CN115029359B

CN115029359B - Improvement method considering both crop yield and quality

Info

Publication number: CN115029359B
Application number: CN202210657091.0A
Authority: CN
Inventors: 尚轶; 黄三文; 张博; 仲阳; 薛晓锋
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2022-06-10
Filing date: 2022-06-10
Publication date: 2023-03-21
Anticipated expiration: 2042-06-10
Also published as: CN115029359A

Abstract

The present application relates to an improved method for achieving both crop yield and quality. The method realizes translation control by modifying a sequence near an upstream open reading frame of a GGP enzyme gene, obtains potatoes with high yield and high vitamin C content, and is a high-efficiency method for improving potato varieties without introducing exogenous genes.

Description

Improvement method considering both crop yield and quality

Technical Field

The application relates to an improvement method considering both crop yield and quality, and belongs to the technical field of biology.

Background

In recent years, more and more people have come to pay attention to the content of ingredients beneficial to the human body, particularly ascorbic acid (vitamin C), in potatoes. Ascorbic acid is by far the most abundant low molecular weight antioxidant in photosynthetic organisms and is also an essential nutrient for the human body. Because of the lack of L-glucono-1, 4-lactone oxidase (Wheeler et al, 1998), humans cannot synthesize ascorbic acid autonomously, and 90% of the vitamin C required by humans comes from fresh vegetables and fruits. Thus, the vitamin C content of crops is an important nutritional quality trait. However, due to the relatively low vitamin C content of potatoes, researchers have attempted to breed new varieties with high vitamin C levels by manipulating the vitamin C biosynthetic pathway. Although in other crops, manipulation of the vitamin C biosynthetic pathway can produce new varieties with 8-10 fold increased vitamin C content, in potatoes, the increased vitamin C content is not as great as reported by the inventors, e.g., overexpression of the vitamin C recycling gene dehydroascorbate reductase (DHAR) in potatoes can also increase its vitamin C content (Qin et al, 2011); arabidopsis thaliana or potato self-GGP genes were transferred into potatoes using either a strong promoter (35S) or a tuber-specific Promoter (PAT), with tuber ascorbic acid content increased 2.7-fold (35S-AtVTC 2) and 4.7-fold (PAT-StVTCA) over wild-type (bulletey et al, 2012). Thus, it would be desirable to obtain potatoes having higher vitamin C content.

Meanwhile, the yield and quality traits of crops are difficult to be considered, for example, in a high-yield anthocyanin rice material, the rice yield is obviously lower than that of a control material, which shows that the improvement of the nutritional quality reduces the yield of rice (Zhu et al, 2017); for another example, the content of vitamin C in the P17C5 tomato mutant generated by EMS mutagenesis is 2-5 times higher than that of the wild type, and the plant phenotype is abnormal flowers and fruits, male sterility, little or no seeds (Deslous et al, 2021), which indicates that the high content of vitamin C also affects the normal growth of crops. Therefore, in practical breeding, the cultivation of high-quality potatoes with high yield and high vitamin C content has a great challenge.

In addition, how to reduce or remove the bitterness of plants is a problem that breeding workers have long been trying to solve. Although some major bitter compounds, and genes or proteins closely related to these bitter compounds, etc. have been discovered, since the formation of bitter taste of plants involves complex interactions between components in the metabolic regulatory network of plants, there is still a need for more research in this regard.

Disclosure of Invention

In order to solve the above-mentioned problems of the background art, the present application provides, first of all, a method for producing potato having a high yield and a high vitamin C content, characterized in that at least one nucleotide in the 5' untranslated region of the potato GGP enzyme gene or the region near the upstream open reading frame of the GGP enzyme gene is modified.

In one embodiment, the modifications are deletions, additions and/or substitutions; or the at least one nucleotide is within 151, within 130, or within 100, or within 80, or within 60, or within 30, such as 1-5, 6-9, 10-17, 18-24, 25-44, 45-81, 82-142, 143-148, 149-151 nucleotides.

In one embodiment, the 5' untranslated region of a potato GGPase gene is modified to include the nucleic acid set forth in SEQ ID NO. 5.

The application also provides a method for reducing the content of bitter compounds in plants, or reducing the bitter taste of plants, and/or removing the bitter taste of plants, which is characterized by comprising the step of increasing the content of vitamin C in the plants.

In one embodiment, the step of increasing the vitamin C content comprises modifying the 5' untranslated region of the plant ggpase gene or the region near the open reading frame upstream of the ggpase gene, thereby inhibiting normal translation of the uORF.

In one embodiment, the bitter tasting compound is alpha-solanine, alpha-chaconine, and/or solanidine.

