Application ofbZIP98 gene from Rixianggui in promoting the enlargement of flower organs of Nicotiana tabacum
By cloning the Rixiang cinnamon ofbZIP98 gene and constructing a plant expression vector, it was transformed into large-flower tobacco, and the flower organs were significantly enlarged, the problem of size regulation of osmanthus flowers was solved, and the efficiency of plant breeding was improved.
Patent Information
- Application Number
- CN202310925427.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-26
- Publication Date
- 2024-12-20
- Estimated Expiration
- 2043-07-26
AI Technical Summary
The existing technology is difficult to effectively promote the enlargement of osmanthus organs, which leads to time-consuming and labor-intensive picking of flowers and lacks effective functional gene regulation methods.
By cloning the Rika cinnamon ofbZIP98 gene, the plant expression vector pBI121-ofbZIP98 was constructed and transformed into large-flower tobacco to achieve overexpression ofbZIP98 gene, thereby promoting the enlargement of flower organs.
The successfully obtained genetically modified large-flower tobacco corolla diameter, circumference, area and cylinder diameter have significantly increased, solving the problem of flower size regulation and improving the efficiency of plant molecules.
Smart Images
Figure CN116836999B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant molecular biology, and more specifically, relates to the application ofbZIP98 gene of Rixianggui in promoting the enlargement of flower organs of Nicotiana tabacum. Background Art
[0002] bZIP transcription factors are named for their highly conserved bZIP domain composed of an alpha helix of approximately 40-60 amino acids long. The domain consists of two different functional regions: a highly conserved basic region at the N-terminus and a leucine zipper region at the C-terminus. The two domains are located on adjacent a helices and can bind to DNA through the major grooves on both sides of the double helix.
[0003] bZIP is a transcription factor that can be expressed in all apical meristems of Arabidopsis (including the meristems of inflorescence, flowers, carpel edges, etc.). It can induce the expression of two cell proliferation and cell elongation regulatory genes, JAG and OFP1, and can also interact with regulatory factors related to meristem and reproductive enlargement, such as WUS, JAB and BEL1.
[0004] Osmanthus fragrans, a gardening tree species of the genus Osmanthus in the family Oleaceae, is one of the ten traditional famous flowers in my country. It can also be used in food processing, ointment and perfume manufacturing, and aromatherapy, and has important ornamental and economic value. Osmanthus has been cultivated in my country for more than 2,500 years, and its variety resources are very rich. There are obvious differences in leaf shape, leaf color, flower fragrance, flower color and other traits among different osmanthus varieties. Osmanthus flowers are small, and the picking process is time-consuming and laborious. Therefore, it is of great significance to explore the functional genes that regulate the size of osmanthus flower organs. Summary of the invention
[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide an application of the bZIP98 gene of Nicotiana tabacum in promoting the enlargement of flower organs of Nicotiana tabacum for use in plant molecular improvement breeding.
[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0007] The use ofbZIP98 gene of Rixianggui in promoting the enlargement of flower organs of Nicotiana tabacum, wherein the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1, comprises the following steps:
[0008] (1) constructing a vector ofbZIP98 gene;
[0009] (2) transforming the constructed vector ofbZIP98 gene into Nicotiana tabacum;
[0010] (3) Cultivate and screen transgenic Nicotiana tabacum with enlarged floral organs.
[0011] The vector is a plant expression vector.
[0012] The plant expression vector is pBI121-ofbZIP98.
[0013] The use ofbZIP98 gene of Rixianggui in increasing the corolla diameter of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.
[0014] The use ofbZIP98 gene of Rixianggui in increasing the circumference of the corolla of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.
[0015] The use ofbZIP98 gene of Rixianggui in increasing the corolla area of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.
[0016] The use ofbZIP98 gene of Rixianggui in increasing the diameter of the corolla tube of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.
