CN116836999A - Application of cinnamomum japonicum ofbZIP98 gene in promotion of flower organ enlargement of large flower tobacco - Google Patents

Abstract

The invention discloses the application of the ofbZIP98 gene of N. grandis in promoting the enlargement of flower organs of Nicotiana grandiflorum, and belongs to the field of plant molecular biology. The nucleotide sequence of the ofbZIP98 gene of the present invention is shown in SEQ ID NO.1; the present invention uses the flowers of the Osmanthus in bloom as materials, obtains the ofbZIP98 gene through cloning, and constructs its overexpression on this basis The vector pBI121‑ofbZIP98 was transferred into Nicotiana grandiflorum to obtain transgenic plants. The floral organs of the ofbZIP98 transgenic line are significantly larger than those of the N. grandiflora strain transferred into the pBI121 empty vector and the wild-type N. grandiflora strain; the ofbZIP98 transgenic line is significantly larger than the N. grandiflora strain transferred into the pBI121 empty vector. Compared with the wild-type Nicotiana grandiflorum strain, the corolla diameter, corolla circumference, corolla area, and corolla tube diameter were significantly increased.

Description

Application of ofbZIP98 gene in promoting flower organ enlargement of Nicotiana grandiflorum

Technical field

The invention belongs to the field of plant molecular biology, and more specifically, relates to the application of the ofbZIP98 gene in promoting the enlargement of Nicotiana grandiflorum flower organs.

Background technique

bZIP transcription factors are named for their highly conserved bZIP domain consisting of an α-helix approximately 40-60 amino acids long, which consists of two different functional regions: a highly conserved basic region located at the N-terminus and Located in the C-terminal leucine zipper region, the two domains are located on adjacent a-helices and can bind to DNA through the main grooves on both sides of the double helix.

bZIP is a transcription factor that can be expressed in all apical meristems of Arabidopsis (including meristems in inflorescences, flowers, carpel edges, etc.) and can induce JAG and OFP1 to regulate cell proliferation and cell elongation. Gene expression can also interact with regulatory factors related to meristem and reproductive enlargement such as WUS, JAB and BEL1.

Osmanthus fragrans, a landscaping tree species of the genus Osmanthus in the family Oleaceae, is one of the top ten traditional famous flowers in my country. It can also be used in food processing, balm and perfume manufacturing, aromatherapy, etc., and has important ornamental value. value and economic value. Osmanthus has been cultivated in my country for more than 2,500 years. The variety resources are very rich, and there are obvious differences in leaf shape, leaf color, floral fragrance, flower color and other traits between different Osmanthus varieties. Osmanthus flowers are small and the picking process is time-consuming and laborious. Therefore, it is of great significance to discover functional genes that regulate the size of Osmanthus flower organs.

Contents of the invention

In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide the application of the ofbZIP98 gene of Nicotiana grandiflorum in promoting the enlargement of flower organs of Nicotiana grandiflorum for plant molecular improvement breeding.

In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

Application of ofbZIP98 gene in promoting the enlargement of flower organs of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO. 1 and includes the following steps:

(1) Construct the vector of ofbZIP98 gene;

(2) Transform the constructed vector of ofbZIP98 gene into Nicotiana grandiflorum;

(3) Breed and screen to obtain transgenic Nicotiana grandiflorum with enlarged flower organs.

The vector is a plant expression vector.

The plant expression vector is pBI121-ofbZIP98.

Application of the ofbZIP98 gene of Nicotiana grandiflorum in increasing corolla diameter of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.

Application of the ofbZIP98 gene of Nicotiana grandiflorum in increasing the corolla circumference of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.

Application of the ofbZIP98 gene in increasing the corolla area of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.

Application of the ofbZIP98 gene of Nicotiana grandiflorum in increasing the corolla tube diameter of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.

The application of the ofbZIP98 gene of Nicotiana grandiflorum in increasing the corolla diameter, corolla circumference, corolla area, and corolla tube diameter of Nicotiana grandiflorum. The nucleotide sequence of the ofbZIP98 gene is shown in SEQ ID NO.1.

Compared with the existing technology, the beneficial effects of the present invention are:

The present invention uses the flowers of Osmanthus niger in full flowering stage as materials to obtain the ofbZIP98 gene through cloning. On this basis, the overexpression vector pBI121-ofbZIP98 is constructed and transferred into Nicotiana grandiflorum to obtain transgenic plants. The floral organ tissues of ofbZIP98 transgenic lines are significantly larger than those of N. grandiflora lines (EV) and wild-type N. grandiflora lines (WT) transferred into pBI121 empty vector; ofbZIP98 transgenic lines are significantly larger than those transferred into pBI121 empty vector. Compared with the wild-type Nicotiana grandiflorum strain (EV) and the wild-type Nicotiana grandiflorum strain (WT), the corolla diameter, corolla circumference, corolla area, and corolla tube diameter were significantly increased.

