Method for increasing anthocyanin content of purple sweet potato tuberous roots

Abstract

The invention relates to the technical field of agricultural biology, and discloses a method for increasing the anthocyanin content of purple sweet potato tuberous roots. According to the method, the endogenous IbMYB27 gene of the purple sweet potato is subjected to interference or knockout or expression weakening, so that the genetic engineering purple sweet potato with the root tuber anthocyanin content increased is obtained. Researches show that the IbMYB27 gene interferes and expresses that the content of anthocyanin in sweet potato peel is 179.2 mg / 100g, the content of anthocyanin in sweet potato pulp is 35.41 mg / 100g, and the content of anthocyanin in sweet potato peel and the content of anthocyanin in sweet potato pulp are obviously higher than those of an Aya wild type. The results show that the IbMYB27 can inhibit the biosynthesis of the anthocyanin induced by the IbMYB1.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a method for increasing the anthocyanin content of purple sweet potato root tubers. Background Art

[0002] Sweet potato is the seventh largest food crop in the world. It is rich in starch, vitamins, minerals and other nutrients. It is also an important industrial raw material. It has the advantages of high yield, strong adaptability, simple reproduction and extensive cultivation. In addition to the nutrients of ordinary sweet potatoes, purple sweet potatoes are also rich in anthocyanins, and have become an important natural pigment source and nutritional health food. Studies have shown that purple sweet potato anthocyanins have the strongest antioxidant and free radical scavenging functions (Furuta et al., 1998). Anthocyanins have a good effect in the prevention and treatment of cardiovascular diseases and anti-tumor (Wang Guanlin et al., 2005). In addition, sweet potato anthocyanins also have many functions such as maintaining blood sugar balance (Jiang et al., 2018), improving liver function (Wang et al., 2017), and inhibiting bacterial growth (Wang Guanlin et al., 2005). Compared with other crops containing anthocyanins, purple sweet potatoes have high yield, strong adaptability, high anthocyanin content, high anthocyanin yield, low cost, and good light stability and thermal stability of anthocyanins (Fang Zhongxiang et al., 2002; Xu et al., 2015), and have broad application prospects in the food, cosmetics and pharmaceutical industries (Li et al., 2019). However, the anthocyanin content of purple sweet potatoes is 32-1390 mg / 100 g DW (Xu et al., 2015; Ji et al., 2015), which is relatively low compared to plants such as blueberries, and the high cost of deep processing limits its further application.

[0003] References Furuta, S., Suda, I., & Nishiba, Y.. (1998). High tert-butylperoxylradical scavenging activities of sweet potato cultivars with purple flesh. Food Science and Technology International, (1), 4. Jang, H. H., Kim, H. W., Kim, S. Y., Kim, S. M., Kim, J. B., & Lee,Y. M.. (2018). In vitro and in vivo hypoglycemic effects of cyanidin 3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside, an anthocyanin isolatedfrom purple-fleshed sweet potato. Food Chemistry, 272(JAN.30), 688-693. Ji, H., Zhang, H., Li, H., & Li, Y.. (2015). Analysis on thenutrition composition and antioxidant activity of different types of sweetpotato cultivars. Food & Nutrition Sciences , 6(1), 161-167. Li, A., Xiao, R., He, S., An, X., He, Y., & Wang, C., et al. (2019).Research advances of purple sweet potato anthocyanins: extraction,identification, stability, bioactivity, application, and biotransformation. Molecules , 24(21). Liu, Y., Lin-Wang, K., Espley, R. V., Wang, L., Yang, H., & Yu, B.,et al. (2016). Functional diversification of the potato r2r3 myb anthocyaninactivators an1, myba1, and myb113 and their interaction with basic helix-loop-helix cofactors. Journal of experimental botany , 67(8), 2159-76. Wang, L., Zhao, Y., Zhou, Q., Luo, CL, Deng, AP, & Zhang, ZC, et al. (2017). Characterization and hepatoprotective activity ofanthocyanins from purple sweet potato (ipomoea batatas l. cultivar eshu no.8). Journal of Food and Drug Analysis , 25(3), 607-618. Xu, J., Su, Food Chemistry , 186(), 90–96. Fang Zhongxiang, Ni Yuanying, Li Hongmin. (2002). Study on the stability of anthocyanins in purple sweet potatoes under different environmental conditions. Food and Fermentation Industries (10), 31-34. Sun Rongqin & Guo Huachun. (2008). Comparison of anthocyanin content in roots of seven purple sweet potato varieties. Crop Journal (06), 56-59. Wang Guanlin, Yue Jing, Li Hongyan, Fang Hongjun. (2005). Extraction of anthocyanins from sweet potatoes and analysis of their antibacterial effects. Chinese Agricultural Science (11), 178-183. Summary of the invention

