significantly reduce the content of cytokinins (trans-zeatin (tZT), isopentenyl adenine (iP), cis-zeatin (cZT)) while significantly improving the photosynthetic efficiency of Brassica napus, and can increase and deepen the leaf fissures of rapeseed leaves, and increase the chlorophyll content, indicating that the BnaPE1 gene plays an important role in regulating photosynthesis and leaf shape of Brassica napus. Therefore, isolating the BnaPE1 gene from Brassica napus and identifying its biological functions in enhancing photosynthesis and changing leaf shape of Brassica napus are of great practical significance for breeding new varieties of Brassica napus with enhanced photosynthetic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0021] Figure 5 Leaf phenotype of BnaPE1-OE transgenic Brassica napus at the seedling stage, where J9712 is the control plant and BnaPE1-OE is the BnaPE1 overexpressing transgenic Brassica napus.

[0022] Figure 6 Chlorophyll content of BnaPE1-OE transgenic Brassica napus, where J9712 is the control plant and BnaPE1-OE is the BnaPE1 overexpressing transgenic Brassica napus.

[0023] Figure 7 Photosynthetic efficiency of BnaPE1-OE transgenic Brassica napus during the bolting period, where J9712 is the control plant and BnaPE1-OE is the BnaPE1 overexpressing transgenic Brassica napus. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

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

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

[0027] The semi-winter pure line material J9712 of Brassica napus was planted in the field, and the tissues and organs of Brassica napus (including Seeding root, Seeding leaf, Axillary bud, Shoot apical meristem, Stem, Adult leaf, Bud, Ovary, Seed (14 DAP) at 14 days after fertilization, Silique wall (14 DAP) at 14 days after fertilization, Seed (24 DAP) at 24 days after fertilization, Silique wall (24 DAP) at 24 days after fertilization, Seed (34 DAP) at 34 days after fertilization, Silique wall (34 DAP) at 50 days after fertilization, and Seed (50 DAP) at 50 days after fertilization) were measured at different stages of growth and development. Wall (50DAP)) samples were sampled and frozen in liquid nitrogen. The total RNA of each tissue and organ of Brassica napus was extracted using the RNAisolater Total RNAExtraction Reagent (Cat.No.: R401-01) reagent of Nanjing Novozyme. After the qualified results were detected by agarose gel electrophoresis and UV spectrophotometer, the RNAHiScript Q RT SuperMix for qPCR (+gDNAwiper) kit (Cat.No.: R223-01) of Nanjing Novozyme was used for reverse transcription. The system is as follows:

[0028] Table 1

[0029]

[0030] After reacting at 42°C for 2 min, add the following reagents.

[0031] Table 2

[0032]

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

[0034] Real-time fluorescence quantitative PCR (qPCR) technology was used to analyze the relative expression level of BnaPE1 in various tissues and organs of Brassica napus. qPCR was performed using the ABI StepOnePlus fluorescent quantitative PCR system and the AceQ Universal SYBR qPCR Master Mix (Cat. No.: P112-03) system of Nanjing Novozyme Biotechnology Co., Ltd., using cDNA of various tissues and organs of Brassica napus plants at various growth and development stages as templates, BnaPE1-specific quantitative primers were BnaPE1-QF (5'-GACCCCAAATCAACAACGTTTA-3') (SEQ ID NO: 5) and BnaPE1-QR (5'-CTATCCTTGCCCTGGTGATTAT-3') (SEQ ID NO: 6), and the internal reference 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 are as follows:

[0035] Table 3

[0036]

[0037]

[0038] Exploit 2 ˉ△△Ct Figure 1 The results showed that the background expression of BnaPE1 was relatively low, and the expression level in tissues and organs such as the stem apical meristem, axillary buds and silique peel was relatively high.