Description of the drawings

Figure 1 is an agarose gel electrophoresis diagram of the amplification product of the target gene ofbZIP98;

Figure 2 shows the detection of positive single colonies after the amplification fragment of the target gene ofbZIP98 was connected to the vector and transformed;

Figure 3 is an agarose gel electrophoresis picture of ofbZIP98 recombinant vector after double enzyme digestion;

Figure 4 is an agarose gel electrophoresis diagram of bacteria after transformation with Agrobacterium GV3101;

Figure 5 shows the positive detection chart of ofbZIP98 transgenic plants;

Figure 6 shows the flower phenotype observation of ofbZIP98 transgenic Nicotiana grandiflorum;

Figure 7 shows the gene expression profile of ofbZIP98 transgenic Nicotiana grandiflorum.

Detailed ways

The present invention will be further described below with reference to specific embodiments. In the following examples, operations not described in detail are all routine biological experimental operations, which can be performed with reference to the molecular biology experimental manual and existing published journal documents, or according to the kit and product instructions. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

The materials used in this application are Osmanthus japonica flowers grown in the Osmanthus National Germplasm Resource Bank in the blooming stage. In October 2020, the Osmanthus japonica flowers were put into sterilized centrifuge tubes, immediately put into liquid nitrogen and quickly frozen, and then placed Store in -80℃ refrigerator.

The Nicotiana grandiflorum seedlings used were provided by Wang Lianggui’s research group at Nanjing Forestry University.

Example 1

1. Extract RNA and reverse-transcribe into cDNA

Using the flowers of Osmanthus nichii in full flowering stage as materials, total plant RNA was extracted using TIANGEN plant RNA extraction kit (DP432). The extracted RNA was reverse transcribed into cDNA using the TaKaRaPrimeScript ^TM RTMasterMix (PerfectRealTime) reverse transcription kit. The final cDNA was diluted 10 times with water and stored in a -20°C refrigerator.

2. Design primers

According to the Osmanthus fragrans genome database (http://117.78.20.255/) published by Wang Lianggui's research group at Nanjing Forestry University, a gene sequence was screened and named ofbZIP98.

The full-length nucleotide sequence of the gene was analyzed using BioXM software, and SmaI and KpnI were selected as restriction endonucleases. Use CE design software to design primers. Fill in the relevant information as required, including the sequence near the restriction site on the vector, the full length of the target gene, fill in the restriction site in the order of the 5' end and the 3' end, and you can get the amplification primers and design them. The sequence was synthesized by Jerui Biotech. The primer sequences are as follows:

ofbZIP98-F:

5′-acgggggactctagaggatccATGAATAATTTCTTTGCGCCTCA-3′,

ofbZIP98-R：

5′-ataagggactgaccacccgggGTGAAGAAGACTTGTGGCAGTAGTG-3′.

3. Vector double enzyme digestion

The pBI21 vector was taken out from the -80°C ultra-low temperature refrigerator in advance for activation and shaking. The pBI21 vector plasmid was extracted according to the kit, and then a double enzyme digestion experiment was performed. The 20 μL system is as follows:

X (μL)=1000ng/vector plasmid concentration (ng/μL). Shake the centrifuge tube slightly to mix it evenly, centrifuge briefly for 6 seconds, and place it in a 37°C water bath for 1 hour. The obtained double-digested vector was subjected to agarose electrophoresis, and then the gel was recovered using a kit.

4. Target gene amplification

Use the 10-fold diluted ofbZIP98 cDNA as a template to perform PCR amplification. The 20 μL system is as follows:

ofbZIP98-F 1μL ofbZIP98-R 1μL cDNA 1μL Prime STAR 10μL ddH ₂ O 7μL

The PCR reaction program was: 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.

The amplification product was detected by agarose electrophoresis (Figure 1), and then the target gene fragment was cut and recovered using a kit.

5. Transform competent cells with the target gene

The 20μL connection system is as follows:

Target gene recovery product 200ng Products recovered from double enzyme digestion 100ng Ligase 2μL Buffer 4μL ddH ₂ O to 20μL

Shake the centrifuge tube slightly to mix it, centrifuge briefly for 6 seconds, place it in a 37°C water bath and incubate for 30 minutes, and keep it on ice for 2 minutes.