[0004] The inventor's previous research shows that the synthesis of anthocyanins in purple sweet potato tubers is regulated by the activation of IbMYB1, but IbMYB1 not only regulates the expression of anthocyanin synthesis structural genes, but also regulates the expression of IbMYB27, and IbMYB27 in turn inhibits the activation function of IbMYB1, reducing the synthesis of anthocyanins, so that cells do not synthesize too much anthocyanins, so that anthocyanins in the body are maintained at a reasonable level. Therefore, interfering with the expression of IbMYB27 can increase the synthesis of anthocyanins and improve the content of anthocyanins in tubers.

[0005] The technical solution provided by the present invention is: A method for increasing the anthocyanin content of purple sweet potato roots, which comprises interfering with, knocking out or weakening the expression of the endogenous IbMYB27 gene of the purple sweet potato to obtain a genetically engineered purple sweet potato with increased anthocyanin content in the roots; The amino acid sequence encoded by the IbMYB27 gene is shown in SEQ ID NO: 2.

[0006] Specifically, the nucleotide sequence of the IbMYB27 gene is shown in SEQ ID NO:2.

[0007] More specifically, it uses a gene editing method to knock out the endogenous IbMYB27 gene in the purple sweet potato; or interferes with the expression of the endogenous IbMYB27 gene in the purple sweet potato through a gene interference method.

[0008] In a specific embodiment, the target of interference is 1-351 bp, including the R2 domain and the R3 domain.

[0009] In a specific embodiment, the steps of the specific interference method are as follows: first, clone the 1-351bp forward sequence of the IbMYB27 gene with BamHI (5') and SpeI (3') restriction sites and the 1-351bp reverse sequence of the IbMYB27 gene with SacI (5') and KpnI (3') restriction sites respectively; then, connect the two fragments into the interference expression vector pCAMBIA1300-35S-X respectively through enzyme digestion and recombination; finally, transform the constructed vector into the purple sweet potato variety Aya through Agrobacterium-mediated method to obtain transgenic plants with higher tuber anthocyanins of IbMYB27 interference expression.

[0010] The present invention provides application of endogenous IbMYB27 gene of purple sweet potato in increasing anthocyanin content of purple sweet potato root tubers; The amino acid sequence encoded by the IbMYB27 gene is shown in SEQ ID NO: 2.

[0011] Specifically, the nucleotide sequence of the IbMYB27 gene is shown in SEQ ID NO:2.

[0012] More specifically, the application is achieved by interfering with, knocking out or expressing a weakened endogenous IbMYB27 gene in purple sweet potato using a genetic engineering method.

[0013] The present invention shows that the anthocyanin content in the sweet potato peel with IbMYB27 gene interference expression is 179.2 mg / 100 g, and the anthocyanin content in the potato flesh is 35.41 mg / 100 g, both significantly higher than that of the wild type of Aya. The above results indicate that IbMYB27 can inhibit the anthocyanin biosynthesis induced by IbMYB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Stable transformation of Arabidopsis thaliana verified the function of IbMYB27.

[0015] Figure 2 Schematic diagram of restriction sites of pCAMBIA1300-35S-X vector.

[0016] Figure 3 For callus induction.

[0017] Figure 4 For the propagation of embryonic callus.

[0018] Figure 5 Callus suspension culture.

[0019] Figure 6 Agrobacterium infection (co-cultivation).

[0020] Figure 7 Screening for callus resistance.

[0021] Figure 8 For resistance callus regeneration.

[0022] Fig. 9 For transgenic seedlings.

