IsMYBL3 gene for negative regulation of anthocyanin synthesis and application of IsMYBL3 gene

Abstract

The invention discloses an IsMYBL3 gene derived from phallus impudicus petals. The invention provides an IsMYBL3 gene capable of realizing negative regulation and control of anthocyanin synthesis. An overexpression vector of the IsMYBL3 gene is constructed, the overexpression vector is transferred into wild tobacco through agrobacterium-mediated genetic transformation, and the anthocyanin content of transgenic tobacco can be obviously reduced. The IsMYBL3 gene can be applied to genetic engineering genetic breeding of plants and cultivation of white flower varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a sansevieria iris IsMYBL3 gene for negatively regulating anthocyanin synthesis and an application thereof. Background Art

[0002] Iris sanguinea is a perennial herbaceous flower of the genus Iris in the family Iridaceae. It has a thick, oblique rhizome, linear leaves, and unclear midribs. It blooms from May to June and fruits from July to September. Iris sanguinea has large, rich, elegant, cold-resistant, and high ornamental value flowers. It is also a precious Chinese herbal medicine. Its rhizome is used as medicine to clear away heat and detoxify. It is a wild flower with high economic value that combines ornamental and medicinal uses. It is mainly distributed in northeast my country and grows in swamps, wet grasslands, or sunny land. In addition, Iris sanguinea has a natural variant, white-flowered Iris sanguinea f.albiflora, which can provide a basis for screening negatively regulating flower color genes.

[0003] Flower color is an important ornamental characteristic of ornamental plants. Its quality is directly related to the ornamental value and commercial value of ornamental plants. With the vigorous development of the flower industry, ornamental plants with unique flower colors have broad market prospects. In nature, flowers bloom in various colors. Iris plants are mainly blue-purple and yellow, lacking white. Therefore, the study of negative regulation of flower color is of great value to enrich the flower color of Iris plants.

[0004] The biosynthesis of anthocyanins belongs to the flavonoid group of the phenylpropanoid biosynthesis pathway. It is well known that anthocyanin biosynthesis genes are controlled by the MBW transcription complex formed by MYB, bHLH and WD40. MYB is the most core transcription factor regulating anthocyanin biosynthesis. MYB transcription factors participate in regulating various complex networks, growth, development and stress resistance throughout the plant life cycle, including anthocyanin biosynthesis pathway, peel coloring, flavonoid / phenylpropanoid metabolism, secondary wall biosynthesis, sugar signaling and response to drought and waterlogging stress. In terms of anthocyanin synthesis, PAP1 and PAP2 in Arabidopsis thaliana (L.) Heynh. and MdMYB10 in apple (Malus pumila Mill.) are positive regulators of anthocyanin biosynthesis. In contrast, other R2R3-MYBs act as anthocyanin biosynthesis inhibitors, such as AtMYB4 and AtMYB3 in Arabidopsis. Summary of the invention

[0005] The purpose of the present invention is to provide a IsMYBL3 gene of Iris sanguinea that negatively regulates anthocyanin synthesis and its application, which has a certain effect on cultivating white flower species plants.

[0006] The invention obtains the IsMYBL3 gene by screening the metabolome and transcriptome of 5 kinds of iris flower colors and the protein group of 3 kinds of flower colors. The nucleotide sequence of the IsM YBL3 gene is SEQ ID NO.1 in the sequence table, the nucleotide sequence is 633bp in length, and encodes 210 amino acids, and the amino acid sequence is SEQ ID NO.2 in the sequence table. A pCAMBIA1300-IsMYBL3-GFP plant overexpression vector is constructed. pCAMBIA1300-IsMYBL3-GFP is transferred into Agrobacterium GV3101 by freezing transfer method, and transferred into tobacco by leaf disc method. PCR identification confirms that the target gene is successfully inserted into the tobacco genome, and the screening is continued to T3 generation. The high expression strain of tobacco T3 generation is screened by RT-qPCR method, and the flower color of the overexpressed IsMYBL3 plant is observed. As a result, the anthocyanin content of the IsMYBL3 gene-transfected tobacco is significantly reduced, and the color becomes white.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A gene IsMYBL3 that negatively regulates the flower color of Iris strychnifolia has a nucleotide sequence of SEQ ID NO.1 in the sequence table.

[0009] The present invention provides a vector containing the above encoding gene.

[0010] The present invention provides application of the above gene in negatively regulating flower color.