In one embodiment, reducing bitter taste in a plant, and/or debittering a plant is down-regulating expression of a key gene involved in the metabolism of solanidine.

In one embodiment, the key gene is GAME4,6,7,8a,8b,11, 12, SGT1 and/or SGT3.

In one embodiment, the plant is a monocot or dicot, preferably potato.

In one embodiment, the method comprises the step of mutating the 5 'untranslated region or the region near the upstream open reading frame of the GGP1 gene and/or the 5' untranslated region or the region near the upstream open reading frame of the GGP2 gene of potato

The outstanding contributions of the present application are:

(1) the uORF of the potato GGP enzyme gene can not be translated normally, so that the vitamin C content can be improved, and high-quality potatoes with the vitamin C content improved by 24.12 times are obtained.

(2) The modified fragment sizes of 5' UTR region or sequences near uORF of GGP gene are divided into two types: small segment modification and large segment modification; the larger the modified segment is, the higher the vitamin C content in the plant is, i.e. the size of the modified segment is positively correlated to the vitamin C content in the plant.

(3) The fact that when the content of the vitamin C in the potato blocks is greatly increased (for example, 8.72 times higher than that of wild type potato blocks), the growth of plants and the development of the potato blocks can be obviously inhibited, and when the content of the vitamin C in the potato blocks is moderately increased (1.46-8.72 times), the influence is avoided, so that the potatoes with high yield and high content of the vitamin C are obtained.

(4) It was found that increasing vitamin C content reduces the bitterness of plants. By modifying the 5' UTR region or the sequence near uORF of the GGP gene, the content of vitamin C is increased, the content of bitter compounds is reduced, and the expression of genes closely related to bitter compounds is downregulated.

Drawings

In figure 1, the content of vitamin C in leaves, stems, potato blocks and cooked potato blocks of the potato with different degrees of mutation of urorf-StGGP is higher than that of the wild type.

FIG. 2 modification of uorf-StGGP 1-A/-B. Where the uORF sequence is started from ATC in box (blue), sgRNA targets are underlined in bold, deleted nucleotides are hyped, and the uORF start codon is shown in red box.

FIG. 3, the content of vitamin C in leaves, stems, potato pieces and steamed potato pieces of the uorf-StGGP1-A/-B homozygous mutant material is obviously improved.

FIG. 4, aboveground/underground phenotypic assay of uorf-StGGP1-A/-B homozygous mutant material.

FIG. 5, the SGA biosynthetic pathway metabolites (A), gene pathway maps (B) and QP results (C) in uorf-StGGP1-A/-B potato leaves and tubers. * Values indicating metabolite content were determined by t-test to be significantly different (P < 0.05). Metabolites labeled red indicate an increase in relative levels relative to the control, while metabolites labeled blue indicate a decrease in relative levels relative to the control.

FIG. 6 illustrates the modification pattern of the uorf-StGGP series.

Detailed Description

In the following examples, specific tests are exemplified, and the experimental methods used are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In higher plants, the L-galactose pathway is considered to be the main pathway for the synthesis of vitamin C. The pathway takes D-glucose as a precursor, and finally synthesizes the L-ascorbic acid through ten-step enzymatic reactions. Among them, GDP-L-galactose phosphatase (GGP) plays a very important role in regulating vitamin C synthesis (Macknight et al, 2017).

uORFs (upstream reading frames) are an open reading frame of part of eukaryotic genes at the 5' end of mRNA, are common cis-acting elements, and can negatively regulate the transcription of downstream mRNA sequences (Calvo et al, 2009, von Arnim et al, 2014). The application provides a uORFs polypeptide, and the amino acid sequence of the uORFs polypeptide is shown in SEQ ID NO.1 or SEQ ID NO. 2. The application also provides nucleic acids encoding the uORFs polypeptides.

The sequences of the 5' untranslated regions of two GGP genes, namely StGGP1 and StGGP2, identified from potatoes are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

The present application further provides a method for increasing vitamin C content in potatoes, or reducing bitter taste in potatoes, and/or removing bitter taste from potatoes, or a method for growing potatoes with high vitamin C content, or low bitter taste, and/or no bitter taste, comprising inhibiting normal translation of the above polypeptides; preferably, the inhibition is performed by modifying the sequence of the nucleic acid.

The present application also provides a method for identifying potatoes with high vitamin C content, low bitter taste and/or no bitter taste, detecting the presence of modifications in the 5' untranslated region of potato GGPase gene (StGGP) or the nucleic acids.

In some embodiments, the modification is a deletion, addition, and/or substitution of at least one nucleotide; preferably, the modification inhibits the normal translation of the ggpase gene uarf polypeptide; preferably, the "at least one nucleotide" is 1-5, 6-9, 10-17, 18-24, 25-44, 45-81, 82-142, 143-148, 149-152, or 153 or more.