[0023] Fig.10 This is the identification diagram of Ri-IbMYB3 / pCAMBIA1300-X transgenic line L1 (M is DL2000).

[0024] Fig.11 These are the phenotypes of wild-type Aya plants and IbMYB27-interfered transgenic plants. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] Example 1: Function of IbMYB27 in overinhibiting anthocyanin synthesis 1 g of fresh potato flesh from wild-type Aya plants was taken and quickly ground into dry powder by adding liquid nitrogen. Total RNA was extracted using a kit method (Beijing Qingke, China). Reverse transcription was used to synthesize cDNA (Beijing Tiangen, China). IbMYB27-RFP-F and IbMYB27-RFP-R were used as cloning primers, and cDNA was used as a template to clone the full-length IbMYB27 fragment. The homologous recombination kit method (Beijing Zhuangmeng, China) was used to construct an overexpression vector containing IbMYB27, which was then transferred into GV3101 Agrobacterium competent cells for use (Beijing Qingke, China). The same steps were used to construct Agrobacterium containing an overexpression vector containing IbMYB1.

[0027] SEQ ID NO: 1 (IbMYB27 gene sequence, 516 bp) ATGAGGAAGGCTTCCTGCGACCATTCTCATCATCATCATGAGATCAACAAGGGTGCTTGGTCCAAGCAGGAAGACCAGAAGCTCCTAGATTACATCCGAAAACACGGCGAGGGCGGCTGGCGTGACCTGCCCAAGGCCGCAGGTCTGCTTCGTTGCAGTAAAAGTTGTAGGCTTAGATGGATGAATCATTTAAAGCAAACTGCCAAACGAGGGAACTTTGGTGATGATGAGGAGGATCTCATCATCAAGCTTCATGCC TTGCTTGGCGACAGGTGGTCGTTGATCGCCGGGAGATTGCCGGGGCGCACCGATGAAGAAGTGAAGAATTACTGGAATTCCCATATCAAAAAAAAGCTTTTGGATATGGGAATTGATCCTAATAATCAC CGCCTTTCTTGCACTCATAACACcgccgccCAaacTTCGGGCGGGAAGAGCCGTGTTACTTCGCCGGAAAAGCAACGGGTCGAGAGCGACGGTGAAGTTTCAGATGCCGGAAGTAGTAACGTCCGTTAG.

[0028] SEQ ID NO: 2 (amino acid sequence encoded by IbMYB27 gene) MRKASCDHSHHHHEINKGAWSKQEDQKLLDYIRKHGEGGWRDLPKAAGLLRCSKSCRLRWMNHLKQTAKRGNFGDDEEDLIIKLHALLGDRWSLIAGRLPGRTDEEVKNYWNSHIKKKLLDMGIDPNNHRLSCTHNTAAQTSGGKSRVTSPEKQRVESDGEVSDAGSSNVR*.

[0029] Vector construction information:

[0030] Will have IbMYB27 Agrobacterium overexpressing the vector was transformed into Arabidopsis thaliana to obtain IbMYB27 Arabidopsis thaliana, the specific steps are as follows: Preliminary preparations: Grow Arabidopsis thaliana (Columbia wild type) seedlings in advance, select strong and uniform seedlings and transplant them into small pots, unify water and fertilizer management, and grow them in an artificial climate room with low light and short daylight.

[0031] Select wild-type Arabidopsis plants that have fruit pods and are growing healthily, cut off the fruit pods, and water them thoroughly overnight.

[0032] (1) Thaw the Agrobacterium solution stored in a -80 ℃ refrigerator in advance and spread it on an LB plate containing Rif (50 mg / L) and Kan (50 mg / L) antibiotics. Incubate the plate in a 28 ℃ incubator to grow Agrobacterium colonies.

[0033] (2) Select a single Agrobacterium colony and place it in LB culture medium with Rif and Kan antibiotics. Shake the culture overnight at 28°C and 180 rpm until the OD600 value reaches 1.5.

