Gene for regulating and controlling taro flavone biosynthesis and application thereof

Abstract

The invention discloses a gene for regulating and controlling taro flavone biosynthesis and application thereof, and belongs to the technical field of biology. The gene comprises a CeHCT1 gene and a CeHCT2 gene; the nucleotide sequence of the CeHCT1 gene is as shown in SEQ ID NO. 9; and the nucleotide sequence of the CeHCT2 gene is as shown in SEQ ID NO. 10. The invention also provides application of the gene in regulation and control of taro flavone content and improvement of plant nutritional quality. The CeHCT1 and / or CeHCT2 genes of the main cultivated variety Xinhua Longxiang taro are / is subjected to multi-target knockout through a gene editing technology, the flavonoid content in the taro can be effectively increased, the nutritional quality of the taro is efficiently enhanced, and the method has a great significance in accelerating development and utilization of taro genes and has a great application prospect. The method for improving the nutritional quality of the taros by applying a biological enhancement technology has important theoretical and practical significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a gene for regulating taro flavonoid biosynthesis and an application thereof. Background Art

[0002] Taro is a perennial herb of the genus Taro of the Araceae family, often cultivated as an annual. The corms, flowers, petioles and other parts of taro are edible, playing a vital role in food security in many countries. In view of the importance of taro in food security and food health, previous research in this field has mainly focused on starch, protein, non-starch polysaccharides, vitamins and mineral elements in taro corms, but there are few research reports on taro regarding important functional factors such as bioflavonoids. Existing studies have shown that flavonoids play an important role in the nutritional fortification of tuber crops such as sweet potatoes, potatoes, and cassava. Therefore, studying the nutritional fortification technology of taro, increasing the flavonoid content in taro corms, and improving the nutritional quality of taro also provide technical support for the further development and utilization of functional taro products with deep processing potential.

[0003] Taro is a crop that mainly reproduces asexually. The flowering of taro depends not only on suitable environmental conditions, but also on the type of variety, the state of the mother taro and the child taro, and the stage of vegetative growth. Therefore, taro rarely blooms under natural conditions, which limits the sexual hybridization of taro. At present, it is difficult to achieve gene transfer in taro through traditional hybridization. Gene editing technology is precisely the most effective way to overcome this problem. Synthetic mutations through gene editing technology can fill in and replace natural genetic mutations in some cases, modify target genes efficiently and accurately, and improve the efficiency of genetic improvement. However, this technology has not yet been applied to the genetic improvement of taro. And because the genome is large and heterozygous, the research on taro at the molecular level is relatively lagging. In the past two decades, taro genetic research has mainly focused on diversity analysis and genetic positioning, and taro genomics research is still in its infancy. Summary of the invention

[0004] To solve the above technical problems, the present invention proposes a gene for regulating the biosynthesis of taro flavonoids and its application. The results of the present invention show that the flavonoid content in the knockout mutant of the CeHCT1 gene is 1.79-1.87 times that of the wild type, and the flavonoid content in the knockout mutant of the CeHCT2 gene is 2.78-3.50 times that of the wild type.

[0005] The present invention provides the following technical solutions:

[0006] Technical solution 1: A gene for regulating the biosynthesis of taro flavonoids, the gene includes CeHCT1 gene and CeHCT2 gene; the nucleotide sequence of the CeHCT1 gene is shown in SEQ ID NO.9; the nucleotide sequence of the CeHCT2 gene is shown in SEQ ID NO.10.

[0007] Furthermore, the nucleotide sequence of the gene also includes a nucleotide sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID NO.9 or SEQ ID NO.10, and also includes mutants, alleles or derivatives generated by adding, substituting, inserting or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.9 or SEQ ID NO.10.

[0008] Technical solution 2: Application of the gene in regulating the flavonoid content of taro; the regulation is negative regulation.

[0009] The present invention verifies that the CeHCT1 and CeHCT2 genes in taro can negatively regulate the biosynthesis of taro flavonoids, and by using gene editing technology, the CeHCT1 and / or CeHCT2 genes of the main cultivated variety Xinghua Longxiang taro are knocked out, which can effectively increase the flavonoid content in the taro corm.