In some embodiments, the modification is as set forth in SEQ ID No. 3: deletion of nucleotide 234 to 257 (deletion of 24 bp); or the nucleotides 207 to 358 were deleted (deletion of 152 bp).

The application provides a modified nucleic acid which is a 5' untranslated region of a modified StGGP1 gene and has a sequence of SEQ ID NO.5 or SEQ ID NO.6.

The application provides a nucleic acid which is a modified 5'UTR region (i.e. 5' untranslated region) of a potato GGP enzyme gene, wherein the modification inhibits normal translation of a polypeptide encoded by uORF and the sequence of which is shown in SEQ ID No.1 or SEQ ID No. 2.

In one embodiment, the potato GGP enzyme gene is StGGP1 and/or StGGP2.

In one embodiment, the sequence of the region of the 5' UTR of the potato GGP enzyme gene is SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and/or SEQ ID No.6.

The present application provides a method for producing potato having a high vitamin C content, reduced bitterness and/or no bitterness, which comprises making the 5' UTR region of potato GGP enzyme gene the modified nucleic acid.

A method for obtaining potato with high yield and high vitamin C content and reduced bitterness and/or no bitterness, which comprises making the 5' UTR region of potato GGP enzyme gene the modified nucleic acid.

The present application also provides biological materials related to all of the above nucleic acids, which are expression cassettes containing the nucleic acid molecules; or a vector containing said nucleic acid molecule; or a cell containing said nucleic acid molecule; or a plant (e.g., potato) tissue or organ (root, stem, leaf, flower or fruit) containing the nucleic acid molecule; or the entire plant or germplasm containing said nucleic acid molecule, which plant or germplasm (e.g. potato) may be heterozygous or homozygous for the mutation; diploid or tetraploid; diploid for heterozygous or homozygous mutation, or tetraploid for heterozygous or homozygous mutation.

The application provides a method for increasing vitamin C content of potatoes, or reducing bitter taste of the potatoes, and/or removing bitter taste of the potatoes, or cultivating potatoes with high vitamin C content, low bitter taste, and/or no bitter taste, and a 5' untranslated region of a potato GGPase gene is modified into a nucleic acid represented by SEQ ID NO.5 or SEQ ID NO.6.

The application provides a method for identifying potatoes with high vitamin C content, low bitter taste and/or no bitter taste, and detecting whether a nucleic acid with a sequence of SEQ ID NO.5 or SEQ ID NO.6 exists in a 5' untranslated region of a potato GGP enzyme gene. "present" means either contained or is itself.

The present application further provides the use of a modified nucleic acid or a biological material related to the modified nucleic acid in potato breeding; preferably, the breeding is to breed potatoes with high vitamin C content, reduced bitterness, and/or no bitterness.

The application provides a method for cultivating potatoes with high yield, high vitamin C content, reduced bitter taste and/or no bitter taste, and modifies a 5' untranslated region of a potato GGP enzyme gene into a nucleic acid represented by SEQ ID NO. 5.

The application provides a method for reducing the content of bitter compounds in potatoes, which comprises the step of increasing the content of vitamin C.

The present application provides a method for reducing or removing the bitterness of potatoes comprising the step of increasing the vitamin C content.

The application provides a method for judging the high and low content of the vitamin C in plants, wherein the 5' untranslated region sequence of the plant GGP enzyme gene or the sequence near the open reading frame of the plant GGP enzyme gene is compared, and the content of the vitamin C in the plants with large-segment modification is higher than that in the plants with small-segment modification or unmodified modification.

The application provides a method for reducing the content of bitter compounds in plants, or reducing the bitter taste of plants, and/or removing the bitter taste of plants, which comprises the step of increasing the content of vitamin C in the plants.

In one embodiment, the key gene is GAME4,6,7,8a,8b,11, 12, SGT1 and/or SGT3.

In one embodiment, the step of increasing vitamin C content comprises modifying the 5' untranslated region of a plant ggpase gene or a region adjacent to the open reading frame upstream of the ggpase gene such that normal translation of the uORF is inhibited.

The plant of the present application is a monocotyledonous plant or dicotyledonous plant (e.g., potato), and may refer to a plant leaf, a plant stem, a plant root, a plant flower, or a plant fruit.

The potato of the present application may refer to a potato leaf, a potato stem, a potato piece, or a cooked potato piece.

In one embodiment, the step of increasing the vitamin C content comprises the step of mutating the 5 'untranslated region or the region near the upstream open reading frame of the GGP1 gene and/or the 5' untranslated region or the region near the upstream open reading frame of the GGP2 gene of potato.