[0034] (3) Centrifuge and discard the supernatant. Pour the resulting bacterial cells into a clean culture dish and adjust the OD600 value to about 0.8 with the staining solution (see Table 1 for the preparation of the staining solution). Leave it at room temperature for 30 min and resuspend it. The co-transduced bacterial liquids are cultured separately, and the centrifuged bacterial cells are poured into a culture dish and resuspended at the same time.

[0035] (4) Immerse the Arabidopsis buds in the staining solution for about 15 seconds, but no longer than 30 seconds. If the staining is prolonged, SilwetL-77 may cause seedling burn or pollen fertility loss.

[0036] (5) Place the infected Arabidopsis plants in a plastic bag with holes punctured in it and place the plants horizontally on a tray. After culturing in the dark for 24 hours, take them out and culture them under normal light.

[0037] (6) Depending on the growth of the plant, infect once every 5-7 days, usually 2 to 3 times, to infect all inflorescences. 0 Store the seeds in a 4°C refrigerator until ready for use.

[0038] Table 1 Infection solution formula

[0039] Experimental Results Agrobacterium transformed with P1300-IbMYB1 and P1300-IbMYB27 plasmids were used to transform Arabidopsis thaliana either alone or in a 1:1 ratio. The phenotypes of the transformed strains identified by screening were quite different (see Figure 1 ). IbMYB1-transformed Arabidopsis plants showed obvious anthocyanin accumulation. The biggest difference between IbMYB27-transformed Arabidopsis plants and wild-type Arabidopsis was the disappearance of pigmentation at the base of the stem and the absence of epidermal hairs. Co-transformed Arabidopsis plants also accumulated a large amount of anthocyanins, but the overall color was lighter than that of IbMYB1-transformed plants, and the anthocyanin distribution in the plants was uneven. These results indicate that IbMYB27 can inhibit anthocyanin biosynthesis induced by IbMYB1.

[0040] IbMYB1 and IbMYB27 As an important gene in the synthesis of anthocyanins in sweet potato tubers, it can also affect the synthesis of proanthocyanidins in the seed coat after transformation into wild-type Arabidopsis. Figure 2 It can be seen that the seed coat of wild-type Arabidopsis is yellow-brown, and a large amount of proanthocyanidins accumulate in the seed coat of IbMYB1 transgenic Arabidopsis plants, showing a dark brown color, which is darker than that of wild-type Arabidopsis. The seed coat of IbMYB27 transgenic Arabidopsis plants is light yellow, and the overall color is lighter. The seed coat of the co-transformed plants is uneven in color, with different shades, and has the phenotypes of IbMYB1 and IbMYB27 transgenic Arabidopsis seeds. These results indicate that IbMYB27 can inhibit the biosynthesis of proanthocyanidins induced by IbMYB1.

[0041] Example 2: IbMYB27 interferes with the function of promoting anthocyanin synthesis The vector synthesis was completed by Xuzhou Sanshu Biological Company. The specific vector information is as follows:

[0042] The schematic diagram of the restriction sites of the pCAMBIA1300-35S-X vector used in this experiment is as follows Figure 2 shown.

[0043] The purple sweet potato Ayazi was transformed with the IbMYB27 interference expression vector to obtain transgenic sweet potato plants. The specific steps are as follows: 1. Obtaining sterile seedlings (1) Take an appropriate amount of wild-type explant stems, place them in a sterilized glass bottle, shake and wash them once with sterile water, pour out the liquid, add 75% alcohol, and shake and wash for 15 seconds.

[0044] (2) Pour off the alcohol, wash once with sterile water, add 0.1% HgCl2 and wash for 2 minutes by shaking. Pour off the HgCl2.

[0045] (3) Wash three times with sterile water.

[0046] (4) Inoculate the plants on Dio 1 medium (in culture bottles) and subculture every 5 weeks.

[0047] 2. Wound healing induction (1) Under a dissecting microscope, remove the axillary buds of the sterile seedlings and place them on Dio2 medium ( Figure 3 ), subculture once every 5 weeks. If embryonic callus is found, transfer it to Dio3 medium for subculture and expand it every 4 weeks ( Figure 4 ).

[0048] 3. Suspension culture The sweet potato callus propagated on Dio3 medium was crushed through a 10-mesh sieve, and 30 ml of Dio4 medium was added to each bottle. The culture was cultured on a shaker at 150 rpm, and the medium was changed every 3 days ( Figure 5 ).

