Ismybl3 gene for negatively regulating anthocyanin synthesis and application thereof

Abstract

The application discloses an IsMYBL3 gene from petals of Flickingeria japonica. The application provides an IsMYBL3 gene capable of negatively regulating anthocyanin synthesis. An overexpression vector of the IsMYBL3 gene is constructed, the overexpression vector is transformed into wild-type tobacco through a method of agrobacterium-mediated genetic transformation, and the anthocyanin content of the transgenic tobacco can be obviously reduced. The IsMYBL3 gene can be applied to genetic engineering breeding of plants, and white-flower varieties can be bred.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a negative regulation of anthocyanin synthesis of IsMYBL3 gene of Iris sanguinea and application thereof. BACKGROUND

[0002] Iris sanguinea is a perennial herbaceous flower of Iridaceae and Iris, the rhizome is thick and obliquely extends, the leaf is linear, the midrib is not obvious, the flowering period is from May to June, and the fruiting period is from July to September. The Iris sanguinea flower is large, dense, elegant, cold-resistant, and has high ornamental value. The rhizome is a kind of wild flower with high economic value, which is used as medicine for clearing heat and detoxification. The Iris sanguinea is mainly distributed in the northeast of China and grows in marshy land, wet grassland or sunny land. In addition, the Iris sanguinea has a natural variety of white flower (I. sanguinea f. albiflora), which can provide a basis for screening negative regulation of flower color genes.

[0003] As an important ornamental feature of ornamental plants, the flower color is directly related to the ornamental value and commercial value of the ornamental plants. With the vigorous development of the flower industry, ornamental plants with unique flower colors have broad market prospects. There are various flower colors in nature, and the Iris plants are mainly blue and yellow, lacking white. Therefore, the research on the negative regulation of flower color has important value for enriching the flower color of Iris plants.

[0004] The biosynthesis of anthocyanins belongs to the flavonoid group of the phenylpropanoid synthesis pathway. It is clear that the anthocyanin biosynthesis genes are controlled by the MBW transcription complex formed by MYB, bHLH and WD40. MYB is the most core transcription factor for regulating anthocyanin biosynthesis. MYB transcription factors participate in regulating various complex networks, growth, development and stress resistance in the whole plant life cycle, including anthocyanin biosynthesis pathway pericarp coloring, flavonoid / phenylpropanoid metabolism, secondary wall biosynthesis, sugar signal transduction and response to drought and waterlogging stress. In terms of anthocyanin synthesis, PAP1, PAP2 in Arabidopsis thaliana (L.) Heynh. and MdMYB10 in Malus pumila Mill. are positive regulators of anthocyanin biosynthesis. On the contrary, other R2R3-MYB acts as an inhibitor of anthocyanin biosynthesis, such as AtMYB4 and AtMYB3 in Arabidopsis thaliana. SUMMARY

[0005] The purpose of the present application is to provide a negative regulation of anthocyanin synthesis of IsMYBL3 gene of Iris sanguinea and application thereof, which has a certain effect on breeding white flower plants.

[0006] This invention identified the IsMYBL3 gene through metabolomics and transcriptomics analysis of five different flower colors and proteomics analysis of three different flower colors. The nucleotide sequence of the IsMYBL3 gene is SEQ ID NO.1 in the sequence listing, with a full length of 633 bp encoding 210 amino acids, and its amino acid sequence is SEQ ID NO.2 in the sequence listing. A plant overexpression vector, pCAMBIA1300-IsMYBL3-GFP, was constructed. pCAMBIA1300-IsMYBL3-GFP was transformed into Agrobacterium GV3101 via freeze-transformation, and then into tobacco using the leaf disc method. PCR confirmed successful insertion of the target gene into the tobacco genome. Screening continued until the T3 generation, and high-expression lines in the T3 generation of tobacco were obtained using RT-qPCR. The flower color of the IsMYBL3-overexpressing plants was observed. Results showed that the anthocyanin content in tobacco plants transfected with the IsMYBL3 gene was significantly reduced, and the flower color turned white.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A gene, IsMYBL3, that negatively regulates flower color in *Fragranceia spp.*, has its nucleotide sequence as SEQ ID NO.1 in the sequence listing.

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

[0010] This invention provides the application of the above-mentioned gene in the negative regulation of flower color.

[0011] The benefits of this invention are as follows:

[0012] This invention successfully cloned the IsMYBL3 gene of *Fungiformis pilosa*, further demonstrating that the IsMYBL3 gene can negatively regulate the flower color of *Fungiformis pilosa*, which is of great significance for a comprehensive understanding of the biological functions of MYB transcription factors in plants.

