CN114752698A

CN114752698A - SNP locus causing tomato ascorbic acid synthesis difference, screening method and identification method

Info

Publication number: CN114752698A
Application number: CN202210318922.1A
Authority: CN
Inventors: 张余洋; 陈卫芳; 叶志彪
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2022-07-15
Anticipated expiration: 2041-07-15
Also published as: CN114752698B; CN113528496A; CN113528496B

Abstract

The invention relates to 4 SNP loci causing tomato ascorbic acid synthesis difference, a screening method and an identification method. The 4 SNP sites are respectively positioned at 286bp, 847bp, 1193bp and 1981bp upstream of the initiation codon of the isocitrate lyase coding gene, the gene is positioned on chromosome 7 of tomato No.7 SL2.50ch07:60973492.60970926, the sequence of the gene is the NCBI serial number NM-001246949.2, and the sequence number of the SGN gene is Solyc07g052480.2. The SNP locus can provide technical support for identification or auxiliary identification of high-content ascorbic acid tomato planting resources.

Description

SNP locus causing tomato ascorbic acid synthesis difference, screening method and identification method

The application is a Chinese patent application with the application date of 2021, month 07 and day 15: application of the ICL gene in the regulation of the accumulation of tomato ascorbic acid (application No. CN 202110802319.6).

Technical Field

The invention relates to the technical field of SNP (single nucleotide polymorphism) detection for synthesizing tomato ascorbic acid, in particular to SNP sites, a screening method and an identification method for causing the synthesis difference of the tomato ascorbic acid.

Background

In plants, ascorbic acid can be used as a coenzyme of some enzymes to regulate and control enzymatic reaction in plants and improve stress resistance of cells; protecting plant photosynthesis; delaying aging; ascorbic acid may also act as a plant growth regulator. Ascorbic acid coordinates jasmonic acid and ethylene ways to induce system disease resistance by regulating redox level; ascorbic acid affects the ripening of tomato fruits by regulating the oxidation/anti-oxidation balance. At present, the reported ascorbic acid biosynthesis pathways mainly include mannose/galactose synthesis pathway, AsA synthesis pathway, galacturonic acid pathway of AsA synthesis, gulose pathway of AsA biosynthesis, inositol pathway of AsA biosynthesis. After the ascorbic acid is synthesized, the ascorbic acid is oxidized and degraded by APX, AO catalytic enzyme and the like, and is regenerated by a DHAR reduction regeneration system.

Isocitrate lyase (ICL), also called Isocitrate lyase, catalyzes the cleavage of Isocitrate into glyoxylate and succinate, wherein the glyoxylate enters the glyoxylate cycle and the succinate synthesizes glucose through a series of reactions. Isocitrate lyase is a key enzyme in the glyoxylate cycle pathway (Kamal et al, 2021), but ICL has been studied less in plants to date, especially in tomato.

Disclosure of Invention

In view of the above, the present invention aims to search for the relevant metabolic pathways of the ICL gene in tomato so as to help the growth and development of tomato and improve yield and quality.

In the first aspect, the embodiment of the invention discloses 4 SNP sites causing the difference of tomato ascorbic acid synthesis, which are respectively positioned at 286bp, 847bp, 1193bp and 1981bp upstream of the initiation codon of an isocitrate lyase coding gene, the gene is positioned on the tomato No.7 chromosome SL2.50ch07:60973492..60970926, the NCBI sequence number of the gene is NM-001246949.2, and the Soxhlet number of the SGN gene is Solyc07g052480.2.

In a second aspect, the invention also discloses a method for screening SNP sites causing the difference of tomato ascorbic acid synthesis, which comprises the following steps:

selecting 5.5M high-quality SNP with MAF of more than 0.05 and minimum allele variety of not less than 6 for whole genome association analysis;

GWAS correlation analysis of ascorbic acid content by compression mixing linear model in TASSEL 4.0, threshold p ≦ 1.8 × 10^-7(P is 1/n, n is the total number of SNPs), genes in a range of 50kb upstream and downstream of the significantly related SNP locus are possible candidate genes;

analyzing SNP sites of the candidate gene in different tomato materials according to the tomato core germplasm resource re-sequencing data and first-generation sequencing to serve as candidate SNP sites;

eliminating SNP sites taken by the candidate gene codes to obtain the SNP sites causing the difference of the tomato ascorbic acid synthesis;

wherein the candidate gene is based on tomato chromosome 7 SL2.50ch07:60973492..60970926, the NCBI sequence number is NM-001246949.2, and the SGN gene sequence number is Solyc07g052480.2;

the SNP sites causing the synthesis difference of the tomato ascorbic acid are respectively positioned at the 286bp, 847bp, 1193bp and 1981bp upstream of the initiation codon of the candidate gene.