[0049] 4. Agrobacterium Preparation (1) Activation: Take Agrobacterium stored at -80℃ and streak it on a resistant medium (YEB+Rif+Chl+Kan). Incubate it upside down at 28℃ for 36-48h.

[0050] (2) Small shaking: Pick a plump single colony and add it to a 50 ml centrifuge tube containing 5 ml of liquid resistance culture medium. Incubate at 28°C at 200 rpm / min for 16-24 hours.

[0051] (3) Large shaking: Pipette 1 ml of the bacterial solution from the small shaking tube and inoculate it into a conical flask containing resistance culture medium at a ratio of 1:50~1:100. Incubate at 28℃ 200rpm for 8~14h or overnight until OD600 = 1.0~1.2.

[0052] (4) Collect bacteria: Transfer the bacterial solution to a sterilized 50 ml centrifuge tube and centrifuge at 4000 rpm for 10 min. Discard the supernatant, add M medium, centrifuge at 4000 g for 10 min. Discard the supernatant, add M+AS medium to suspend the bacterial solution to an OD600 value of OD600 = 0.8, and set aside (can be cultured at 80 rpm).

[0053] 5. Agrobacterium infection (1) Take the suspension culture callus, drain the liquid, pour the callus into the bacterial solution, and culture at 80 rpm for 30 min. Figure 6 ).

[0054] (2) Place the infected callus on sterile filter paper, absorb the bacterial solution, and spread it on Dio5 medium. Incubate at 22℃ under weak light for three days.

[0055] 6. Screening and regeneration (1) Move the callus to a sterile culture bottle, wash it 2-3 times with Dio4+cef (500 mg / L), dry it with sterile filter paper, and transfer it to Dio6 medium. Culture at 28℃, 16 / 8h light intensity ( Figure 7 ).

[0056] (2) When the negative callus tissue is killed and the positive callus survives, transfer the positive callus to Dio7 medium and continue culturing. Change the medium every 4 weeks. Figure 8 ).

[0057] (3) After the callus tissue grows obvious small buds, transfer the material to Dio8 medium. Change the medium every 4 weeks ( Fig. 9 ).

[0058] 7. Culture medium preparation (per liter) YEB medium: 1g yeast extract, 5g beef extract, 5g sucrose, 0.5g MgSO4•7H2O, 15g agar, pH 7.2. Liquid M: 4.43g MS salts and vitamins (PhytoTech, #M519) + 30g sucrose, pH 5.7.

[0059] Dio 1 for tissue culture seedling propagation: MS salt and vitamins 4.43g + sucrose 30g + agar powder 7g, pH 5.8.

[0060] Callus induction Dio2: MS salt and vitamins 4.43g + sucrose 30g + agar powder 7g + 2,4-D 2mg, pH 5.8.

[0061] Callus propagation Dio3: MS salt and vitamins 4.43g + sucrose 30g + agar powder 7g + 2,4-D 2mg, pH 5.8.

[0062] Suspension culture Dio4: MS salts and vitamins 4.43 g + sucrose 30 g + 2,4-D 2 mg + 6-BA 0.1 mg, pH 5.8.

[0063] Co-culture medium Dio 5: MS salt and vitamin 4.43g + sucrose 30g + agar powder 7g + 2,4-D 2mg + 6-BA 0.1mg, pH 5.3, add 200uM AS after sterilization.

[0064] Screening medium Dio 6: MS salt and vitamin 4.43g + sucrose 30g + agar powder 7g + 2,4-D 2mg + 6-BA0.1mg, pH 5.8, add Tim 200mg, Hgy50mg after sterilization.

[0065] Regeneration medium Dio 7: MS salt and vitamin 4.43g + sucrose 30g + agar powder 7g, pH 5.8, add Tim 200mg, Hgy 50mg after sterilization.

[0066] Rooting medium Dio 8: MS salt and vitamin 4.43g + sucrose 30g + agar powder 7g, pH 5.8, add Tim 200mg, Hgy 50mg after sterilization.