[0017] The use ofbZIP98 gene of Rixianggui in increasing the corolla diameter, corolla circumference, corolla area and corolla tube diameter of Nicotiana tabacum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The invention uses the flowers of the osmanthus sinensis in full bloom as materials, obtains the osmanthus sinensis ofbZIP98 gene by cloning, constructs its overexpression vector pBI121-ofbZIP98 on this basis, and transfers it into Nicotiana tabacum to obtain transgenic plants. The flower organ tissue of the ofbZIP98 transgenic strain is significantly larger than that of the Nicotiana tabacum strain (EV) transferred into the pBI121 empty vector and the wild-type Nicotiana tabacum strain (WT); the corolla diameter, corolla circumference, corolla area, and corolla tube diameter of the ofbZIP98 transgenic strain are significantly increased compared with the Nicotiana tabacum strain (EV) transferred into the pBI121 empty vector and the wild-type Nicotiana tabacum strain (WT). BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Agarose gel electrophoresis of the amplified product of the target gene ofbZIP98;
[0021] Figure 2 This is the positive single colony detection image after the target gene ofbZIP98 amplified fragment was connected to the vector and transformed;
[0022] Figure 3 This is the agarose gel electrophoresis diagram of the recombinant vector ofbZIP98 after double restriction digestion;
[0023] Figure 4 This is the agarose gel electrophoresis diagram of bacterial test after transformation of Agrobacterium GV3101;
[0024] Figure 5 This is the positive detection image ofbZIP98 transgenic plants;
[0025] Figure 6 This is the phenotype observation diagram of the flower ofbZIP98 transgenic Nicotiana tabacum;
[0026] Figure 7 This is a diagram of the gene expression ofbZIP98 transgenic Nicotiana tabacum. DETAILED DESCRIPTION
[0027] The present invention is further described below in conjunction with specific examples. In the following examples, the operations not described in detail are all routine biological experimental operations, which can be performed with reference to the molecular biology experiment manual and existing published journals, or according to the kit and product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.
[0028] The materials used in this application are flowers of Osmanthus fragrans in full flowering period growing in the National Osmanthus Fragrance Germplasm Resource Bank. In October 2020, the flowers of Osmanthus fragrans were placed in sterilized centrifuge tubes, immediately quick-frozen in liquid nitrogen, and then stored in a -80°C refrigerator.
[0029] The Nicotiana tabacum seedlings used were provided by Wang Lianggui's research group at Nanjing Forestry University.
[0030] Example 1
[0031] 1. Extract RNA and reverse transcribe it into cDNA
[0032] The flowers of Osmanthus fragrans in full bloom were used as materials to extract total RNA using the TIANGEN Plant RNA Extraction Kit (DP432). TM The extracted RNA was reverse transcribed into cDNA using the RT Master Mix (Perfect Real Time) reverse transcription kit. The resulting cDNA was diluted 10 times with water and stored in a -20°C refrigerator.
[0033] 2. Design primers
[0034] According to the Osmanthus fragrans whole genome database published by Wang Lianggui's research group at Nanjing Forestry University (http: / / 117.78.20.255 / ), a gene sequence was screened and named ofbZIP98.
[0035] BioXM software was used to analyze the restriction sites of the full-length nucleotide sequence of the gene, and SmaI and KpnI were selected as restriction endonucleases. Primers were designed using CE design software. Fill in the relevant information as required, including the sequence near the restriction site on the vector, the full length of the target gene, and the restriction site in the order of the 5' end and 3' end, and the amplification primers can be obtained. The designed sequence was synthesized by Jierui Biotechnology Co., Ltd. The primer sequence is as follows:
[0036] ofbZIP98-F:
[0037] 5′-acgggggactctagaggatccATGAATAATTTCTTTGCGCCTCA-3′,
[0038] ofbZIP98-R:
[0039] 5′-ataagggactgaccacccgggGTGAAGAAGACTTGTGGCAGTAGTG-3′.
[0040] 3. Double digestion of vector
[0041] Take the pBI21 vector out of the -80℃ ultra-low temperature freezer in advance for activation and shaking, extract the pBI21 vector plasmid according to the kit, and then perform a double enzyme digestion experiment. The 20μL system is as follows:
[0042]
[0043]
[0044] X (μL) = 1000ng / vector plasmid concentration (ng / μL). Slightly shake the centrifuge tube to mix it, centrifuge it for 6 seconds, and incubate it in a 37°C water bath for 1 hour. Perform agarose electrophoresis on the double-enzyme-digested vector, and then use the kit to cut the gel for recovery.
[0045] 4. Target gene amplification
[0046] PCR amplification was performed using 10-fold diluted ofbZIP98 cDNA as a template. The 20 μL system was as follows:
[0047] ofbZIP98-F 1μL ofbZIP98-R 1μL cDNA 1μL Prime STAR 10μL <![CDATA[ddH2O]]> 7μL
[0048] The PCR reaction program was as follows: denaturation at 98°C for 10 s; annealing at 58°C for 15 s; extension at 72°C for 2 min, 35 cycles; total extension at 72°C for 10 min; and termination of the reaction at 16°C.
[0049] The amplified products were detected by agarose electrophoresis ( Figure 1 ), and then use the kit to excise the target gene fragment.
[0050] 5. Transformation of target gene into competent cells
[0051] The 20 μL ligation system is as follows:
[0052] Target gene recovery product 200ng Double enzyme digestion recovery product 100ng Ligase 2μL Buffer 4μL <![CDATA[ddH2O]]> to 20μL
[0053] The centrifuge tube was shaken slightly to mix, centrifuged for 6 seconds, and incubated in a 37°C water bath for 30 minutes, and placed on ice for 2 minutes.