In a third aspect, the invention also discloses an identification or auxiliary identification method of high-content ascorbic acid tomato germplasm resources, which comprises the following steps:

detecting SNP sites at 286bp, 847bp, 1193bp and 1981bp upstream of the initiation codon of the isocitrate lyase coding gene, wherein the gene is located on tomato No.7 chromosome SL2.50ch07:60973492..60970926, the sequence number of the NCBI of the gene is NM-001246949.2, and the sequence number of the SGN gene soxhlet is Solyc07g052480.2;

if the nucleotide bases of 4 SNP sites of the gene at 286bp, 847bp, 1193bp and 1981bp are A, T, G, C in sequence, the tomato material is judged to be a haplotype high-content ascorbic acid tomato germplasm resource.

Compared with the prior art, the invention has at least the following beneficial effects:

the embodiment of the invention determines 4 SNP sites causing ascorbic acid synthesis difference in tomato candidate genes by a screening method, and the SNP sites can provide technical support for identification or auxiliary identification of high-content ascorbic acid tomato planting resources.

Drawings

FIG. 1 is a graph of the results of the distribution of ascorbic acid content in a natural population of tomatoes provided in accordance with the present invention; FIG. 1a is a graph of ascorbic acid content in different natural populations; FIG. 1b is a normal distribution of ascorbic acid content in different natural populations; FIG. 1c is a chart of the classification of ascorbic acid content in different populations.

FIG. 2 is a mGWAS correlation diagram of the relative ascorbic acid content of natural population of tomato ripe fruit of red fruit provided by the embodiment of the invention; the correlation results of ascorbic acid GWAS in 2013 are Manhattan graph (a) and QQ plot (b); results of correlation of ascorbic acid GWAS in 2016 manhattan graph (c) and QQ plot (d); the lowest partial map in FIG. 2 is the highest point chr07: 60983724131 kb interval SNP locus map.

FIG. 3 is a diagram showing the alignment of ICL amino acid sequences of various tomatoes with other species according to the embodiment of the present invention; in the figure; boxes indicate the ARM tripeptide of the Peroxisome Targeting Signal (PTS) (Ala-Arg-Met), the LKP motif (Leu-169, Lys-170, Pro-171) and the TKK motif (Thr-210, Lys-211, Lys-212), respectively; tomato: solyc07g052480.2.1, tobacco: XP _016500530.1, XP _009615982.1, XP _009790446.1, XP _019249474.1, cacao: XP — 007045605.2, almond: XP — 034206574.1, wild apricot: AIU64852.1, sweet cherry: XP — 021822841.1, poppy: XP — 026435746.1, thunder god vine: XP — 038705040.1, apple: XP — 008379623.2, cassava: XP _021630157.1, XP _031103078.1, XP _019192878.1, AAG44479.1, mei: XP — 008230083.1, potato: XP — 006367277.1, pepper: KAF3674797.1, KAF 3680818.1.

FIG. 4 is a graph showing the results of ICL haplotype analysis and ascorbic acid content analysis in the natural population of tomato according to the present invention; FIG. 4a is the result of an ICL sequence haplotype analysis in the natural population; FIG. 4b shows the ascorbic acid content corresponding to the ICL haplotype; FIG. 4c shows the different tomato genotypes represented by the SNP site 286bp upstream of the ICL promoter (chr07:60973535) and their ascorbic acid content; in FIG. 4a, the thick rectangular rectangles represent exons, introns are between the exons, the left-most exon is preceded by a promoter region, and the outside of the right-most exon is the 5' UTR region; the SNP sites of the four genotypes are all positioned in a promoter region, and the quantity and the total quantity of the four genotypes in different varieties of tomatoes in the embodiment of the invention are respectively represented in a right square box.