Transformation: In the ultra-clean workbench, use a pipette to add 5 μL of the ligation product to 50 μL of Trelief ^TM 5α competent cells, flick to mix, then bathe in ice for 5 minutes, then bathe in 42°C water for 60 seconds, then bath in ice for 2 minutes, and add 250 μL of liquid LB (excluding Kana), incubate in a shaker at 37°C and 200 rpm for 30 minutes.

Plate: Take 200 μL of the incubated bacterial solution, spread it evenly on the LB solid medium (containing 50 mg/L Kana) with a sterilized glass rod and dry it, cover it with a sealing film and invert it in a 37°C constant temperature incubator. Incubate for 12-14 hours.

6. Positive single colony screening

When bacteria grow on the culture medium, a single colony test is performed in a clean workbench. Pick 8 full single colonies for each gene, back them up on LB solid culture medium containing Kana resistance in sequence, and use a sterile toothpick to dip the corresponding single colonies into the following 20 μL system for bacterial detection:

35sF 1μL ofbZIP98-R 1μL Green Mix 10μL ddH ₂ O 8μL

35s-F: 5’-GACGCACAATCCCACTATCC-3’.

PCR reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 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. The obtained amplification product was subjected to agarose electrophoresis (Figure 2), and three positive single bacterial colonies with the correct length were selected and sent for testing. The nucleotide sequence of the ofbZIP98 gene was determined as shown in SEQ ID NO. 1.

7. Double enzyme digestion verification

The plasmid with the correct sequence obtained by sequencing was verified by double enzyme digestion. The 20 μL ligation system is as follows:

Restriction endonuclease 1 1μL Restriction endonuclease 2 2μL Buffer 2μL vector plasmid XμL ddH ₂ O 6μL

X (μL)=1000ng/vector plasmid concentration (ng/μL). Shake the centrifuge tube slightly to mix it evenly, centrifuge briefly for 6 seconds, and place it in a 37°C water bath for 1 hour. The obtained double-digested vector was subjected to agarose electrophoresis (Figure 3). Two bands were observed, and the length of the bands was consistent with the target fragment, indicating that the target gene OfbZIP98 had been successfully connected to the pBI121 vector.

Example 2

1. Transformation of Agrobacterium with recombinant plasmid

Take out the GV3101 competent cells stored in the -80°C ultra-low temperature refrigerator and place them on ice to thaw; add 1 μL of plasmid for every 33 μL of competent cells, mix well by pipetting, and then ice bath for 20 minutes, liquid nitrogen quick freezing for 5 minutes, 37°C water bath for 5 minutes, and ice bath. 5min; add 500μL of non-resistant LB liquid culture medium, culture on a 200rpm shaker at 28°C for 1h; after the culture is completed, centrifuge the bacterial solution at 6000r for 1min, discard part of the supernatant, and leave 100μL to evenly spread on the LB solid culture base (containing 50mg/LKana), seal with parafilm, and place upside down in a 28°C incubator for 40-48h.

The results are shown in Figure 4. The target bands in the bacterial test are correct and have consistent brightness. Pick the backup corresponding colonies into LB liquid culture medium (containing 50mg/LKana) and shake them. Then mix the bacterial solution and 50% glycerol at a volume ratio of 3:7 to preserve the bacteria. Quick freeze in liquid nitrogen and store at -80 ℃ ultra-low temperature refrigerator.

2. Agrobacterium-mediated transformation of Nicotiana grandiflorum

Take the pBI121 empty vector and the vector bacterial liquid connected to 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 liquid to 20 mL of LB liquid culture medium (containing Kana 10 μg/mL), 28 Cultivation at ℃, 200rpm, dark shaking for about 12-16h, until the OD600 is 0.4-0.5; select healthy young leaves of Nicotiana tabacum that have not bloomed and are free of diseases and insect pests as explants. First, clean the gray layer on the surface of the leaves with detergent and rinse with running water. After 30 minutes, transfer to the ultra-clean workbench for disinfection. First, pour 75% ethanol into the beaker and shake to make the ethanol fully contact with the surface of the young leaves. The time is 30 seconds. Wash with sterile water 3 times. Then soak in 5% NaClo for 10 min, rinse 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 remove the leaf edges and veins, and then cut the remaining leaves into small pieces of 0.5×0.5cm for infection; quickly add Agrobacterium bacteria to the cut Nicotiana grandiflorum leaves. Infect the liquid in the liquid for about 10 minutes. During this period, gently shake the bacterial liquid every 2 minutes, and then use sterile filter paper to dry the bacterial liquid on the surface of the leaves; place the dried Nicotiana grandiflorum leaves on the symbiotic medium with the front side facing up. , cultivated in the dark at 25°C for 3 days; transfer the co-cultured materials to the screening medium, cultivate in the light at 25°C, and replace the medium every 15 days until resistant callus and resistant buds grow; strong bud culture When the callus around the leaf disk differentiates into resistant buds, transfer it to the bud-strengthening medium, culture it under light at 25°C, and replace the medium every 15 days; when the adventitious buds grow to about 5cm, remove them from the tissue. Cut and remove the callus connected to the stem tissue, and then transfer it to the rooting medium to induce rooting; when the main root of the transgenic seedlings extends to about 5cm and 7 to 8 leaves grow, add N. grandiflora Take the seedlings out of the culture medium, rinse away the agar gel remaining between the root tissues without harming the root system, place it in a tissue culture bottle filled with deionized water, and acclimate it for 2 days. During the acclimation period, change the water regularly to prevent contamination. Damage the Nicotiana grandiflorum seedlings; after completing the domestication, mark the Nicotiana grandiflorum seedlings with numbers and transplant them into sterilized matrix soil to continue culturing.

