A gene for regulating flavonoid biosynthesis in taro and its application

Abstract

The present invention discloses a gene for regulating the biosynthesis of flavonoids in taro and its application, belonging to the field of biotechnology. The gene includes the CeHCT1 gene and the CeHCT2 gene; the nucleotide sequence of the CeHCT1 gene is as shown in SEQ ID NO.9; the nucleotide sequence of the CeHCT2 gene is as shown in SEQ ID NO.10. The present invention also provides the application of the gene in regulating the flavonoid content of taro and improving the nutritional quality of plants. By gene editing technology, multi-target knockout of the CeHCT1 and / or CeHCT2 genes of the main cultivated variety Xinghualongxiangyu can effectively increase the flavonoid content in taro and efficiently enhance the nutritional quality of taro, which has important theoretical and practical significance for accelerating the development and utilization of taro genes and improving the nutritional quality of taro by using biofortification technology.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a gene for regulating the biosynthesis of flavonoids in taro and its application. Background Art

[0002] Taro is a perennial herbaceous plant of the genus Colocasia in the Araceae family and is often cultivated as an annual. The corms, flowers, petioles and other parts of taro can be eaten and play a crucial role in the food security of many countries. Given 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. However, there are few research reports on this important functional factor, bioflavonoids, in taro. Existing research has shown that flavonoids play an important role in the nutritional enhancement of tuber crops such as sweet potato, potato, and cassava. Therefore, studying the nutritional enhancement 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 mainly propagated asexually. Taro flowering depends not only on suitable environmental conditions but also on variety type, the state of mother corms and daughter corms, and the vegetative growth stage. Therefore, taro rarely flowers under natural conditions, which limits its sexual hybridization. Currently, it is very difficult to transfer genes in taro through traditional hybridization. Gene editing technology is precisely the most effective way to overcome this problem. By gene editing technology, synthetic mutations can fill and in some cases replace natural genetic variations, efficiently and precisely modify target genes, and improve the efficiency of genetic improvement. However, this technology has not been applied to the genetic improvement of taro. Moreover, due to its large and highly heterozygous genome, the research on taro at the molecular level lags behind. In the past two decades, taro genetic research has mainly focused on diversity analysis and genetic mapping, and the genomics research of taro is still in its infancy. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a gene for regulating the biosynthesis of flavonoids in taro and its application. The results of the present invention show that the flavonoid content in the knockout mutants of the CeHCT1 gene is 1.79 - 1.87 times that of the wild type, and the flavonoid content in the knockout mutants 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, which includes the CeHCT1 gene and the CeHCT2 gene; the nucleotide sequence of the CeHCT1 gene is as shown in SEQ ID NO.9; the nucleotide sequence of the CeHCT2 gene is as 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: The application of the said gene in regulating the content of taro flavonoids; the regulation is negative regulation.

[0009] It is verified in the present invention that the CeHCT1 and CeHCT2 genes in taro can negatively regulate the biosynthesis of taro flavonoids. By gene editing technology, knocking out the CeHCT1 and / or CeHCT2 genes of the main cultivated variety Xinghua Longxiangyu can effectively increase the flavonoid content in taro corms.

[0010] Technical solution 3: The application of the said 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, which includes the step of multi-target knocking out of the said CeHCT1 and / or CeHCT2 genes.

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

[0014] In the present invention, the CRISPR-Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP of the CeHCT1 gene and the CeHCT2 gene are transferred into Longxiangyu, and 4 knockout mutant materials of the HCT gene (CeHCT1 gene and CeHCT2 gene) of Longxiangyu are identified.

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

[0016] The present invention discloses the structural genes CeHCT1 and CeHCT2 related to flavonoid metabolism in taro corms, which 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, and screens and obtains stable genetic materials, providing technical support for the subsequent development and utilization of genes and the genetic improvement of taro. The present invention also uses the genetic transformation technology of Longxiang taro 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 4 knockout mutant materials of the HCT gene of Longxiang taro. The color of the roots and petioles of the knockout mutant materials of the HCT gene of Longxiang taro deepens. Further detection finds that flavonoids are extremely significantly enriched in the roots of the mutants. Among them, the flavonoid content in the knockout mutants of the CeHCT1 gene is 1.79 - 1.87 times that of the wild type, and the flavonoid content in the knockout mutants of the CeHCT2 gene is 2.78 - 3.50 times that of the wild type.