[0067] NOTE: All antibiotics and hormones are at final concentrations.

[0068] 8. Positive verification Take 1g of fresh potato flesh from wild-type Aya and IbMYB27 gene interference expression plants, add liquid nitrogen and grind into dry powder quickly, and extract total DNA using kit method (Beijing Qingke, China). Use Master Mix (Beijing Qingke, China) to run PCR to detect positive plants. PCR system 20 ul:

[0069] The PCR program is as follows:

[0070] Agarose gel electrophoresis results ( Fig.10 ). As shown in the figure, IbMYB1-3 bands were detected in the IbMYB27 gene interference expression plants, while no bands were found in the control and wild type. This indicates that the IbMYB27 gene interference vector has indeed been transferred into the plant genome.

[0071] 9. Phenotypic identification of transgenic plants The transgenic plants were planted in pots and the phenotypes of the wild-type and IbMYB27 gene interference expression plants were observed, especially the color of the plants and tubers ( Fig.11), and the anthocyanin content of the root skin and flesh was determined (Table 1). The extraction and quantification of anthocyanins were based on the method described by Sun (Sun Rongqin, 2008). The skin and flesh of wild-type sweet potatoes and sweet potatoes with IbMYB27 gene interference expression were purple, with obvious anthocyanin accumulation, and the purple color of IbMYB27 gene interference expression plants was darker than that of the wild type. The stem color of the aboveground IbMYB27 gene interference expression plants was purple, while the stem color of the wild-type plants was green. We further determined the anthocyanin content of the wild-type and IbMYB27 gene interference expression plants. The results are shown in Table 1.

[0072] Table 1 Anthocyanin content in the root tubers of Aya wild type and IbMYB27-interfered transgenic plants

[0073] The results showed that the anthocyanin content in the skin of wild-type Aya was 33.94 mg / 100 g, and the anthocyanin content in the flesh was 18.13 mg / 100 g; while the anthocyanin content in the skin of sweet potatoes with IbMYB27 gene interference expression was 179.2 mg / 100 g, and the anthocyanin content in the flesh was 35.41 mg / 100 g, both significantly higher than that of wild-type Aya. The above results indicate that IbMYB27 can inhibit the anthocyanin biosynthesis induced by IbMYB1.

Claims

1. A method for increasing the anthocyanin content of purple sweet potato root tubers, characterized in that: The method involves interfering, knocking out or weakening the expression of the endogenous IbMYB27 gene of purple sweet potato to obtain a genetically engineered purple sweet potato with increased anthocyanin content in the root tuber; The amino acid sequence encoded by the IbMYB27 gene is shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that The nucleotide sequence of the IbMYB27 gene is shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that The method uses a gene editing method to knock out the endogenous IbMYB27 gene of the purple sweet potato; or uses a gene interference method to interfere with the expression of the endogenous IbMYB27 gene of the purple sweet potato.

4. The method according to claim 3, characterized in that The target of interference is 1-351 bp, including the R2 domain and the R3 domain.

5. The method according to claim 4, characterized in that The specific interference method steps are as follows: first clone the 1-351bp forward sequence of the IbMYB27 gene with BamHI (5') and SpeI (3') restriction sites and the 1-351bp reverse sequence of the IbMYB27 gene with SacI (5') and KpnI (3') restriction sites respectively; then connect the two fragments into the interference expression vector pCAMBIA1300-35S-X by enzyme digestion and recombination; finally, transform the constructed vector into the purple sweet potato variety Aya through Agrobacterium-mediated method to obtain transgenic plants with higher tuber anthocyanins of IbMYB27 interference expression.

6. Application of endogenous IbMYB27 gene in purple sweet potato to increase anthocyanin content in purple sweet potato root tubers; in, The amino acid sequence encoded by the IbMYB27 gene is shown in SEQ ID NO:

2.

7. The use according to claim 6, characterized in that The nucleotide sequence of the IbMYB27 gene is shown in SEQ ID NO:

2.

8. The use according to claim 7, characterized in that The application is achieved by interfering with, knocking out or expressing a weakened endogenous IbMYB27 gene of purple sweet potato through a genetic engineering method.

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