[0010] Technical solution three: Application of the gene in improving the nutritional quality of plants. The nutritional quality includes increasing the flavonoid content in taro.

[0011] Furthermore, the plant includes taro or Arabidopsis thaliana.

[0012] Technical Solution 4: A method for increasing the flavonoid content of taro, comprising the step of performing multi-target knockout on the CeHCT1 and / or CeHCT2 gene.

[0013] Technical solution 5: A genetic transformation technology for taro, comprising the step of transferring the taro explants into an infection solution of Agrobacterium tumefaciens EHA105 for incubation to obtain infected explants.

[0014] The present invention transfers CRISPR-Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP of CeHCT1 gene and CeHCT2 gene into taro, and identifies and obtains 4 knockout mutant materials of HCT gene (CeHCT1 gene and CeHCT2 gene) of taro.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] The present invention discloses structural genes CeHCT1 and CeHCT2 related to flavonoid metabolism in taro corms, and accelerates the development and utilization of taro genes. The present invention applies gene editing technology to create knockout mutant materials of CeHCT1 and CeHCT2 genes, screens and obtains stable genetic materials, and provides technical support for the subsequent development and utilization of genes and the genetic improvement of taro. The present invention also uses the Longxiang taro genetic transformation technology to transfer the CRISPR-Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP of CeHCT1 and CeHCT2 genes into Longxiang taro, and identifies and obtains 4 knockout mutant materials of Longxiang taro HCT genes. The color of the roots and petioles of the knockout mutant material of the HCT gene of the dragon taro deepened. Further testing found that flavonoids were significantly enriched in the roots of the mutants. The flavonoid content in the knockout mutant of the CeHCT1 gene was 1.79-1.87 times that of the wild type, and the flavonoid content in the knockout mutant of the CeHCT2 gene was 2.78-3.50 times that of the wild type.

[0017] The present invention performs biofortification on taro with strong pertinence and high fortification efficiency, and at the same time provides new ideas and methods for the biofortification of taro and other crops, and efficiently enhances the nutritional quality of taro, which has important theoretical and practical significance for improving the nutritional quality of taro. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Phylogenetic analysis of CeHCT1 and CeHCT2 proteins;

[0020] Figure 2 Prediction of the secondary structure of CeHCT1 protein;

[0021] Figure 3 Prediction of the secondary structure of CeHCT2 protein;

[0022] Figure 4 is the tissue expression profile of CeHCT1 and CeHCT2 genes;

[0023] Figure 5 This is the plasmid map of the pHG expression vector of CeHCT1 gene;

[0024] Figure 6This is the plasmid map of the pHG expression vector of CeHCT2 gene;

[0025] Figure 7 For subcellular localization analysis of CeHCT1 and CeHCT2 genes;

[0026] Figure 8 The electrophoresis diagram of HPT gene; M: DL 2,000 Marker; 1-8: CeHCT1 Arabidopsis transgenic plants; 9-16: CeHCT2 Arabidopsis transgenic plants; 17: wild type;

[0027] Fig. 9 is the expression level of CeHCT1 gene and CeHCT2 gene in Arabidopsis transgenic plants;

[0028] Fig.10 Phenotypic observation of Arabidopsis transgenic plants, A: wild type; B: CeHCT1 transgenic plants; C: CeHCT2 transgenic plants;

[0029] Fig.11 Comparison of total flavonoids content in transgenic Arabidopsis plants, where A: T1 stems and leaves; B: T2 leaves; C: T2 stems;

[0030] Fig.12 This is the CRISPR-Cas9 multi-target vector plasmid map of the CeHCT1 gene;

[0031] Fig.13 This is the CRISPR-Cas9 multi-target vector plasmid map of the CeHCT2 gene;

[0032] Fig.14 This is the electrophoresis diagram of HPT gene, where M: DL 2,000 Marker; 1: wild type; 2-9: CeHCT2 Longxiangtaro transgenic plants; 10-17: CeHCT1 Longxiangtaro transgenic plants;