[0011] The present invention has the following benefits:

[0012] The present invention successfully cloned the IsMYBL3 gene of Iris sanguinea, further proving that the IsMYBL3 gene can negatively regulate the flower color of Iris sanguinea, which is of great significance for a comprehensive understanding of the biological functions of MYB transcription factors in plants.

[0013] The invention not only enriches the flower color regulation theory of Ilex strychnifolia, but can also be used for regulating flower color in gene breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Attached Figure 1 It is the agarose gel electrophoresis diagram of the cloning of IsMYBL3 gene; wherein: lane 1 is DL2000 marker, lane 2 is water as a control, and lanes 3 and 4 are the PCR results of IsMYBL3 gene.

[0015] Attached Figure 2 The flower color phenotypes of wild-type and IsMYBL3-overexpressing tobacco are shown in the figure; WT is wild-type tobacco, and IsMYBL3 is transgenic IsMYBL3 tobacco.

[0016] Attached Figure 3This is a graph of total anthocyanin content in wild-type and IsMYBL3-overexpressing tobacco corollas. DETAILED DESCRIPTION

[0017] The following examples are provided to further describe the technical solution of the present invention in detail, but are not intended to limit the protection scope of the present invention.

[0018] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and researchers in this field can achieve the results by following conventional molecular biology techniques; the materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0019] The relevant culture medium preparation method in the embodiment is as follows:

[0020] YEP liquid medium (100 mL): 1 g yeast extract + 1 g tryptone + 0.5 g sodium chloride.

[0021] YEP solid medium (100 mL): 1 g yeast extract + 1 g tryptone + 0.5 g sodium chloride + 1.5 g bacterial agar.

[0022] The anthocyanin content in the example was determined by the hydrochloric acid-ethanol extraction method, refer to (Tang M, Xue W, LiX, et al. Mi totically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemu m[J]. New Phytol, 2022, 236(3): 1075-1088.).

[0023] Example 1: IsMYBL3 gene cloning and construction of overexpression vector.

[0024] The overexpression vector used in the present invention is pCAMBIA1300-GFP, into which the IsMYBL3 gene fragment is inserted to obtain the pCAMBIA1300-IsMYBL3-GFP plant overexpression vector.

[0025] IsMYBL3 gene cloning. Using the cDNA of the petals of Ilex strychnifolia as a template, the upstream primer IsMYBL3-F: 5'-CTCCAAGAGTTCATAGAGCCTAAG-3' and the downstream primer IsMYBL3-R: 5'-CATTCTCCAACTACTGCATCG-3' were designed according to the sequence of IsMYBL3. The target fragment ( Figure 1), PCR program annealing temperature 55°C, detailed method refer to KOD FX instruction manual. Cloning vector selection blunt end cloning vector TRAN company -Blunt Zero Cloning Kit, 4 μL of purified PCR amplified fragments were mixed with 1 μL -Blunt Zero Cloning Kit vector, ligated at 30°C for 10 min, then 5 μL of ligation product was transformed into E. coli competent DH5α at 42°C, positive single colonies were picked for testing and the plasmid was returned.

[0026] Construction of plant overexpression vector pCAMBIA1300-IsMYBL3-GFP. Analyze and design the homology arm sequence of seamless cloning of pCAMBIA1300-GFP vector, and design upstream homology arm primer tIsMYBL3-F: 5'- TTGATACATATG CCCGTCGAC ATGATGAGCCTAAGCTCAG-3', downstream homology arm primer tIsMYBL3-R: 5'- CCCTTGCTCACCATGG ATCC TAATTCCAAGCATATATGATCAAT-3', the above primers retain the BamHI and SalI restriction sites of pCAMBIA1300-GFP. The underline in the primers indicates the homology arms of pCAMBIA1300-GFP.

[0027] The correct cloned plasmid identified by sequencing of IsMYBL3 from C. sanguinea was used as a template, and the target gene fragment with homology arms of pCAMBIA1300-GFP vector was obtained after PCR amplification using KOD FX. The pCAMBIA1300-GFP vector plasmid was linearized using restriction endonucleases BamHI and SalⅠ from TaKaRa to obtain the linearized pCAMBIA1300-GFP vector. The homology arm recombination method was used to construct the pCA MBIA1300-IsMYBL3-GFP plant overexpression vector. The recombinase was ExnaseⅡ from Vazyme. The specific amount of the vector and the target fragment was calculated according to the formula provided in the instruction manual of the ExnaseⅡ product. The PCR instrument was used to control the temperature at 37℃ for 30 minutes and then transformed into the competent Escherichia coli DH5α. The single cloning point was selected for bacterial liquid PCR and sequencing verification. The upstream verification primer was y1300-F: 5'-TTGATACATA TGCCCGTCGAC-3', and the downstream verification primer was y1300-R: 5'-CCCTTGCTCACCATGGATCC-3'. Finally, the correct plant overexpression vector pCAMBIA1300-IsMYBL3-GFP was obtained. The recombinant plasmid pCAMBIA1300-IsMYBL3-GFP was transferred into the competent cells of Agrobacterium GV3101 by freezing.