[0017] The biological fortification of taro in the present invention is highly targeted and has high fortification efficiency. At the same time, it provides new ideas and methods for the biological fortification of taro and other crops, and efficiently fortifies 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 is the phylogenetic analysis of CeHCT1 and CeHCT2 proteins;

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

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

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

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

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

[0025] Figure 7 It is the subcellular localization analysis of the CeHCT1 and CeHCT2 genes;

[0026] Figure 8 It is the electrophoresis map of the HPT gene; M: DL 2,000 Marker; 1-8: Arabidopsis thaliana transgenic plants of CeHCT1; 9-16: Arabidopsis thaliana transgenic plants of CeHCT2; 17: Wild type;

[0027] Figure 9 It is the expression levels of the CeHCT1 gene and the CeHCT2 gene in Arabidopsis thaliana transgenic plants;

[0028] Figure 10 It is the phenotype observation of Arabidopsis thaliana transgenic plants, where A: Wild type; B: CeHCT1 transgenic plants; C: CeHCT2 transgenic plants;

[0029] Figure 11 It is the comparison of the total flavonoid content in Arabidopsis thaliana transgenic plants, where A: Shoots and leaves of the T1 generation; B: Leaves of the T2 generation; C: Stems of the T2 generation;

[0030] Figure 12 It is the plasmid map of the CRISPR-Cas9 multi-target vector for the CeHCT1 gene;

[0031] Figure 13 It is the plasmid map of the CRISPR-Cas9 multi-target vector for the CeHCT2 gene;

[0032] Figure 14 It is the electrophoresis map of the HPT gene, where M: DL 2,000 Marker; 1: Wild type; 2-9: Longxiang taro transgenic plants of CeHCT2; 10-17: Longxiang taro transgenic plants of CeHCT1;

[0033] Figure 15 It is the target mutation types of gene-edited plants;

[0034] Figure 16 It is the phenotype observation (roots) of the Longxiang taro HCT gene knockout mutant plants, where A: Wild type; B: CR-HCT1-2; C: CR-HCT2-1;

[0035] Figure 17 It is the phenotype observation (petioles) of the Longxiang taro HCT gene knockout mutant plants, where A: Wild type; B: CR-HCT1-2; C: CR-HCT2-1;

[0036] Figure 18Comparison of total flavonoid content in the HCT gene knockout mutant plants of Longxiang taro. Detailed implementation manners

[0037] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0038] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0041] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all 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 the taro variety 'Xinghua Longxiang taro' (provided by Xinghua Meihua Vegetable Professional Cooperative) and the new taro line 'Ty22-01' (bred by Taizhou Agricultural Science Institute of Jiangsu Academy of Agricultural Sciences); the tobacco is Nicotiana benthamiana; the Arabidopsis thaliana is the Col-0 ecotype. The used strains Escherichia coli DH5α and TOP10, Agrobacterium tumefaciens GV3101 and EHA105 can all be obtained commercially.

[0046] 1.2 Experimental treatments and sample preparation

[0047] 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 total RNA of taro peel and flesh was extracted using a polysaccharide and polyphenol plant RNA extraction kit (Nanjing Genscript Biotech Co., Ltd.); the concentration and purity of the extracted total RNA were measured. After passing the detection, 1 μL of total RNA was taken, and genomic DNA was removed according to the instructions of a reverse transcription kit (Nanjing Novoprotein Scientific Inc.), followed by reverse transcription reaction 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] Analysis of the CeHCT1 and CeHCT2 gene sequence information and blast were completed using DNAMAN software. Phylogenetic tree analysis was completed using MEGA 7.0 software, with the neighbor-joining method and Bootstrap set to 1000 times. The online software ProtParam (https: / / web.expasy.org / protparam / ) was used to analyze the CeHCT1 and CeHCT2 protein sequences. 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 structure of the CeHCT1 and CeHCT2 proteins.