[0033] Fig.15 is the target mutation type of the gene-edited plant;

[0034] Fig.16 Phenotypic observation of HCT gene knockout mutant plants of Longxiangtaro (roots), where A: wild type; B: CR-HCT1-2; C: CR-HCT2-1;

[0035] Fig.17 Phenotypic observation of HCT gene knockout mutant plants of Longxiangtaro (petiole), among which, A: wild type; B: CR-HCT1-2; C: CR-HCT2-1;

[0036] Fig.18Comparison of total flavonoids content in HCT gene knockout mutant plants of Longxiang Taro. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1 Materials and methods

[0044] 1.1 Test materials

[0045] The plant materials are taro varieties 'Xinghua Longxiangyu' (provided by Xinghua Meihua Vegetable Professional Cooperative) and taro new line 'Ty22-01' (bred by Taizhou Agricultural Science Institute of Jiangsu Academy of Agricultural Sciences); tobacco is Nicotiana benthamiana; Arabidopsis thaliana is Col-0 ecotype. The strains used are Escherichia coli DH5α and TOP10, Agrobacterium tumefaciens GV3101 and EHA105, which can be obtained commercially.

[0046] 1.2 Experimental processing and sample preparation

[0047] The total RNA of taro roots, stems, leaves and buds was extracted using a plant total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the total RNA of taro peel and taro flesh was extracted using a polysaccharide and polyphenol plant RNA extraction kit (Nanjing Jisi Huiyuan Biotechnology Co., Ltd.); the concentration and purity of the extracted total RNA were determined. After the test was qualified, 1 μL of total RNA was taken, and the genomic DNA was removed according to the instructions of the reverse transcription kit (Nanjing Novozyme Biotechnology Co., Ltd.) and then reverse transcription reaction was performed to obtain the corresponding cDNA, which was stored at -20°C and used as a template for vector construction and RT-qPCR.

[0048] 1.3 Bioinformatics analysis

[0049] The analysis and blast of CeHCT1 and CeHCT2 gene sequence information were completed using DNAMAN software. Phylogenetic tree analysis was completed using MEGA 7.0 software, using the neighbor-joining method, with Bootstrap set to 1000 times. The online software ProtParam (https: / / web.expasy.org / protparam / ) was used to analyze the protein sequences of CeHCT1 and CeHCT2. The online software SOPMA (https: / / npsa.lyon.inserm.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_sopma.html) was used to predict the secondary structures of CeHCT1 and CeHCT2 proteins.

[0050] 1.4 Vector construction

[0051] Using taro cDNA as template, PCR amplification of the full length of CeHCT1 and CeHCT2 gene ORF was performed using gene-specific primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 (see Table 1). 50 μL of PCR reaction system: 10×PCR Buffer 5 μL, dNTP 5 μL, forward and reverse primers 1.5 μL each, cDNA template 2 μL, DMSO 2 μL, KOD DNA polymerase 1 μL and ddH 2O 32μL. PCR reaction program: 94℃ pre-denaturation for 4min; 94℃ denaturation for 30s, 59℃ annealing for 30s, 68℃ extension for 2min, 40 cycles; 68℃ insulation for 8min. Purify and recover the amplified target gene fragment. The target vector pHG was double-digested with SacI and SpeI, and the CeHCT1 and CeHCT2 gene fragments were connected to the pHG vector by homologous recombination to obtain pHG-CeHCT1 and pHG-CeHCT2 recombinant plasmids. After detection, they were transferred into Agrobacterium tumefaciens GV3101 for use.

[0052] According to the GC abundance and the sequence corresponding to the protospacer adjacent motif (PAM) site, the specific guide RNA (sgRNA) on the targeting strand of CeHCT1 and CeHC T2 genes was selected, and its off-target effect was verified by the web server CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Specific primers SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 (see Table 1) were designed to PCR amplify the dual-target sgRNA fragments. The PCR reaction system and reaction procedure were the same as above. The amplified dual-target sgRNA fragments were connected to the pRGEB32-GFP binary vector digested with HindIII and SbfI to obtain the recombinant plasmids of CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP, which were transferred into Agrobacterium tumefaciens EHA105 for use after detection.