FIG. 5 is a chart of the ICL haplotype distribution of each variety in the natural population according to the embodiment of the present invention.

FIG. 6 shows ascorbic acid content (a) and ICL expression level (b) of ascorbic acid extreme materials of natural populations, which are provided by the present invention, and is divided by a dotted line, the left side is an extremely low material, and the right side is an extremely high material.

FIG. 7 is a diagram showing the results of GUS vector-based assay provided in the example of the present invention, in which the first lane is the proS1ICL^(AC)GUS, in the second lane, proS1ICL^(TS158)::GUS。

FIG. 8 is a diagram showing the results of GUS staining and GUS expression of promoters of TS158 tomato and AC tomato material provided in the examples of the present invention; FIG. 8a is a GUS staining map of promoter of TS158 and AC material in tobacco; FIG. 8b is a diagram showing the result of GUS gene expression level of promoters of TS158 and AC materials in tobacco; FIG. 8c is a GUS staining diagram of transgenic material seedlings of TS158 and AC material promoters.

FIG. 9 is a diagram showing the result of detection of 35S ICL-YFP fusion expression vector according to the present invention; from left to right in the figure, lanes 1 to 10 are 35S:: ICL-YFP fusion expression vector PCR detection bands, and lane 11 is marker (the size of the marker band is 5000bp, 3000bp, 2000 bp, 1500bp, 750bp, 500bp, 300bp, 200bp, 100bp from top to bottom).

FIG. 10 is a chart of the results of ICL subcellular localization provided by an embodiment of the present invention; 35S, exciting ICL-YFP by green light; MTBR is a mitochondrial marker showing red light.

FIG. 11 is a graph showing the results of detection of ICL-pro35S-OE vector provided in accordance with an embodiment of the present invention; from left to right, the 1 st lane is marker (the size of the marker band is 5000bp, 3000bp, 2000, 1500bp, 750bp, 500bp, 300bp, 200bp and 100bp from top to bottom), and the 2 nd to 9 th lanes are ICL-pro35S-OE overexpression vector PCR detection bands.

FIG. 12 is a graph showing the results of detection of ICL-pro158-OE vector provided in accordance with an embodiment of the present invention; from left to right, lanes 1 to 10 are PCR detection bands of ICL-pro158-OE self promoter overexpression vector, and lane 11 is marker (the size of the marker band is 5000bp, 3000bp, 2000 bp, 1500bp, 750bp, 500bp, 300bp, 200bp, 100bp from top to bottom).

FIG. 13 is a schematic diagram of the ICL CRISPR knockout target (FIG. 13a) and analysis of the target knockout sequence (FIG. 13b, FIG. 13c) provided in embodiments of the invention.

FIG. 14 is a diagram showing the results of ICL expression analysis in ICL transgenic plants driven by different promoters according to the present invention; FIG. 14a shows that the CaMV35S promoter drives the expression of excess transgenic material ICL; FIG. 14b shows the amount of ICL expression of TS158 from the promoter-transgenic material; FIG. 14c shows ICL expression level of AC self-promoter over-transgenic material.

FIG. 15 is a graph showing the results of ascorbic acid content in ICL excess transgenic material provided by an embodiment of the present invention; FIGS. 15a-15d are graphs sequentially showing the results of the ascorbic acid content of leaves of transgenic material driven by CaMV35S, TS158, CaMV35S and TS158 promoters.

FIG. 16 is a graph showing the results of the ascorbic acid content of ICL CRISPR knockout transgenic material provided in examples of the present invention; FIGS. 16a-16b are sequential ICL CRISPR knock-out transgene leaf ascorbate content against AC and TS 158; FIGS. 16c-16d are ICL CRISPR knock-outs of transgenic ripe fruit for ascorbic acid content against AC and TS158 in sequence.

Fig. 17 is a graph of GC-MS analysis results of ripe red fruit of knockout transgenic material with TS158 as background according to an embodiment of the present invention, wherein related components include mannose, inositol, galacturonic acid, galactose, glucose, sucrose, citric acid, and malic acid.