[0050] 1.4 Vector construction

[0051] Using the cDNA of taro as a template, the full-length ORFs of CeHCT1 and CeHCT2 genes were amplified by PCR with gene-specific primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 (see Table 1). The 50 μL PCR reaction system: 5 μL of 10×PCR Buffer, 5 μL of dNTP, 1.5 μL of each forward and reverse primer, 2 μL of cDNA template, 2 μL of DMSO, 1 μL of KOD DNA polymerase, and 32 μL of ddH2O. The PCR reaction procedure: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, extension at 68 °C for 2 min, 40 cycles; incubation at 68 °C for 8 min. The amplified target gene fragments were purified and recovered. The target vector pHG was double-digested with SacI and SpeI, and the CeHCT1 and CeHCT2 gene fragments were ligated into the pHG vector by homologous recombination method to obtain the recombinant plasmids pHG-CeHCT1 and pHG-CeHCT2. After correct detection, they were transferred into Agrobacterium tumefaciens GV3101 for standby.

[0052] According to the GC content and the sequences corresponding to the protospacer adjacent motif (PAM) sites, specific guide RNAs (sgRNAs) on the targeting strands of CeHCT1 and CeHC T2 genes were selected respectively, and their off-target effects were verified by the web server CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Specific primers SEQ ID NO.5, SEQ IDNO.6, SEQ ID NO.7, and SEQ ID NO.8 (see Table 1) were designed to amplify the double-target sgRNA fragments by PCR. The PCR reaction system and reaction procedure were the same as above. The amplified double-target sgRNA fragments were ligated into the pRGEB32-GFP binary vector digested with HindIII and SbfI to obtain the recombinant plasmids CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP. After correct detection, they were transferred into Agrobacterium tumefaciens EHA105 for standby.

[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 tumefaciens GV3101 containing the plasmid vectors pHG-CeHCT1 and pHG-CeHCT2 was activated and then cultured on a large scale. After collecting the bacterial cells, they were resuspended with a resuspension solution (containing 10 mmol / L MgCl2, 10 mmol / L MES, and 0.1 mmol / L acetosyringone) to make the OD 600nm about 0.5, and then left standing for 2 - 3 h. Then, it was injected into tobacco leaves that had grown for 3 - 4 weeks through a syringe. Observation could be carried out 2 days after infection. The leaves transformed with the empty vector pHB-eGFP were used as a control. GFP fluorescence was observed using a laser confocal microscope 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. The reagent used was Ready-to-use premix for the first-strand cDNA synthesis of Ⅲ qPCR (Yeasen, China). It was completed using a fluorescence quantitative PCR instrument (BIOER, China). The program was set as follows: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, for 40 cycles; 72℃ for 30 s. The internal reference gene was CeGAPDH. The relative expression level of the target gene was calculated using the 2 －ΔΔCt method.

[0059] 1.7 Arabidopsis thaliana genetic transformation

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

[0061] 1.8 Genetic transformation of Longxiang taro

[0062] Using the leaves of sterile seedlings of Longxiang taro as explants, in the pre-culture medium (the components were 4.4 g﹒L -1 MS medium + 30 g﹒L -1Sucrose + 7 g·L -1 Agar + 2 mg·L -1 2,4-D + 1 mg·L -1 6-BA + 0.5 mg·L -1 Pre-culture was carried out on (NAA), and then transferred to the infection solution of Agrobacterium tumefaciens EHA105 containing the CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFPAS vectors for incubation to obtain the infected explants. After co-culturing for 3 d, they were transferred to the resistant dedifferentiation medium (component 4.4 g·L -1 MS medium + 30 g·L -1 Sucrose + 7 g·L -1 Agar + 2 mg·L -1 2,4-D + 1 mg·L -1 6-BA + 0.5 mg·L - 1 NAA + 200 mg·L -1 Glutamine + 7.5 mg·L -1 Hygromycin + 300 mg·L -1 Timentin) to screen for resistant calli. The resistant calli differentiated into T0 generation transgenic plants, and 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 0.1 g of the sample was weighed and 1 mL of 60% (v / v) ethanol solution was added. Ultrasonic extraction was carried out at 60 °C for 30 min, centrifuged at 8000 g and 25 °C for 10 min, and the supernatant was taken and diluted for determination. 108 μL of the test solution was taken into a 96-well plate, 6 μL of 5% sodium nitrite was added, mixed well, and left standing at room temperature for 6 min; then 6 μL of 10% Al(NO3)3 solution was added, mixed well, and left standing at room temperature for 6 min. Then 80 μL of 4% NaOH solution was added, mixed well, and left standing at room temperature for 15 min, and the absorbance was measured 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 research on the anthocyanin accumulation in taro bulbs, 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 sequence, and cloned 2 HCT genes, named CeHCT1 and CeHCT2 respectively. Among them, the full-length CDS of the CeHCT1 gene consists of 1314 bases and encodes 437 amino acids; the full-length CDS of the CeHCT2 gene consists of 1305 bases and encodes 434 amino acids.