[0053] Table 1

[0054]

[0055] 1.5 Subcellular localization analysis

[0056] The subcellular localization of CeHCT1 and CeHCT2 proteins was analyzed by transient expression in tobacco leaf epidermal cells. Agrobacterium GV3101 containing pHG-CeHCT1 and pHG-CeHCT2 vector plasmids were activated and expanded, and the cells were collected and resuspended with a resuspension solution (containing 10 mmol / L MgCl 2 , 10mmol / L MES and 0.1mmol / L acetosyringone) and resuspended to OD 600nm The pHB-eGFP was transformed into leaves with an empty vector as control. GFP fluorescence was observed by laser confocal microscopy with an excitation wavelength of 488 nm and an emission wavelength of 507 nm.

[0057] 1.6 RT-qPCR analysis

[0058] The cDNA sample to be tested was diluted 10 times as a template, and the reagents were The first-strand cDNA synthesis ready-to-use premix of ⅢqPCR (Yisheng, China) was used for the quantitative PCR (BIOER, China) with the program set at 95℃ for 5min; 95℃ for 10s, 60℃ for 30s, 40 cycles; 72℃ for 30s. The internal reference gene was CeGAPDH. The relative expression of the target gene was expressed as 2 －ΔΔCt Method calculation.

[0059] 1.7 Arabidopsis genetic transformation

[0060] The plant expression vectors pHG-CeHCT1 and pHG-CeHCT2 were introduced into Agrobacterium tumefaciens competent cells GV3101, and positive clones were used for genetic transformation after identification by colony PCR. Arabidopsis thaliana (Col-0 ecotype) was transformed by the floral dipping method, and T0 generation seeds were harvested. After disinfection, the T0 generation seeds were sown on 1 / 2MS medium (containing 30μg / ml hygromycin) for resistance screening, and the genomic DNA of the resistant seedlings was extracted for PCR identification. The RNA of the obtained positive seedlings was further extracted and qRT-PCR was performed, and the seeds of the T1 generation positive plants were harvested. The T1 generation seeds continued to be screened using hygromycin resistance plates, and the resistant seedlings were transplanted into nutrient soil and further identified by PCR to obtain T2 generation positive plants. After growing to the flowering stage, the leaves and stems of the T2 generation positive plants were collected according to the T1 generation for the determination of physiological and biochemical indicators.

[0061] 1.8 Genetic transformation of Longxiang taro

[0062] The leaves of sterile seedlings of Longxiang taro were used as explants and cultured in a pre-cultured medium (component: 4.4 g﹒L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1 Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L -1 NAA) and then transferred to the infection medium of Agrobacterium tumefaciens EHA105 containing CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFPAS vectors for incubation. The infected explants were obtained and co-cultured for 3 days before being transferred to the resistance dedifferentiation medium (component 4.4 g﹒L -1 MS medium + 30g﹒L -1 Sucrose +7g﹒L -1Agar + 2 mg L -1 2,4-D+1mg﹒L -1 6-BA+0.5mg﹒L - 1 NAA+200mg﹒L -1 Glutamine +7.5mg﹒L -1 Hygromycin + 300mg﹒ L -1 Resistant calli were screened on Timentin, and the resistant calli differentiated into T0 generation transgenic plants. The stems, leaves and roots were taken for determination of relevant indicators.

[0063] 1.9 Determination of total flavonoid content

[0064] The sample was ground into powder with liquid nitrogen, about 0.1g of sample was weighed, and 1mL of 60% ethanol solution was added. Ultrasonic extraction was performed at 60℃ for 30min, and centrifuged at 8000g at 25℃ for 10min. The supernatant was diluted and tested. 108μL of the test solution was placed in a 96-well plate, 6μL of 5% sodium nitrite was added, mixed, and allowed to stand at room temperature for 6min; 6μL of 10% Al(NO 3 ) 3 Solution, mix well, let stand at room temperature for 6 min, then add 80 μL 4% NaOH solution, mix well, let stand at room temperature for 15 min, and then measure the absorbance at a wavelength of 510 nm.