The CRISPR-Cas9 vector for knocking out the target gene is constructed, and agrobacterium EHA105 containing the target gene knock-out vector is utilized to infect potato stem segments which are pre-cultured for 2 days and are 4 weeks old and do not have axillary buds. Infected explants were cultured on regeneration-inducing medium containing antibiotics for 8 weeks, during which the medium was changed once every two weeks. And screening the regenerated seedling for rooting resistance to obtain a positive plant. And carrying out PCR detection on the exogenous DNA fragment of the positive plant, amplifying the sequence of the target site, and determining the mutation condition of the target site by Sanger sequencing. And (3) planting the successfully mutated plants, and detecting the expression quantity of the target gene through qPCR (quantitative polymerase chain reaction) to finally determine that the target gene is knocked out. In order to further study the biological mechanism of dynamic coordination of potato yield and vitamin C content, transcriptome and metabolome studies were also performed. The specific embodiment is as follows.

Example 1 cloning of uORF and 5' UTR of StGGP1 and StGGP2

Genes StGGP1 and StGGP2 were cloned from diploid potato clones S.tuberosum group Phureja S15-65 and quadruple system D é e. Wherein S15-65 is provided by the international potato center, and D é e is provided by the cultivation and research laboratory of Yunnan Master university. The uORF and 5' UTR of the StGGP1 and StGGP2 genes were found and confirmed, and they were subjected to sequence cloning and sequencing using primers. And (3) sequencing results: the polypeptide sequences coded by uORF of StGGP1 and StGGP2 are respectively shown in SEQ ID NO.1 or SEQ ID NO. 2; sequences of 5' UTR of StGGP1 and StGGP2 are respectively shown in SEQ ID NO.3 or SEQ ID NO. 4.

Example 2 mutations in the vicinity of the uORF of StGGP1 and StGGP2

By gene editing techniques, one-way guide RNAs (sgrnas) targeting the initiation codon of uORF-StGGP1 or uORF-StGGP2 were designed. Random insertions and deletions may be introduced due to uncertainty in the self-repair of gene editing techniques; furthermore, it is uncertain which positions of GGP-uORF of potato are susceptible to gene editing, whether the translation level can be greatly increased after gene editing, and the like. Therefore, the final result will be confirmed in a variety of ways as follows.

The single guide RNA sequence of StGGP1uORF and the 19-nt single guide RNA sequence of StGGP2uORF were constructed using pKSE402-AtU626 vector. Among these, pKSE402 was digested with BsaI enzyme for insertion of sgRNA, the final binary vector was used for transformation of potato internodes by agrobacterium tumefaciens strain EHA105, and GFP fluorescence was used for identification of mutant plants.

All Wild (WT) and mutant lines were grown normally in fall in a controlled greenhouse with 16h light/8 h dark cycle at 25 ℃ daytime/18 ℃ nighttime temperature.

Example 3 genotyping and ploidy of plants

Leaf samples were collected from GFP fluorescence positive potato plants and genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. Target sequences were amplified from all regenerated plants using specific primers and sequenced to identify mutations in the target region. Genotyping individual lines by sequencing the cloned PCR products, wherein 8 monoclonal sequences are measured per sample for diploid plants and at least 20 monoclonal sequences are required per sample for tetraploid plants. Chromosome doubling occurs at a very high frequency during regeneration of potato callus, and thus ploidy of mutant plants can be determined by flow cytometry assay (BD, aria SORP).

Example 4 Dual luciferase reporter Gene assays

To generate constructs for dual-luciferase reporter assay, the 35S promoter and the wild-type and mutant forms of the 5' leaders (or untranslated regions) of StGGP1 and StGGP2 were cloned into pGreenII0800-LUC vector with BamHI and NcoI. Both the Wild (WT) and mutant constructs were transiently expressed in tobacco leaves by Agrobacterium-mediated transformation. Agrobacterium tumefaciens strain GV3101 containing the dual luciferase reporter vector was resuspended in 500mM MES buffer containing 1M MgCl 2 and 100mM acetosyringone to a final OD600 of 0.6. The buffer was infiltrated into fully expanded leaves of 4 week old tobacco using a needleless syringe. After infiltration, plants were grown in the dark for 12 hours, then in 16 hours light/8 hours dark for 2 days. LUC/REN activity was measured using a dual luciferase reporter assay system.