[0069] Sequence information of the CeHCT1 gene:

[0070]

[0071] Sequence information of the 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] From the tissue expression profiles of the CeHCT1 and CeHCT2 genes ( Figure 4 and Figure 5 ), it can be seen that there are differences in the expression levels and expression patterns of the CeHCT1 and CeHCT2 genes in taro varieties with different flavonoid contents. The expression level of the CeHCT2 gene in taro is higher than that of the CeHCT1 gene; in the variety 'Ty22-01' with high flavonoid content, the overall expression levels of the two genes are higher than those in the variety 'Longxiangyu' with low flavonoid content.

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

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

[0083] In order to analyze the functions of the CeHCT1 and CeHCT2 genes, the full-length CDSs of the cloned CeHCT1 and CeHCT2 genes were used as templates in this invention. Specific primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 (see Table 1) were designed to amplify the target fragments with SacI and SpeI restriction sites. The target fragments were inserted into the double-digested pHG expression vector by homologous recombination to construct binary expression vectors pHG-CeHCT1 and pHG-CeHCT2 respectively. This vector carries both the target gene and the 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 the CeHCT1 and CeHCT2 proteins, in this invention, Agrobacterium tumefaciens GV3101 carrying the fusion GFP vectors (pHG-CeHCT1 and pHG-CeHCT1) of CeHCT1 and CeHCT2 respectively was transformed into tobacco leaves by transient transformation of tobacco leaves, and the fluorescence positions were observed by a laser confocal microscope, with the pHB-GFP empty vector as a control. The leaves transformed with the empty vector had strong green fluorescence in the nucleus, cytoplasm and cell membrane. The leaves transformed with CeHCT1 had strong green fluorescence in the nucleus and weak green fluorescence in the cell membrane. The leaves transformed with the CeHCT2 protein had strong green fluorescence in the nucleus, cytoplasm and cell membrane. Figure 7) These results indicate that the CeHCT1 protein is mainly localized in the nucleus, and the CeHCT2 protein is localized in the nucleus, cytoplasm and cell membrane.

[0086] 2.5 Identification of Arabidopsis thaliana Transgenic Plants with CeHCT1 and CeHCT2 Genes

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

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

[0089] 2.6 Effects of Transgenic CeHCT1 and CeHCT2 Genes on Arabidopsis thaliana

[0090] 2.6.1 Effects of Transgenic CeHCT1 and CeHCT2 Genes on the Phenotype of Arabidopsis thaliana Plants

[0091] The phenotypes of Arabidopsis thaliana transgenic plant seedlings were observed ( Figure 10 ), and no obvious phenotypic differences were found compared with the wild-type.

[0092] 2.6.2 Effects of Transgenic CeHCT1 and CeHCT2 Genes on the Flavonoid Content in the Leaves of Arabidopsis thaliana Plants

[0093] Three lines were selected from the transgenic Arabidopsis thaliana offspring with CeHCT1 and CeHCT2 respectively, and the total flavonoid contents in their leaves and stems were detected. Compared with the wild-type, the total flavonoid contents in the transgenic lines were significantly decreased ( Figure 11 ). The results indicate that the CeHCT1 and CeHCT2 genes inhibit the synthesis of flavonoids in the stems and leaves of Arabidopsis thaliana plants.