[0065] 2 Results and analysis

[0066] 2.1 Cloning and sequence analysis of CeHCT1 and CeHCT2 genes

[0067] 2.1.1 Cloning of CeHCT1 and CeHCT2 genes

[0068] Based on the omics study of anthocyanin accumulation in taro corms, the present invention designed specific primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 (see Table 1) according to the candidate HCT gene sequences, and cloned two HCT genes, named CeHCT1 and CeHCT2, respectively. The full-length CDS of the CeHCT1 gene consists of 1314 bases, encoding 437 amino acids; the full-length CDS of the CeHCT2 gene consists of 1305 bases, encoding 434 amino acids.

[0069] Sequence information of CeHCT1 gene:

[0070]

[0071] Sequence information of CeHCT2 gene:

[0072]

[0073] 2.1.2 Sequence analysis of CeHCT1 and CeHCT2 genes

[0074] Phylogenetic analysis showed that CeHCT1 gene had high homology with HCT genes in species such as sunflower (QBM78938.1), lettuce (XP023733842.1), Xinjiang cirsium (QQH14907.1), cardoon (APT68193.1), etc.; CeHCT2 gene had high homology with HCT genes in plants such as aconite (XP018682107.1), thrush (KAF5177976.1), jujube (XP024928964.1), sesame (XP011082506.1), etc. The protein sequences of CeHCT1 and CeHCT2 were analyzed using online software ProtParam. The molecular formula of CeHCT1 protein was C 2143 H 3341 N 587 O 597 S 20 The molecular weight of CeHCT1 protein is 47521.88Da. CeHCT1 protein is composed of 20 amino acids, of which alanine is the most abundant (Ala, 12.1%) and tryptophan is the least abundant (Trp, 1.4%) (Table 2). The molecular formula of CeHCT2 protein is C 2162 H 3345 N 589 O 614 S 15 , with a molecular weight of 47893.82Da, the CeHCT2 protein is composed of 20 amino acids, of which leucine is the most abundant (Leu, 9.4%) and cysteine ​​is the least abundant (Cys, 0.9%) (Table 2).

[0075] Table 2

[0076]

[0077]

[0078] The online software SOPMA was used to predict the secondary structures of CeHCT1 and CeHCT2 proteins. The prediction showed that the main structures of the two proteins were α-helix, extended strand and random coil. α-helix, extended strand and random coil run through the entire amino acid chain (see Figure 2 and Figure 3 ).

[0079] 2.2 Analysis of tissue expression patterns of CeHCT1 and CeHCT2 genes

[0080] The tissue expression profiles of CeHCT1 and CeHCT2 genes ( Figure 4 and Figure 5 ) It can be seen that the expression levels and expression patterns of CeHCT1 and CeHCT2 genes are different in taro varieties with different flavonoid contents. The expression level of CeHCT2 gene in taro is higher than that of CeHCT1 gene; the overall expression level of the two genes in the variety 'Ty22-01' with high flavonoid content is higher than that in the variety 'Longxiangyu' with low flavonoid content.

[0081] In 'Ty22-01', the expression level of CeHCT1 gene was low overall, and the main expression sites were roots and petioles; except for the high expression level in roots, the expression level of CeHCT2 gene in other parts was also low overall. In 'Longxiangyu', CeHCT1 gene was mainly expressed in buds, leaves and roots, while CeHCT2 gene was mainly expressed in petioles, roots and taro flesh.

[0082] 2.3 Construction of binary expression vectors of CeHCT1 and CeHCT2 genes

[0083] In order to analyze the functions of CeHCT1 and CeHCT2 genes, the present invention uses the cloned full-length CDS of CeHCT1 and CeHCT2 genes as templates, designs specific primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 (see Table 1), amplifies the target fragment with SacI and SpeI restriction sites, inserts the target fragment into the pHG expression vector after double restriction digestion by homologous recombination, and constructs binary expression vectors pHG-CeHCT1 and pHG-CeHCT2, respectively, which carry the target gene and eGFP reporter gene ( Figure 5 and Figure 6 ).