Example 5 determination of ascorbic acid content

The mature potato leaves, stems, tuber tissue and the potato pieces after 30 minutes of cooking were thoroughly frozen in liquid nitrogen. The powder was dissolved using extraction buffer containing 74.45mg EDTA, 286.65mg TCEP and 5ml 98% orthophosphoric acid in a final volume of 100ml Milli-Q water. The suspension was vortexed for 30 seconds. The sample was then left at 4 ℃ for 30 minutes. Subsequently, they were centrifuged at 12,000g for 30 minutes at 4 ℃, the supernatant was retained and filtered using a 4mm hydrophilic PTFE syringe filter. Ascorbic acid concentration was measured using liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS Agilent 6460). The filtered samples were analyzed using Aquality CSH C18 (waters). The chromatographic separation was carried out with a gradient elution at a flow rate of 0.2ml/min using 0.1% formic acid in water as mobile phase A and methanol and 0.1% formic acid as mobile phase B. The gradient is as follows: 5-45% B as analytical run, then 50 seconds up to 95% B, then 2 minutes at 5% B after rebalancing, the injection volume is 15 microliters. During the separation, the analytical column was kept at a temperature of 35 ℃ in a thermostatted column oven. Identification of parent and product ions was performed for all analytes based on mass abundance ratios. The LC eluate was introduced into the Agilent 6490 system in negative ESI mode. The detailed experimental conditions were: ion spray voltage 3500v, air curtain 35psi, collision gas medium, temperature 350 ℃. Analyte ions are detected in a Multiple Reaction Monitoring (MRM) mode.

Example 6 modification results

For uORF-StGGP2, we designed two sgRNAs targeting sequences away from the uORF-StGGP2 start codon, and finally obtained four chimeric lines (uORF-StGGP 2-A/-B/-C/-D) in the Desir é background. Each line has a combination of 2 to 44bp deletions in the sequence of uORF-StGGP2, with an insertion of 1bp in addition to the deletion mutation of uORF-StGGP 2-B. LUC/REN analysis confirmed that all uORF-StGGP2 disruptions identified from these mutants were effective in manipulating transcription of downstream reporter genes and increasing potato vitamin C content (FIG. 1).

For uORF-StGGP1, a diploid was obtained and the mean vitamin C content in the StGGP1-uORF mutant material potato pieces was 1.03mg/g FW, about 2-fold that of the StGGP2-uORF mutant lines. Therefore, uORF-StGGP1 was mainly studied as follows.

The observation of the phenotypes of a series of potato StGGP-uORF mutations and potato seed materials with increased vitamin C content (figure 1 and figure 6) created by the inventor shows that the vitamin C content in leaves, stems and potato blocks of the materials is increased to different degrees, and the deletion size of the StGGP-uORF fragments is positively correlated with the vitamin C content in plants, namely the larger the mutated fragments are, the higher the vitamin C content is. Of these, two homozygous mutant germplasm uORF-StGGP1-A/-B (or written as: GGP1-uORF-A, GGP 1-uORF-B) containing deletions of 24bp and 152bp, respectively, were the most representative potatoes finally screened (FIG. 2).

Compared with the control, the content of the vitamin C in the mutant material leaves is improved by 1.20-2.71 times in terms of the mutant as a whole; the increase multiple in the stem is 1.50-6.14 times; the content of vitamin C in the potato blocks is obviously improved, and the amplification is 1.51-24.12 times. It is particularly surprising that the level of vitamin C in the leaves, stems and tubers of the uorf-StGGP1-B mutant with the deletion of the largest fragment (152 bp) is significantly increased by 2.71, 5.78, 24.12 fold, respectively, compared to the control S15-65 and other diploid mutants.

In addition, given that vitamin C is highly susceptible to degradation during cooking, we further examined the change in vitamin C content in these material potato pieces at high temperatures. The results show that after 30 minutes of cooking, vitamin C in the control and mutant potato pieces is lost to some extent. However, compared to the control (80% -90% loss), the vitamin C loss of the potato pieces of the gene-edited material was about 20% -50%, and still sufficient nutritional components were retained (fig. 1).

For the most representative potato uorf-StGGP1-a and uorf-StGGP1-B, the vitamin C content in leaves, stems and potato pieces of the uorf-StGGP1-a material (5' utr region nucleic acid sequence shown in SEQ ID No. 5) with small fragment deletion (24 bp) was increased by 2.02, 2.60 and 8.72 fold, respectively, compared to the control; the vitamin C levels in leaves, stems and potato pieces of the large-fragment (152 bp) deleted uorf-StGGP1-B material (the nucleic acid sequence of the 5' UTR region is shown as SEQ ID NO. 6) are increased by 2.71, 5.78 and 24.12 times respectively. Among them, the increase of vitamin C content in potato tuber of uorf-StGGP1-B is the most significant and the most concentration-changing case of the vitamin C content reported (FIG. 1, FIG. 3). However, the large fragment knockout uorf-StGGP1-B line showed stunted aerial plant, delayed flowering, reduced biomass and reduced tuber yield phenotype compared to control material and uorf-StGGP 1-A. Wherein, the plant height, the overground fresh weight and the weight of the single-plant tuber are respectively reduced by 29.24 percent, 27.53 percent and 69.29 percent. However, the content of vitamin C in the uorf-StGGP1-A potato pieces is obviously improved (8.72 times), and meanwhile, the growth and development inhibition phenomenon does not occur (figure 1 and figure 4).