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

[0040] The three-leaf and one-heart stage seedlings of Brassica napus "Darmor-bzh" were sampled, and the leaf, stem and root samples were placed in liquid nitrogen for quick freezing and then stored in a -70°C refrigerator to extract RNA in time. The total RNA of Brassica napus seedlings was extracted according to the method described in Example 1, and reverse transcribed into cDNA 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, and the BnaPE1 gene in Brassica napus was amplified by PCR. The PCR product was analyzed by agarose gel electrophoresis, and the size of the target fragment recovered by gel cutting was consistent with the expected size. The above-mentioned BnaPE1-T amplified fragment was connected to the TA / Blunt-Zero vector through the 5min TA / Blunt-Zero Cloning Kit (Cat. No.: C601-02) of Nanjing Novozyme Biotechnology Co., Ltd., and transformed into Trans1-T1 Escherichia coli competent cells. White single colonies were picked for colony PCR positive identification, and the positive clones were selected and entrusted to Yangzhou Qingke Biotechnology Co., Ltd. for sequencing verification. The positive clones with correct sequences were named BnaPE1-T.

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

[0042] To explore 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 conditions. The overexpression vector construction method is as follows: the BnaPE1-T intermediate vector plasmid and the pMDC83 backbone vector are double-digested with Spe I+AscI, and then the target DNA and vector fragments are recovered using the Gel DNA Extraction Mini Kit (Lot: 23122923) of Yangzhou Wanhe Biotechnology Co., Ltd. The target fragment and the vector fragment are connected to construct an overexpression vector of the BnaPE1 gene (the construction method refers to Jian Li et al. (2021)), named pMDC83-BnaPE1 ( Figure 2 ).

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

[0044] The pMDC83-BnaPE1 overexpression vector was transformed into Agrobacterium competent cells according to the following electroporation transformation method: a tube of GV3101 Agrobacterium competent cells was placed on ice to melt, 1 μL of the target plasmid was added to the competent cells and gently mixed; the mixture of plasmid and Agrobacterium competent cells was pipetted and added to the bottom of a pre-cooled sterile electroporation cup, 500 μL of LB liquid culture medium was quickly added to the electroporation cup after 1800V electroporation to resuspend the bacteria, and then transferred to a new sterile 1.5mL centrifuge tube, and revived at 200rpm at 28℃ for 30min; centrifuged at 6000rpm at room temperature for 5min, 450 μL of supernatant was discarded and the bacteria were resuspended with the remaining culture medium; an appropriate amount of bacterial liquid was spread on a LA culture medium plate containing 50mg / L Kan and 50mg / L Rif, and cultured inverted at 28℃ for 2d.

[0045] Using the sterile hypocotyls of Brassica napus J9712 seedlings as explants, the exogenous fragments were introduced into Brassica napus for genetic transformation using the Agrobacterium infection method. The specific steps refer to patent application 202410912937X. The transgenic plants obtained were named BnaPE1-OE.

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

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

[0048] The above DNA samples were diluted 10 times, and 1 μL of DNA from each sample was used as a PCR template. The 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 Bn aPE1-F (5'-GACCTCGACTCTAGAACTAGTATGGGATTGACCTCA-3') (SEQ ID NO: 9) and GFP-R (5'-CATCACCTTCACCCTC-3') (SEQ ID NO: 12), respectively. The program was: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, 35 cycles; 72°C for 7 min. Agarose gel electrophoresis was used to detect the PCR products. If a specific target fragment was obtained, it was proved that the corresponding transgenic plant was a positive plant and the pMDC83-BnaPE1 vector had been integrated into the genome of Brassica napus. The PCR system was as follows:

[0049] Table 4

[0050]

[0051] Subsequently, the expression level of the BnaPE1 gene in the negative control and some positive transgenic Brassica napus plants was analyzed using qPCR technology. Positive transgenic plants and control plants with the same growth status were selected, leaves of the same size and position were taken and quickly frozen in liquid nitrogen, and 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 of BnaPE1 in most transgenic positive plants increased to varying degrees, among which the expression of BnaPE1 in BnaPE1-OE-1, BnaPE1-OE-5, BnaPE1-OE-8, BnaPE1-OE-16, BnaPE1-OE-24, and BnaPE1-OE-30 increased most significantly. We then measured the content of cytokinins in BnaPE1-OE and J9712 plants (sent to Myvi Metabolism Company for testing), and found that compared with the control J9712, the content of trans-zeatin (tZT), isopentenyl adenine (iP), and cis-zeatin (cZT) in BnaPE1-OE transgenic plants was significantly reduced ( Figure 4 ).