[0013] This invention not only enriches the theory of flower color regulation in *Fructus scutellariae*, but can also be used for gene breeding to regulate flower color. Attached Figure Description

[0014] Appendix Figure 1 This is an agarose gel electrophoresis image of the cloned IsMYBL3 gene; where: lane 1 is the DL2000 marker, lane 2 is water as a control, and lanes 3 and 4 are the PCR results of the IsMYBL3 gene.

[0015] Appendix Figure 2 These are flower color phenotypes of wild-type and IsMYBL3 overexpressing tobacco; where WT represents wild-type tobacco and IsMYBL3 represents transgenic IsMYBL3 tobacco.

[0016] Appendix Figure 3Figure 1 is a graph of total anthocyanin content of wild type and IsMYBL3 gene overexpressed tobacco corolla. DETAILED DESCRIPTION

[0017] The following examples are used to further illustrate the technical solutions of the present application, but are not used to limit the protection scope of the present application.

[0018] The experimental methods used in the examples are conventional methods, and the results can be achieved by operating according to conventional molecular biology techniques by researchers in the field; the materials, reagents, etc. used in the examples can be obtained from commercial channels, unless otherwise specified.

[0019] The preparation methods of the related culture media used in the examples are as follows:

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

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

[0022] The anthocyanin content determination in the examples adopts the hydrochloric acid-ethanol extraction method, and the reference literature is (Tang M, Xue W, Li X, et al. Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum [J]. New Phytol, 2022, 236(3): 1075-1088.).

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

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

[0025] Cloning of IsMYBL3 gene. The cDNA of the Xishun flower petal was used as a template, and 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 was obtained after PCR amplification using TOYOBO KOD FX. Figure 1), PCR program annealing temperature 55℃, detailed method according to KOD FX instruction manual. Cloning vector selection blunt end cloning vector TRAN company -Blunt Zero Cloning Kit, 4 μL purified PCR amplified fragment mixed with 1 μL of -Blunt Zero Cloning Kit vector mixed, 30℃ connection 10 min, then 5 μL ligation product 42℃ transformed into E. coli competent DH5α, positive single colony picked and sent for testing and return plasmid.

[0026] Construction of plant overexpression vector pCAMBIA1300-IsMYBL3-GFP. Analysis design pCAMBIA1300-GFP vector seamless cloning of homologous arm sequence, according to the correct cloning of IsMYBL3 sequence design upstream homologous arm primer tIsMYBL3-F: 5'- ATGATGAGCCTAAGCTCAG-3', downstream homologous arm primer tIsMYBL3-R: 5'- TAATTCCAAGCATATATGATCAAT-3', the above primer retains pCAMBIA1300-GFP BamH I and Sal I restriction sites. Primer underlined indicates pCAMBIA1300-GFP homologous arm. TTGATACATATG CCCGTCGAC ATGATGAGCCTAAGCTCAG-3', downstream homologous arm primer tIsMYBL3-R: 5'- TAATTCCAAGCATATATGATCAAT-3', the above primer retains pCAMBIA1300-GFP BamH I and Sal I restriction sites. Primer underlined indicates pCAMBIA1300-GFP homologous arm. CCCTTGCTCACCATGG ATCC ATGATGAGCCTAAGCTCAG-3', downstream homologous arm primer tIsMYBL3-R: 5'- TAATTCCAAGCATATATGATCAAT-3', the above primer retains pCAMBIA1300-GFP BamH I and Sal I restriction sites. Primer underlined indicates pCAMBIA1300-GFP homologous arm.

[0027] The correct cloning plasmid identified by sequencing of IsMYBL3 of Isoetes sinensis was used as a template, and KOD FX was used for PCR amplification to obtain the target gene fragment with pC AMBIA1300-GFP vector homologous arm. The pCAMBIA1300-GFP vector plasmid was linearized using the restriction endonuclease BamH I and Sal I of Takara Company, and the linearized pCAMBIA1300-GFP vector was obtained. The pCA MBIA1300-IsMYBL3-GFP plant overexpression vector was constructed by using the homologous arm recombination method, the recombinase was Exnase II of Vazyme Company, the use amount of the vector and the target fragment was calculated according to the formula provided in the product manual of Exnase II, and after 37℃ connection for 30 min by using a PCR instrument, E. coli competent DH5α was transformed, and single colony picking was performed for bacterial liquid PCR and sequencing verification. The upstream verification primer was y1300-F: 5'-TTGATACATATGCCCGTCGAC-3', and the downstream verification primer was y1300-R: 5'-CCCTTGCTCACCATGGATCC-3', and finally the correct plant overexpression vector pCAMBIA1300-IsMYBL3-GFP was obtained. The recombinant plasmid pCAMBIA1300-IsMYBL3-GFP was transformed into the competent cells of Agrobacterium GV3101 by the freeze-thaw method.