Therefore, the uorf-StGGP1-A is a germplasm material which gives consideration to both yield and nutritional quality, namely the potato with high yield and high vitamin C content; and the urorf-StGGP 1-B is a germplasm material for the excessive conversion of energy flow into metabolic flow, and is a potato with ultrahigh vitamin C content.

Example 7 omics analysis

(1) Transcriptome data determination and analysis

After sequencing the samples, reads filtration was performed to remove the adaptor-ligated sequence at the 3' end and Reads with an average mass fraction below Q20. The filtered Reads were compared to a reference genome (http:// spuddb. Ugh.edu/software/HISAT 2/index. Shtml) using HISAT2 (http:// ccb. Jhu. Edu/dm _ v6_ 1. Download. Shtml) software, read Count values on each gene were statistically compared using stringtie as the original expression amount of the gene, and the expression amount was normalized using FPKM values. And carrying out PCA analysis on the gene expression quantity of each sample by using DESeq2 software, carrying out differential expression analysis on normalized gene data, and screening differential genes under the conditions that the significance P-value is less than 0.05 and the | log2 fold change | is 1. And (4) carrying out enrichment analysis by using top GO, and determining Go Term with obviously enriched differential genes by using super-geometric distribution. And (4) performing KEGG pathway enrichment analysis by using R language clusterProfiler to determine a signal path related to the differential expression gene.

(2) Establishing potato metabolism database

A potato metabolism database can be built by using high-quality secondary fragment information and high-resolution mass spectrum data. The structure of the substance is mainly analyzed through four aspects of standard substance comparison, metabolic database matching, spectrogram analysis, reference documents and the like. Firstly, molecular formula calculation is carried out on metabolites with high-resolution data, and then matching of molecular formula and fragment cracking mode is carried out on the metabolites with a public metabolic database, so that preliminary analysis of metabolite structures is realized. For metabolites for which a standard can be referenced, the same substance is identified if all three conditions are the same by aligning the m/z, RT and fragmentation patterns of the standard and unknown substances. For metabolites which can not be referred to by a standard substance, the metabolites are identified mainly through spectrogram analysis, information such as published documents and published metabolic databases is comprehensively applied, elution rules, cracking rules and the like of the metabolites are systematically summarized, and the analysis is applied to unknown substances.

(3) Metabolome data determination and analysis:

the holographic mass spectrometry data collected in the positive and negative ion modes were processed using Agilent MassHunter Qualitative Analysis software. The method mainly comprises the following steps: data import, chromatographic peak alignment, experimental design, peak selection, normalization, deconvolution, compound identification and statistical analysis. And analyzing the introduced sample according to the automatically selected quality control sample. The selected adduction ion mode comprises: [ M-H2O-H ] -, [ M + FA-H ] -, [ M + Na-2H ] -, [ M + K-2H ] -, [2M + FA-H ] -, and [2M-H ] -. The mass spectrum data is deconvoluted, PCA principal component analysis and OPLS-DA analysis are carried out, S-Plot is used for identifying different metabolites of each group, and then a self-made potato metabolism database is used for carrying out qualitative analysis on retention time-accurate mass pair (RT-EM). And finally, performing data filtration through analysis of variance (P-value is less than or equal to 0.05 and Max fold change value is more than or equal to 2) to determine the differential metabolites in the sample.

(4) Gene and metabolite association assay

And carrying out co-expression analysis on the common gene expression data in a plurality of sequencing samples by using weight gene co-expression network analysis (WGCNA) software, using metabolites as characters, and carrying out cluster analysis, wherein different branches of clusters represent different gene modules (modules), and the co-expression degree of genes in the same module is high. Kmeans clustering analysis is carried out according to the different metabolites and the different genes obtained by analyzing samples of different tissues in different periods, and then drawing is carried out according to classification, so that the metabolites and the genes with consistent change trend can be found. In order to further determine which metabolites and genes are highly related to express, a Pearson correlation (pearson) method is used for calculating the correlation between genes with consistent variation trend and metabolites, and genes and metabolites with higher correlation are screened according to the correlation of more than 0.6, and the P-value of less than or equal to 0.05. And (3) utilizing the Cytoscape software to draw a network map for the differential genes and the differential metabolites with higher correlation, and visually displaying the relationship between the metabolites and the genes through the network map.