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

[0053] To investigate the effect of BnaPE1 on the growth and development of Brassica napus, we observed the phenotypes of BnaPE1-OE transgenic positive plants (BnaPE1-OE-1, BnaPE1-OE-5, BnaPE1-OE-8) and control plants (J9712). We found that compared with the control, the leaf fissures of BnaPE1-OE transgenic plants increased and deepened, and the leaf color was significantly darker ( Figure 5 ). The chlorophyll content of BnaPE1-OE transgenic positive plants and control plants was further measured. The results showed that the chlorophyll content of BnaPE1-OE plants was significantly increased compared with the control ( 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] Since the leaves of BnaPE1-OE transgenic Brassica napus turned green, we speculated that BnaPE1 might affect the photosynthesis of Brassica napus. In order to explore the effect of BnaPE1 on photosynthesis of Brassica napus, we used the LI6800 portable photosynthetic instrument to measure the net photosynthetic rate and stomatal conductance of the leaves of BnaPE1-OE transgenic plants and wild-type plants. During the bolting period of rapeseed, three different transgenic lines (BnaPE1-OE-1, BnaPE1-OE-5, BnaPE1-OE-8) and wild-type control (J9712) plants were selected. Each line was set up with 3-5 replicates, and flat fresh leaves were selected as much as possible for measurement. The parameters of the photosynthetic instrument were set as follows: temperature 25℃, relative humidity 60%, CO2 concentration 400μmol / mol, light intensity 1200μmol m -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 materials, indicating that overexpression of BnaPE1 can improve the photosynthetic capacity of Brassica napus leaves ( Figure 7 ). Therefore, we believe that the increased expression of BnaPE1 can enhance the photosynthesis of rapeseed.

[0056] The present invention isolates the Brassica napus gene BnaPE1 related to photosynthesis and leaf shape, which has certain theoretical guiding significance for studying the molecular mechanism of plant growth and development regulation. The photosynthesis and leaf shape related genes isolated by the present invention come from the plant itself and have little impact on the environment. Molecular breeding for genetic improvement of rapeseed photosynthetic efficiency using the isolated gene is of great significance for cultivating new varieties of Brassica napus with relatively improved photosynthetic efficiency.

Claims

1. Application of BnaPE1 protein in regulating photosynthesis and / or leaf shape of Brassica napus, characterized in that: The amino acid sequence of the BnaPE1 protein is shown in SEQ ID NO.

2.

2. Use of the BnaPE1 gene encoding the BnaPE1 protein according to claim 1 in regulating photosynthesis and / or leaf shape of Brassica napus, characterized in that: The nucleotide sequence of the BnaPE1 gene is shown in SEQ ID NO.

1.

3. Use of an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the BnaPE1 gene in regulating photosynthesis and / or leaf shape of Brassica napus, characterized in that: The nucleotide sequence of the BnaPE1 gene is shown in SEQ ID NO.

1.

4. The use according to any one of claims 1 to 3, characterized in that: The leaf shape includes the number and depth of leaf lobes.

5. The use according to claim 3, characterized in that: The method for constructing a recombinant vector containing the BnaPE1 gene comprises the following steps: using the cDNA of Brassica napus as a template, PCR amplifying the BnaPE1 gene and connecting it to a TA / Blunt-Zero vector, transforming competent Escherichia coli cells, performing enzyme digestion on positive colonies, recovering DNA fragments and connecting them to the pMDC83 vector after enzyme digestion to obtain a recombinant vector.

6. The use according to claim 4, characterized in that: The primers used for PCR amplification of the BnaPE1 gene have sequences shown in SEQ ID NOs. 9-10.

7. The use according to claim 3, characterized in that: The transgenic cell line containing the BnaPE1 gene is obtained by introducing the recombinant vector of the BnaPE1 gene into Agrobacterium.

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

2.

9. A method for identifying Brassica napus varieties with high photosynthetic efficiency, characterized in that: The genomic RNA of Brassica napus was extracted and the expression level of BnaPE1 gene was detected.

10. The method for identifying Brassica napus varieties with high photosynthetic efficiency according to claim 9, characterized in that: The primer sequences used for detection are shown in SEQ ID NO:5 and SEQ ID NO:6.

Images