[0054] Transformation: In the clean bench, use a pipette to take 5 μL of the ligation product and add it to 50 μL of Trelief TM 5α competent cells were flicked to mix, placed in an ice bath for 5 min, placed in a 42°C water bath for 60 s, and placed in an ice bath for another 2 min. 250 μL of liquid LB (without Kana) was added, and incubated in a shaker at 37°C, 200 rppm for 30 min.
[0055] Plate coating: Take 200 μL of the incubated bacterial solution and spread it evenly on LB solid culture medium (containing 50 mg / L Kana) with a sterilized glass rod and let it dry. Cover with sealing film and invert it in a 37°C constant temperature incubator for 12-14 hours.
[0056] 6. Screening of positive single colonies
[0057] When bacteria grow on the culture medium, single colony detection is performed in the clean bench. Pick 8 full single colonies for each gene, and backup them in sequence on LB solid culture medium containing Kana resistance, and use a sterile toothpick to dip the corresponding single colony into the following 20μL system for bacterial detection:
[0058] 35sF 1μL ofbZIP98-R 1μL Green Mix 10μL <![CDATA[ddH2O]]> 8μL
[0059] 35s-F: 5'-GACGCACAATCCCACTATCC-3'.
[0060] The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s; 58°C annealing for 30 s; 72°C extension for 2 min, 35 cycles; 72°C total extension for 10 min; 16°C termination. The obtained amplified products were subjected to agarose electrophoresis ( Figure 2), three positive single colonies with the correct length were selected for testing, and the nucleotide sequence of the ofbZIP98 gene was measured as shown in SEQ ID NO.1.
[0061] 7. Double enzyme digestion verification
[0062] The plasmid with the correct sequence obtained by sequencing was double-digested for verification, and the 20μL ligation system was as follows:
[0063] Restriction endonuclease 1 1μL Restriction endonuclease 2 2μL Buffer 2μL Vector plasmid XμL <![CDATA[ddH2O]]> 6μL
[0064] X (μL) = 1000ng / vector plasmid concentration (ng / μL). Slightly shake the centrifuge tube to mix it, centrifuge it for 6 seconds, and incubate it in a 37°C water bath for 1 hour. Figure 3 ), two bands were observed, and the lengths were consistent with those of the target fragment, indicating that the target gene OfbZIP98 had been successfully connected to the pBI121 vector.
[0065] Example 2
[0066] 1. Transformation of Agrobacterium with recombinant plasmid
[0067] Take out the GV3101 competent cells stored in the -80℃ ultra-low temperature refrigerator and thaw them on ice; add 1μL plasmid to every 33μL competent cells, pipette and mix well, then place in an ice bath for 20min, quick-freeze in liquid nitrogen for 5min, bath in 37℃ water for 5min, and bath in ice for 5min; add 500μL of LB liquid culture medium without resistance, and culture on a shaker at 28℃ and 200rpm for 1h; after the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and keep 100μL to evenly spread on LB solid culture medium (containing 50mg / LKana), seal with sealing film, and invert in a 28℃ incubator for 40-48h.
[0068] The results are as follows Figure 4 As shown, the target bands in the bacterial test are correct and have consistent brightness. The corresponding colonies were picked into LB liquid culture medium (containing 50 mg / L Kana) and shaken, and then the bacterial solution and 50% glycerol were preserved at a volume ratio of 3:7, and then quickly frozen in liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator.