[0094] 2.7 Construction of the CRISPR-Cas9 Multi-Target Vector for CeHCT1 and CeHCT2 Genes

[0095] To obtain multi-target knockout mutants of taro CeHCT1 and CeHCT2 genes, in the present invention, the amplified dual-target sgRNA fragment was ligated to the pRGEB32-GFP binary vector digested with HindIII and SbfI to construct CRISPR / Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-T1T2-pRGEB32-GFP in which the OsU3 promoter drives the dual-target sgRNA respectively ( Figure 12 and Figure 13 ).

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

[0097] The CRISPR-Cas9 vectors CR-HCT1-T1T2-pRGEB32-GFP and CR-HCT2-pRGEB32-GFP of CeHCT1 and CeHCT2 genes were transformed into Longxiang taro to obtain transgenic Longxiang taro plants. After PCR detection, 14 transgenic positive Longxiang taro plants were obtained, including 6 transgenic positive plants of CR-HCT1 and 8 transgenic positive plants of CR-HCT2 ( Figure 14 ). DNA was extracted from the leaves of transgenic positive Longxiang taro plants, and specific primers were designed to perform PCR amplification and sequencing of the target gene fragment. The gene editing sites were determined by analyzing the sequencing results. Sequencing found that 4 out of 14 transgenic plants had insertions or deletions of 1-4 bases at the target site sgR1, resulting in frameshift mutations ( Figure 15 ).

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

[0099] Observing the plant phenotypes of the knockout mutants of taro CeHCT1 and CeHCT2 genes, it was found that purple-red depositions appeared in the roots of the knockout mutant plants of CeHCT1 and CeHCT2, and the pigment deposition was more obvious in the CeHCT2 knockout mutant ( Figure 16 ); in addition, purple-red depositions were also visible in the petiole parts of the CeHCT2 knockout mutants ( Figure 17)。Both hydroxycinnamoyl transferase (HCT) and chalcone synthase (CHS) can use coumaroyl-CoA as a substrate to introduce the phenylpropanoid metabolism into the lignin monomer synthesis and flavonoid synthesis pathways respectively. In the knockout mutants of the HCT genes (CeHCT1 gene and CeHCT2 gene) of taro, the synthesis of HCT is blocked. CHS uses coumaroyl-CoA as a substrate to introduce the phenylpropanoid metabolism into the flavonoid synthesis pathway and synthesize flavonoids. In view of this, the flavonoid content in the roots of the knockout mutants of the taro HCT genes was measured in this invention. The results showed that the flavonoid contents in the roots of the knockout mutants CR-HCT1-1 and CR-HCT1-2 of the taro CeHCT1 gene and the knockout mutants CR-HCT2-1 and CR-HCT2-2 of the taro CeHCT2 gene were all extremely significantly increased compared with the wild type. The flavonoid content of CR-HCT1-1 was 1.79 times that of the wild type, the flavonoid content of CR-HCT1-2 was 1.87 times that of the wild type, the flavonoid content of CR-HCT2-1 was 3.50 times that of the wild type, and the flavonoid content of CR-HCT2-2 was 2.78 times that of the wild type ( Figure 18 )。In summary, this invention uses gene editing technology to identify and obtain 4 knockout mutant materials of the taro HCT genes, and the flavonoid content in the mutants is 1.79 - 3.50 times that of the wild type.

[0100] In conclusion, the CeHCT1 and CeHCT2 genes in taro can negatively regulate the biosynthesis of taro flavonoids. By using gene editing technology to perform multi-target knockout of the HCT genes of the main cultivated variety Xinghua taro, the flavonoid content in taro can be effectively increased, and the nutritional quality of taro can be efficiently enhanced, which has important theoretical and practical significance for accelerating the development and utilization of taro genes and improving the nutritional quality of taro by using biofortification technology.

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

Claims

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

10.

2. Use of the gene according to claim 1 in regulating the flavonoid content of taro.

3. The application according to claim 2, wherein The regulation is negative regulation.

4. Use of the gene according to claim 1 in improving the nutritional quality of plants, characterized in that, The plant is taro or Arabidopsis thaliana; the nutritional quality is the flavonoid content.

5. A method for increasing the flavonoid content of taro, characterized in that, It includes the step of performing multi-target knockout on the CeHCT1 gene or CeHCT2 gene described in claim 1.

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