FIG. 18 is a graph showing the results of GC-MS analysis of ripe fruit of ICL excess transgenic material according to the present invention, wherein the relevant components include malic acid, galactose, glucose, citric acid, galactose, inositol, and sucrose.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

According to the embodiment of the invention, the genes related to the ascorbic acid content in the natural population of the tomato are analyzed, the genes are positioned in the ICL gene through analysis, the effect of the ICL gene in the anabolism of the ascorbic acid in the tomato is fully researched, and the result shows that the overexpression or overexpression of the genes can promote the tomato to obtain high-content ascorbic acid, so that technical support is provided for improving the quality of the tomato and obtaining high-quality tomato planting resources. The following will describe the implementation of the present invention in detail.

The sources of the strain and the carrier related by the invention are as follows: e.coli strain Trans T1 (Baiolaibo), C58, GV2260 Agrobacterium tumefaciens strain (Beijing Huayue Americans organisms); the vectors used for genetic transformation include overexpression vector pHelles gate8(Biovector NTCC type culture Collection), CRISPR knockout vector PTX (Henan bioscience), GUS expression vector pMV2 (Sammerfy), subcellular localization vector 101-YFP (Sammerfy). Other related experimental materials and reagents, not described or supplemented in detail, are commercially available.

Example 1 distribution of ascorbic acid content in Natural population of tomato

According to the embodiment, the ascorbic acid content is analyzed according to GC-MS measurement results of 360 parts of tomato natural population red ripe fruits in 2013.

The tomato core germplasm resource red ripe fruit utilized in the embodiment of the invention is subjected to genome-wide association analysis of ascorbic acid content, and tomato materials are respectively from the United States Department of Agriculture (USDA), the Tomato Genetic Resource Center (TGRC), the French national agricultural research Institute (INRA), the European Union Solanaceae project (EU-SOL) and the Chinese academy of agricultural sciences vegetable and flowers research institute (IVF-CAAS). Including 166 BIG fruit tomatoes (BIG tomatoes) (BIG); 17 parts of F1 modern commercial variety (BIG); 112 parts cherry tomato (CER); 53 parts of gooseberry tomato (PIM); 10 parts of wild tomatoes. Species information and sequencing results for these materials are found in Lin et al, 2014. The metabolites of the red ripe fruits are measured by GC-MS, 360 parts of the red ripe fruits are planted in the experimental field of Huazhong agriculture university in 2013, more than 500 parts of the red ripe fruits are planted in the northern park of vegetable research institute in Wuhan City in 2016, biological repetition is carried out twice, and mGWAS correlation analysis is carried out on the ascorbic acid content. The high ascorbic acid content TS158 and the low ascorbic acid content TS9(AC) are selected as genetic transformation background materials for gene function identification.

Tomato sample preparation: the tomato samples are all taken from fruits above the second inflorescence in the red mature period, the mature period of the fruits, the sizes of the fruits and the like are uniform when the samples are taken, and a plurality of plants in the same line are sampled. Quickly freezing the fruit flesh with liquid nitrogen, and storing in a refrigerator at-80 deg.C. The sampling method for all the transgenic materials is the same as the above, and the biological period is consistent. The stored samples were ground to a powder with liquid nitrogen for subsequent assays.

GC-MS determination of ascorbic acid in tomato ripe red fruit:

1. sample pretreatment

Taking samples after liquid nitrogen grinding, weighing about 0.2g of each sample, and recording the weighed weight; adding 750 μ l of 100% chromatographic grade methanol for extraction, mixing uniformly, and vortexing for 15 s; adding 31.5 μ l of ribitol with concentration of 0.2 m/ml as internal standard, and mixing by vortex; shaking at 30 deg.C for 15min, and centrifuging at 14000r/min for 10 min; after centrifugation, 600. mu.l of the supernatant was added to a new 2ml centrifuge tube, 402. mu.l of chromatographic grade chloroform was added, and 803.23. mu.l of ddH was added₂O; the mixture is vortexed for 15s and centrifuged for 15min at 4000 r/min; vacuum drying 200 μ l of the supernatant for 2 hr to get gel; adding 90 mul of 20mg/ml methoxylamine hydrochloride pyridine solution, and reacting for 90min in an incubator at 37 ℃; adding 90 μ l derivatization reagent N-N-N-trimethylsilyl trifluoroacetamide (MSTFA), and reacting in an incubator at 37 ℃ for 90 min; 14000r/min for 10min, 100. mu.l of the supernatant was added to the vial containing the inner cannula and tested on the machine.