(5) Results of omics analysis

The alpha-solanine and the alpha-chaconine are obviously reduced and expressed due to the increase of the content of the vitamin C in the potato. The inventors observed Sub>A similarity of metabolite changes upon vitamin C upregulation in both GGP1-uORF-A and GGP1-uORF-B mutants. For example, compared to WT, both the main bitter compounds α -solanine and α -chaconine in the Solanaceae family were significantly reduced in uorf-StGGP1-A/-B (i.e., uorf-StGGP1-A and/or uorf-StGGP 1-B), approximately 200-fold lower as estimated by peak area (FIG. 5). Furthermore, in uorf-StGGP1-A/-B, all currently known key genes involved in the metabolism of solanidine, such as GAME4,6,7,8a,8B,11, 12, SGT1 and SGT3, down-regulated expression, indicating that vitamin C enhancement in potato tubers would reduce its bitter taste and would be appreciated by consumers. Thus, the fact that increased vitamin C content leads to a reduction in the content of bitter compounds, thereby reducing or eliminating the bitter taste of plants, is a new and important mechanism that the inventors have unexpectedly discovered.

In conclusion, the present application takes a series of StGGP-uORF mutations of potato and seed materials with elevated vitamin C content of potato as examples (as test materials) to deeply study the relationship between the GGP-uORF mutations of plants and the vitamin C content. The inventor finds that after the GGP-uORF is mutated, the vitamin C content in leaves, stems and potato blocks of the materials is improved to different degrees, and the increase of the vitamin C content is in positive correlation with the deletion size of plant GGP-uORF fragments, namely, the increase of the vitamin C content caused by small fragment deletion is far lower than that of plants with large fragment deletion, so that the larger the plant GGP-uORF mutated fragment is, the higher the vitamin C content is.

Particularly, the vitamin C content of potato blocks of the strain (uorf-StGGP 1-B) with the highest vitamin C content obtained by the method is improved by 24.12 times, and the method is the most obvious case for improving the vitamin C content in the currently known report. However, the inventors found that the growth and development of the uorf-StGGP1-B plants and the yield of potato blocks are remarkably inhibited. In the other small fragment-deleted strain uorf-StGGP1-A, the vitamin C content of the potato tuber is improved by 8.72 times, but the growth and development of the plant are not influenced. Therefore, the uorf-StGGP1-B is an extremely mutant material with excessive energy flow conversion to metabolic flow, and the uorf-StGGP1-A is a material with better balance between energy flow and metabolic flow, so that the quality and the yield are both considered.

Experiments are carried out by utilizing the two special materials and metabolome, transcriptome and other omics big data technologies, and the results reveal that the increase of the content of the vitamin C in the potato leads to the extremely obvious down-regulation expression of bitter compounds and key genes in a metabolic network thereof, and suggest that the synthesis of the plant vitamin C can influence the bitter forming process, so that the bitter of the plant can be removed or reduced by increasing the content of the vitamin C (such as mutant GGP-uORF).

While the present application has been described in considerable detail with reference to certain preferred versions thereof, it is not intended to limit the scope of the invention to the exact forms disclosed.

Sequence listing

<110> university of Yunnan Master

<120> an improvement method giving consideration to both crop yield and quality

<130> 20220609

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 62

<212> PRT

<213> Artificial Synthesis ()

<400> 1

Ile Thr Ala Ile His Lys Val Asn Arg Arg Pro Leu Leu His Val Pro

1 5 10 15

Ala Val Arg Arg Lys Gly Cys Val Thr Ala Thr Asn Pro Ala Pro His

20 25 30

Gly Gly Arg Gly Ala Leu Pro Ser Glu Gly Gly Ser Pro Ser Asp Leu

35 40 45

Leu Phe Leu Ala Gly Gly Gly Ser Phe Leu Ser Phe Ser Tyr

50 55 60

<210> 2

<211> 72

<212> PRT

<213> Artificial Synthesis ()

<400> 2

Ile Thr Ala Leu Leu Gly Ser Phe Val Cys Ile Leu Thr Ile His His

1 5 10 15

His Arg Lys Val Leu Thr Leu Asn Pro Thr Leu Leu Val Asp Asp Lys

20 25 30

His Arg Thr Pro Ser Pro His Gly Gly Arg Gly Ala Ser Pro Ala Glu

35 40 45

Gly Gly Cys Pro Ser Asp Leu Leu Phe Leu Ala Gly Gly Gly Pro Ile

50 55 60

Leu Pro Phe Ser Phe Ser Phe Ser

65 70

<210> 3

<211> 512

<212> DNA

<213> Artificial Synthesis ()