[0069] 2. Agrobacterium-mediated transformation of Nicotiana tabacum
[0070] Take out the pBI121 empty vector and the vector bacterial solution with the target gene at -80°C and melt it at room temperature until it is mixed with ice and water, then insert it into ice to melt, add 200 μL of bacterial solution to 20 mL of LB liquid culture medium (containing Kana 10 μg / mL), and culture it at 28°C, 200 rpm, and shake it in the dark for about 12-16 hours until the OD600 is 0.4-0.5; the explants are selected from the healthy young leaves of Nicotiana tabacum that have not bloomed and are free of pests and diseases. First, use detergent to clean the ash layer on the surface of the leaves, rinse with running water for 30 minutes, and then transfer to the clean bench for disinfection. First, pour 75% ethanol into a beaker, shake it, so that the ethanol is fully in contact with the surface of the leaves, and wash it with sterile water for 30 seconds. Then soak it in 5% NaClo for 10 minutes, rinse it with sterile water 4 times, and use sterile filter paper to absorb the moisture on the surface of the leaves. After disinfection, use a sterile scalpel to cut off the leaf edges and veins, and then cut the remaining leaves into small pieces of 0.5×0.5 cm for infection; quickly put the cut Nicotiana tabacum leaves into the Agrobacterium solution for infection for about 10 minutes, gently shake the solution every 2 minutes, and then use sterile filter paper to dry the solution on the surface of the leaves; place the dried Nicotiana tabacum leaves on the symbiotic culture medium, facing up, and culture in the dark at 25℃ for 3 days; transfer the co-cultured materials to the screening culture medium, culture them under light at 25℃, and change the culture medium every 15 days until resistant callus and resistant buds grow; when the callus around the leaf disc differentiates into resistant buds, transfer them to the strong bud culture medium The adventitious buds were cultured on bud culture medium at 25°C under light, and the culture medium was changed every 15 days. When the adventitious buds grew to about 5 cm, they were cut off from the tissues and the callus tissue connected to the stem tissue was removed, and then transferred to the rooting medium to induce rooting. When the main root of the transgenic seedlings extended to about 5 cm and 7 to 8 leaves grew, the Nicotiana tabacum seedlings were taken out of the culture medium, and the agar gel remaining between the root tissues was washed off without damaging the root system. The seedlings were placed in a tissue culture bottle filled with deionized water for acclimation and culture for 2 days. During the acclimation period, the water was changed regularly to prevent contamination and damage to the Nicotiana tabacum seedlings. After the acclimation was completed, the Nicotiana tabacum seedlings were numbered and marked, and transplanted to the sterilized substrate soil for further culture.
[0071] 3. Screening of transgenic positive plants and phenotypic observation
[0072] Three Nicotiana tabacum strains (EV) and wild-type Nicotiana tabacum strains (WT) with the same status and transferred with the pBI121 empty vector were selected as transgenic controls. Ten flowers ofbZIP98 transgenic plants, WT and EV at the same growth stage were collected, and each flower grew until the corolla was fully extended and the stamens and anthers were fully dispersed. The corolla tube diameter, corolla diameter, corolla circumference and flower area were measured by scanner.
[0073] The results are as follows Figure 6As shown, the floral organ tissue of the ofbZIP98 transgenic line was significantly larger than that of WT and EV; the corolla diameter, corolla circumference, corolla area, and corolla tube diameter of the ofbZIP98 transgenic line were significantly increased compared with those of WT and EV.
[0074] 4. qRT-PCR quantification
[0075] The TIANGEN plant RNA extraction kit (DP432) was used to extract the transgenic Nicotiana tabacum ( Figure 5 ) and total RNA from wild-type Nicotiana tabacum leaves. TM The extracted RNA was reverse transcribed into cDNA using RT Master Mix (Perfect Real Time) reverse transcription kit, and the cDNA was diluted 10 times with water. Semi-quantitative test (qRT-PCR) was performed on the transgenic Nicotiana tabacum strains and wild-type transgenic Nicotiana tabacum plants that tested positive.
[0076] The results are as follows Figure 7 As shown, the expression level ofbZIP98 transgenic positive plants was higher than that of wild-type plants.
Claims
1. Application of the ofbZIP98 gene of Rixianggui in promoting the enlargement of flower organs of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.
1.
2. The use ofbZIP98 gene of Rixianggui according to claim 1 in promoting the enlargement of flower organs of Nicotiana tabacum, characterized in that: The following steps are involved: (1) constructing a vector ofbZIP98 gene; (2) transforming the constructed vector ofbZIP98 gene into Nicotiana tabacum; (3) Cultivate and screen transgenic Nicotiana tabacum with enlarged floral organs.
3. The use ofbZIP98 gene of Rixianggui according to claim 2 in promoting the enlargement of flower organs of Nicotiana tabacum, characterized in that: The vector is a plant expression vector.
4. The use ofbZIP98 gene of Rixianggui in promoting the enlargement of Nicotiana tabacum flower organs according to claim 3, characterized in that: The plant expression vector is pBI121-ofbZIP98.
5. Application of the ofbZIP98 gene of Rixianggui in increasing the corolla diameter of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.
1.
6. Application of the ofbZIP98 gene of Rixianggui to increasing the circumference of the corolla of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene being shown in SEQ ID NO.
1.
7. Application of the ofbZIP98 gene of Rixianggui in increasing the corolla area of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene being shown in SEQ ID NO.
1.
8. Use of the ofbZIP98 gene of Rixianggui in increasing the diameter of the corolla tube of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.
1.
9. Use of the ofbZIP98 gene of Rixianggui in increasing the corolla diameter, corolla circumference, corolla area, and corolla tube diameter of Nicotiana tabacum, the nucleotide sequence of the ofbZIP98 gene being shown in SEQ ID NO.1.