3. Screening and phenotypic observation of transgenic positive plants

Three N. grandiflora lines (EV) and wild-type N. grandiflorum line (WT) with the same status and transformed into the pBI121 empty vector were selected as transgenic controls. 10 flowers of ofbZIP98 transgenic plants, WT and EV were collected at the same growth stage. Each flower enlarged until the corolla was fully extended and the stamen anthers were completely dispersed. Corolla tube diameter, corolla diameter, corolla circumference and flower area were measured by scanner.

The results are shown in Figure 6. The floral organs of the ofbZIP98 transgenic line were significantly larger than those of WT and EV. Compared with WT and EV, the corolla diameter, corolla circumference, corolla area, and corolla tube diameter of ofbZIP98 transgenic line were all the same. significantly increased.

4. qRT-PCR quantification

The TIANGEN plant RNA extraction kit (DP432) was used to extract total RNA from the leaves of transgenic N. grandiflorum (Figure 5) and wild-type N. grandiflorum that tested positive. The extracted RNA was reverse transcribed into cDNA using TaKaRa PrimeScript ^TM RT Master Mix (PerfectReal Time) reverse transcription kit, and the final cDNA was diluted 10 times with water. A semi-quantitative test (qRT-PCR) was performed on the positive transgenic N. grandiflora lines and the wild-type transgenic N. grandiflora plants.

The results are shown in Figure 7. The expression level of ofbZIP98 transgenic positive plants was higher than that of wild-type plants.

Claims (9)

1. The application of the cinnamomum japonicum ofbZIP98 gene in promoting the organ enlargement of the tobacco flowers is provided, wherein the nucleotide sequence of the gene ofbZIP98 is shown as SEQ ID NO. 1.

2. Use of the cinnamomum japonicum ofbZIP98 gene according to claim 1, for promoting the enlargement of floral organs of nicotiana macrophylla, comprising the steps of:

(1) Constructing a vector of the ofbZIP98 gene;

(2) Transforming the constructed vector of the ofbZIP98 gene into tobacco with big flowers;

(3) And culturing and screening to obtain the transgenic large flower tobacco with enlarged flower organs.

3. The use of the dixiang cinnamon ofbZIP98 gene according to claim 2, wherein said vector is a plant expression vector for promoting the enlargement of floral organs of nicotiana macrophylla.

4. The use of the gene of cinnamomum japonicum ofbZIP98 according to claim 3, wherein the plant expression vector is pBI121-ofbZIP98.

5. The application of the cinnamomum japonicum ofbZIP98 gene in increasing the diameter of the corolla of the nicotiana macrophylla, wherein the nucleotide sequence of the ofbZIP98 gene is shown as SEQ ID NO. 1.

6. The application of the cinnamomum japonicum ofbZIP98 gene in increasing the circumference of the corolla of the nicotiana macrophylla, wherein the nucleotide sequence of the ofbZIP98 gene is shown as SEQ ID NO. 1.

7. The application of the cinnamomum japonicum ofbZIP98 gene in increasing the crown area of the tobacco with large flowers, wherein the nucleotide sequence of the gene ofbZIP98 is shown as SEQ ID NO. 1.

8. The application of the cinnamomum japonicum ofbZIP98 gene in increasing the diameter of a crown cylinder of the tobacco with large flowers, wherein the nucleotide sequence of the gene ofbZIP98 is shown as SEQ ID NO. 1.

9. The application of the cinnamomum japonicum ofbZIP98 gene in increasing the diameter of the corolla, the circumference of the corolla, the area of the corolla and the diameter of the corolla cylinder of the large flower tobacco is provided, and the nucleotide sequence of the gene ofbZIP98 is shown as SEQ ID NO. 1.