<400> 3

tcctccatta tcccttctcc aatttattat ataaataccc acatctccct gctctttctc 60

ccttatcatc aacaacaaca accaaatttc ttcttttttt cttccacagt agcaatctat 120

caacaaaaca gagaccccat tacaagaatc ttggaatttt agttttgggt ttaagaggaa 180

ggggttattg tatttgcagt tttgagggta aagcccagtt taacaaggtg tagacatcac 240

ggctatacac aaagtaaacc gccgaccact tttacatgtt ccagcagtac gtcgtaaggg 300

ttgtgtaaca gctactaacc ctgcgccgca cggtggacgt ggcgctttgc cctctgaagg 360

tggcagtcct tccgacctcc tcttccttgc cggcggcggt tctttcctct ccttctccta 420

ctagatatag ttatacttac cgtagatctc tagcttatta cgtacagttc tatctagtat 480

tctcttgatt attcgaagaa aaacacaaaa ag 512

<210> 4

<211> 470

<212> DNA

<213> Artificial sequence ()

<400> 4

taagggggtg cttatataaa gttggggagt ctaccaatga gacgaactca ttgaccaaat 60

acgtctgcag gagaaaaacc accggagcac caaacgccac ccaacaacca cccattaaat 120

tcttccagaa aaaaacatct tcctcaaaat tatcgatgaa ggatcgttcc ttagtagttg 180

ttcgttgatc ctacaaattc aatcacggct cttcttggat ctttcgtttg tattctcaca 240

attcatcatc accgcaaagt gttgaccctt aatccaactc ttctggtgga cgataagcac 300

cggacccctt cccctcacgg aggtaggggt gcctcacccg ctgaaggcgg ttgcccctcc 360

gatctcctct tcctcgccgg cggcggtcca attcttcctt tctctttctc cttctcctaa 420

tttttcgtgt aagaattgta tttttgatta tccatccaag aacaggaccg 470

<210> 5

<211> 488

<212> DNA

<213> Artificial sequence ()

<400> 5

tcctccatta tcccttctcc aatttattat ataaataccc acatctccct gctctttctc 60

ccttatcatc aacaacaaca accaaatttc ttcttttttt cttccacagt agcaatctat 120

caacaaaaca gagaccccat tacaagaatc ttggaatttt agttttgggt ttaagaggaa 180

ggggttattg tatttgcagt tttgagggta aagcccagtt taacaaggtg tagaccgccg 240

accactttta catgttccag cagtacgtcg taagggttgt gtaacagcta ctaaccctgc 300

gccgcacggt ggacgtggcg ctttgccctc tgaaggtggc agtccttccg acctcctctt 360

ccttgccggc ggcggttctt tcctctcctt ctcctactag atatagttat acttaccgta 420

gatctctagc ttattacgta cagttctatc tagtattctc ttgattattc gaagaaaaac 480

acaaaaag 488

<210> 6

<211> 360

<212> DNA

<213> Artificial sequence ()

<400> 6

tcctccatta tcccttctcc aatttattat ataaataccc acatctccct gctctttctc 60

ccttatcatc aacaacaaca accaaatttc ttcttttttt cttccacagt agcaatctat 120

caacaaaaca gagaccccat tacaagaatc ttggaatttt agttttgggt ttaagaggaa 180

ggggttattg tatttgcagt tttgagggtg gcagtccttc cgacctcctc ttccttgccg 240

gcggcggttc tttcctctcc ttctcctact agatatagtt atacttaccg tagatctcta 300

gcttattacg tacagttcta tctagtattc tcttgattat tcgaagaaaa acacaaaaag 360

Claims

1. A method of reducing the level of bitter compounds or reducing the bitter taste of potatoes which comprises modifying the 5' untranslated region of the potato GGP enzyme gene to the nucleic acid of SEQ ID No.5 or SEQ ID No.6 such that normal translation of the uORF is inhibited.

2. The method of claim 1, wherein the bitter tasting compound is alpha-solanine, alpha-chaconine, and/or solanidine.

3. The method according to claim 1 or 2, wherein the reduction of bitterness in potatoes is down-regulation of expression of key genes involved in the metabolism of solanidine, said key genes being GAME4, GAME6, GAME7, GAME8a, GAME8b, GAME11, GAME12, SGT1 and/or SGT3.

4. The method of claim 1 or 2, wherein: the potato GGP enzyme gene is a potato GGP1 gene or a potato GGP2 gene.