[0028] Example 2: Genetic transformation of tobacco with the IsMYBL3 gene of Iris sanguinea.

[0029] The Agrobacterium containing the pCAMBIA1300-IsMYBL3-GFP plant overexpression vector obtained in Example 1 was used to genetically transform tobacco using the leaf disc method. The specific method is as follows: (1) Take out the Agrobacterium GV3101 containing the recombinant plasmid from the -80°C refrigerator and activate it. Incubate it in a dark environment at 28°C for 48 hours. h. Pick out the single clone strain for PCR identification, and then expand the culture of the correctly identified bacteria in YEP liquid culture medium. (2) Shake Agrobacterium to OD 600 After reaching 0.7, it is used for infection. (3) Select the sterile tobacco seedlings that are growing well, and cut the young leaves into 1 cm 2 The square leaves were then placed on MS solid medium (containing 20 g·L -1 Sucrose + 1 mg·L -1 6-BA+0.05mg·L -1 of NAA) and cultured under light for 2 days. (4) Collect the shaken bacterial solution and resuspend it in a resuspending solution (1 / 2MS + 20g L -1The pre-cultured tobacco leaves were then immersed in the resuspension and shaken continuously for 5 min. After infection, they were placed on filter paper to dry and then placed back on the culture medium for co-culture in the dark for 2 d. (5) Place the leaves on the screening medium and culture under light. Change the medium every 15 days. The medium composition is MS + 1 mg·L -1 6BA+ 0.05mg·L -1 NAA+20mg·L -1 Hyg+200mg·L -1 Timentin. (6) When adventitious buds differentiated from leaves, they were cut off and transferred to 1 / 2MS solid medium (containing 20 g·L -1 Sucrose + 25 mg·L -1 Hyg+200mg·L -1 Rooting culture was carried out in Timentin. (7) The rooted tobacco seedlings are transferred to nutrient soil for further cultivation, and leaves are cut to extract DNA for PCR verification. The positive tobacco seedlings are further cultivated until the seeds are harvested, and the next generation of positive plants are continuously screened until the T3 generation of positive plants is obtained.

[0030] Example 3: Comparison of flower color between IsMYBL3-overexpressing tobacco and control tobacco.

[0031] The harvested tobacco seeds were sterilized and sown in a soil containing 25 mg·L -1 The 1 / 2MS screening medium of Hyg was sterilized by using 75% alcohol for 1 minute, then washing with sterile water for 3 times, then oscillating and sterilizing with 2% sodium hypochlorite solution for 10 minutes, and finally washing with sterile water for 5 times; after vernalization at 4°C for 2 days, it was cultured normally in the tissue culture room. When the tobacco grew four leaves and was slightly larger, it was moved to nutrient soil (peat soil: vermiculite = 3:1) for culture. The light environment was light / dark 14h / 10h, and watered once a week to observe the changes in tobacco flower color. The results showed that the flower color of tobacco transgenic with IsMYBL3 gene was significantly lighter than that of wild-type tobacco, and the corolla color of tobacco transgenic with IsMYBL3 gene turned white ( Figure 2 ), the total anthocyanin content in corolla was significantly lower than that in wild-type tobacco ( Figure 3 ), indicating that the IsMYBL3 gene can effectively inhibit the anthocyanin synthesis of plants.

[0032] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A transcription factor of IsMYBL3 from Iris strychnifolia, characterized in that Its amino acid sequence is SEQ ID NO.

2.

2. A gene encoding the IsMYBL3 transcription factor of claim 1.

3. The gene encoding the IsMYBL3 transcription factor of Iris strychnifolia according to claim 2, characterized in that: Its nucleotide sequence is SEQ ID NO.

1.

4. Use of the IsMYBL3 transcription factor of claim 1 or the encoding gene of claim 2 in inhibiting anthocyanin synthesis in petals of Sansevieria sansevieria, wherein the use is the use of overexpressing the IsMYBL3 transcription factor of Sansevieria sansevieria in inhibiting anthocyanin synthesis in Sansevieria sansevieria.

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