[0084] 2.4 Subcellular localization analysis of CeHCT1 and CeHCT2 genes

[0085] In order to study the cytological functions of CeHCT1 and CeHCT2 proteins, the present invention uses transient transformation of tobacco leaves, and transforms tobacco leaves with Agrobacterium GV3101 carrying fusion GFP vectors of CeHCT1 and CeHCT2 (pHG-CeHCT1 and pHG-CeHCT1), and observes the fluorescence position by laser confocal microscopy, wherein the pHB-GFP empty vector is used as a control. The leaves transformed with the empty vector have strong green fluorescence in the nucleus, cytoplasm, and cell membrane, the leaves transformed with CeHCT1 have strong green fluorescence in the nucleus and weak green fluorescence in the cell membrane, and the leaves transformed with CeHCT2 protein have strong green fluorescence in the nucleus, cytoplasm, and cell membrane ( Figure 7These results indicate that CeHCT1 protein is mainly localized in the nucleus, and CeHCT2 protein is localized in the nucleus, cytoplasm, and cell membrane.

[0086] 2.5 Identification of CeHCT1 and CeHCT2 Transgenic Arabidopsis

[0087] The binary expression vectors pHG-CeHCT1 and pHG-CeHCT2 were transformed into Arabidopsis thaliana by Agrobacterium-mediated method to obtain transgenic Arabidopsis thaliana plants. DNA extracted from leaves of wild-type Arabidopsis thaliana and transgenic plants was used as template for PCR amplification. Figure 8 It can be seen that a single band of the hygromycin resistance gene HPT can be detected in all Arabidopsis transgenic plants, while there is no band in the wild-type plants.

[0088] qRT-PCR technology was further used to detect the expression levels of CeHCT1 and CeHCT2 genes. Fig. 9 It can be seen that CeHCT1 and CeHCT2 genes are not expressed in the leaves of Arabidopsis wild-type plants, but their expression levels are significantly increased in Arabidopsis transgenic plants. The above results all indicate that CeHCT1 and CeHCT2 genes have been successfully introduced into the model plant Arabidopsis.

[0089] 2.6 Effects of CeHCT1 and CeHCT2 transfection on Arabidopsis

[0090] 2.6.1 Effects of CeHCT1 and CeHCT2 transfection on Arabidopsis thaliana plant phenotypes

[0091] Observation of the phenotype of Arabidopsis transgenic plants at the seedling stage ( Fig.10 ), no obvious phenotypic differences were observed compared with the wild type.

[0092] 2.6.2 Effects of CeHCT1 and CeHCT2 transgenes on flavonoid content in Arabidopsis leaves

[0093] Three lines were selected from the transgenic offspring of CeHCT1 and CeHCT2 Arabidopsis thaliana, and the total flavonoid content in their leaves and stems was detected. Compared with the wild type, the total flavonoid content in the transgenic lines was significantly decreased ( Fig.11 ). The results showed that CeHCT1 and CeHCT2 genes inhibited the synthesis of flavonoids in the stems and leaves of Arabidopsis plants.

[0094] 2.7 Construction of CRISPR-Cas9 multi-target vectors for CeHCT1 and CeHCT2 genes

[0095] In order to obtain multi-target knockout mutants of CeHCT1 and CeHCT2 genes of taro, the present invention connected the amplified dual-target sgRNA fragments to the pRGEB32-GFP binary vector digested by HindIII and SbfI, and constructed CRISPR / Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP ( Fig.12 and Fig.13 ).

[0096] 2.8 Identification of knockout mutants of CeHCT1 and CeHCT2 genes in taro

[0097] The CRISPR-Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-pRGEB32-GFP of CeHCT1 and CeHCT2 genes were used to transform Longxiangtaro to obtain Longxiangtaro transgenic plants. After PCR detection, 14 Longxiangtaro transgenic positive plants were obtained, including 6 transgenic positive plants of CR-HCT1 and 8 transgenic positive plants of CR-HCT2 ( Fig.14 ). DNA was extracted from the leaves of the transgenic positive plants of Longxiangyu, and specific primers were designed to perform PCR amplification and sequencing of the target gene fragments. The site where gene editing occurred was determined by analyzing the sequencing results. Sequencing revealed that 4 of the 14 transgenic plants had an insertion or deletion of 1-4 bases at the target site sgR1, resulting in a frameshift mutation ( Fig.15 ).