[0028] Example 2: Genetic transformation of tobacco by IsMYBL3 gene of Isoetes sinensis.

[0029] The Agrobacterium containing the pCAMBIA1300-IsMYBL3-GFP plant overexpression vector obtained in Example 1 was used for leaf disc genetic transformation of tobacco, and the specific method was as follows:

[0030] (1) The Agrobacterium GV3101 containing the recombinant plasmid was taken out from the-80℃ refrigerator, activated, and cultured in a 28℃ incubator in the dark for 48

[0031] h, and single colony picking was performed for PCR identification, and the correct bacterial liquid was further cultured in YEP liquid medium.

[0032] (2) The Agrobacterium was shaken to OD 600 0.7, and then used for infection.

[0033] (3) The well-growing tobacco sterile seedlings were selected, the tender leaves were cut into 1cm 2 square leaves, and then placed on MS solid medium (containing 20g·L -1 sucrose+1mg·L -1 6-BA+0.05mg·L -1 NAA) for light culture for 2d.

[0034] (4) Collect the bacteria liquid, resuspend with resuspension liquid (1 / 2MS+20g·L -1 sucrose), then immerse the pre-cultured tobacco leaves in the resuspension liquid and shake for 5 minutes, dry on filter paper after infection, and then re-cultivate in the medium for 2 days in the dark.

[0035] (5) Place the leaves on the screening medium and cultivate under light, replace the medium every 15 days, and the medium composition is MS+1mg·L -1 6BA+0.05mg·L

[0036] 0.05mg·L -1 NAA+20mg·L -1 Hyg+200mg·L -1 Timentin.

[0037] (6) When the leaves differentiate into adventitious buds, cut off the adventitious buds and transfer them to 1 / 2MS solid medium (containing 20g·L -1 sucrose+25mg·L -1 Hyg+200mg·L -1 Timentin) for rooting culture.

[0038] (7) Move the rooted tobacco seedlings to nutrient soil for further culture, cut off the leaves to extract DNA for PCR verification, and continue to culture the positive tobacco seedlings until the seeds are harvested, continue to screen the next generation of positive plants, and continue to T3 generation of positive plants.

[0039] Example 3: Comparison of overexpression IsMYBL3 tobacco and control tobacco flowers.

[0040] Harvested tobacco seeds are sterilized and sown on 1 / 2MS screening medium containing 25mg·L -1 Hyg, the sterilization method is: sterilized with 75% alcohol for 1 minute, then washed with sterile water for 3 times, then oscillated with 2% sodium hypochlorite solution for 10 minutes, and finally washed with sterile water for 5 times; after being vernalized at 4°C for 2 days, then normally cultured in the tissue culture room, when the tobacco grows four leaf blades, move to nutrient soil (grass carbon soil: vermiculite = 3:1) for culture, the light environment is light / dark 14h / 10h, water once a week, and observe the color change of tobacco. The results show that the flower color of IsMYBL3 gene transformed tobacco is significantly lighter than that of wild type tobacco, the flower color of IsMYBL3 gene transformed tobacco is white( Figure 2 ), the total anthocyanin content of the flower crown is significantly lower than that of wild type tobacco( Figure 3 ), which shows that IsMYBL3 gene can effectively inhibit the synthesis of anthocyanin in plants.

[0041] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. It is intended that the scope of the application be limited not with this detailed description, but rather by the claims appended hereto.

Claims

1. A Isoetes IsMYBL3 transcription factor, characterized in that, The amino acid sequence is SEQ ID NO.

2.

2. A gene encoding the IsMYBL3 transcription factor of the S. japonicum according to claim 1. 3.The gene encoding the IsMYBL3 transcription factor of S.ichinensis according to claim 2, wherein, The nucleotide sequence is SEQ ID NO.

1.

4. The IsMYBL3 transcription factor of the S. japonicum according to claim 1 or the gene encoding the same according to claim 2 is applied to inhibit anthocyanin synthesis of the S. japonicum, and the application is overexpression of the IsMYBL3 transcription factor of the S. japonicum to inhibit anthocyanin synthesis.

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