[0098] 2.9 Analysis of changes in flavonoid content in knockout mutants of CeHCT1 and CeHCT2 genes

[0099] The phenotypes of the knockout mutants of CeHCT1 and CeHCT2 genes of Longxiangtaro were observed, and purple-red pigmentation appeared in the roots of the knockout mutants of CeHCT1 and CeHCT2, among which the pigmentation was more obvious in the knockout mutants of CeHCT2 ( Fig.16 ); In addition, purple-red deposits were also observed in the petioles of CeHCT2 knockout mutants ( Fig.17). Both hydroxycinnamoyltransferase (HCT) and chalcone synthase (CHS) can use coumaroyl-CoA as substrate to introduce phenylpropanoid metabolism into the lignin monomer synthesis and flavonoid synthesis pathways, respectively. In the HCT gene (CeHCT1 gene and CeHCT2 gene) knockout mutant of Longxiangtaro, HCT synthesis is blocked, and CHS uses coumaroyl-CoA as substrate to introduce phenylpropanoid metabolism into the flavonoid synthesis pathway to synthesize flavonoids. In view of this, the present invention determines the flavonoid content in the roots of the knockout mutants of the HCT gene of the dragon taro. The results show that the flavonoid content in the roots of the knockout mutants CR-HCT1-1 and CR-HCT1-2 of the dragon taro CeHCT1 gene and the knockout mutants CR-HCT2-1 and CR-HCT2-2 of the dragon taro CeHCT2 gene are significantly increased compared with the wild type. The flavonoid content of CR-HCT1-1 is 1.79 times that of the wild type, the flavonoid content of CR-HCT1-2 is 1.87 times that of the wild type, the flavonoid content of CR-HCT2-1 is 3.50 times that of the wild type, and the flavonoid content of CR-HCT2-2 is 2.78 times that of the wild type ( Fig.18 ). In conclusion, the present invention uses gene editing technology to identify and obtain 4 knockout mutant materials of the HCT gene of Longxiangtaro, and the flavonoid content in the mutants is 1.79-3.50 times that of the wild type.

[0100] In summary, CeHCT1 and CeHCT2 genes in taro can negatively regulate the biosynthesis of taro flavonoids. Through gene editing technology, multi-target knockout of HCT genes in the main cultivated variety Xinghua Longxiang taro can effectively increase the flavonoid content in taro and efficiently enhance the nutritional quality of taro. This has important theoretical and practical significance for accelerating the development and utilization of taro genes and using biofortification technology to improve the nutritional quality of taro.

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene for regulating taro flavonoid biosynthesis, characterized in that: The genes include CeHCT1 gene and CeHCT2 gene; the nucleotide sequence of the CeHCT1 gene is shown as SEQ ID NO.9; the nucleotide sequence of the CeHCT2 gene is shown as SEQ ID NO.

10.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene also includes a nucleotide sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID NO.9 or SEQ ID NO.10, and also includes mutants, alleles or derivatives generated by adding, replacing, inserting or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.9 or SEQ ID NO.

10.

3. Use of the gene described in claim 1 or 2 in regulating the flavonoid content of taro.

4. The use according to claim 3, characterized in that: The regulation is negative regulation.

5. Use of the gene according to claim 1 or 2 in improving the nutritional quality of plants.

6. The use according to claim 5, characterized in that: The plant includes taro or Arabidopsis thaliana.

7. A method for increasing the flavonoid content of taro, characterized in that: The method comprises the step of performing multi-target knockout on the CeHCT1 and / or CeHCT2 gene described in claim 1.

8. A genetic transformation technology for taro, characterized in that: The method comprises the step of transferring the taro explants into an infection solution of Agrobacterium tumefaciens EHA105 for incubation to obtain infected explants.

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