2、GC-MS：

The metabolite content was determined using gas chromatography-mass spectrometry (Thermo Finnigan, Manchester, UK) with an HP-5MS capillary column (100% dimethylpolysiloxane, 30mm x 0.25mm i.d. x 0.25 μm film thickness) (agilent, usa), an EI ion source (70eV), a scanning range of 50-600m/z, an ion source temperature of 280 ℃, an injection port temperature of 230 ℃, an interface temperature of 250 ℃, a carrier gas of helium, and a flow rate of 1.2 ml/min; the temperature rising procedure is as follows: keeping at 70 deg.C for 5min, heating to 300 deg.C at 5 deg.C/min, and keeping for 3 min. Finally balancing for 1min at 70 ℃, and then feeding the next sample; the split ratio is 50: 1; mu.l of each sample was taken, one sample took 60min and was taken automatically.

The relative ascorbic acid content of these 4 tomato varieties was analyzed based further on the variety classification of 360 materials (PIM: currant tomato, CER: cherry tomato, BIG: BIG fruit tomato, F1) showing that the PIM tomatoes have significantly higher ascorbic acid content than CER, BIG and F1; the CER-th, F1 lowest. This result is consistent with tomato evolution, with PIM evolving to CER and then to BIG, indicating that the ascorbic acid content is progressively reduced.

Example 2 tomato ascorbic acid content Whole genome Association analysis

The ascorbic acid is greatly different in natural population and is controlled by a micro-effective polygene. The tomato genotype data used in this example is re-sequencing data (Lin et al, 2014Nature Genetics) in published papers, MAF was selected>The genome-wide association analysis was performed using 5.5M high-quality SNP of 0.05 and minimum allele size ≧ 6. GWAS correlation analysis of ascorbic acid content by Compression Mixed Linear Model (CMLM) in TASSEL 4.0, with a threshold of p ≦ 1.8 × 10^-7(P ═ 1/n; n ═ total number of SNPs), genes within 50kb upstream and downstream of the significantly related SNP site were possible candidates.

According to the GWAS correlation result, the embodiment discovers that a SNP site exceeding a threshold exists on the chromosome 7 and the chromosome 9, and the SNP site of the chromosome 9 is identified as bHLH59 transcription factor which can be combined with promoters of SlPMM, SlGMP2 and SlGMP3 genes to regulate the content of tomato ascorbic acid (Ye et al, 2019). Therefore, this implementation focuses on the locus on chromosome 7 and this locus is available in both GWAS associations (fig. 2). Now the Lead SNP at this site is Ch 07-60983724, and the p value is 4.56 × 10^-10(FIG. 2).

According to the results of the association analysis, the present example further analyzed the genes in the 50kb region upstream and downstream of the SNP site, found that there are 12 genes in the region, and primarily used Solyc07g052480 as candidate genes based on the annotation information of the genes, the sequence analysis of the promoter and coding region, the amount of gene expression in the extreme materials, and the like. The candidate gene was annotated as Isocitrate lyase (ICL) and was only 10232bp away from the major SNP (see Table 1).

Table 1 AsA mGWAS analysis gave gene in 50kb range upstream and downstream of major SNP of chromosome 7

Gene	Distance from major SNP	Gene annotation
			Solyc07g052420	47690	Chromosome 15 contig 1 DNA sequence
Solyc07g052430	42493	Transmembrane protein 97
			Solyc07g052440	40800	Transmembrane protein 97
Solyc07g052450	38198	Phosphate translocator
			Solyc07g052460	31879	Genomic DNA chromosome 3 P1 clone
Solyc07g052470	12741	Syntaxin
			Solyc07g052480	10232	Isocitrate lyase
Solyc07g052490	-20510	Myb family transcription factor
			Solyc07g052500	-27593	Polyubiquitin
Solyc07g052510	-38188	Peroxidase
			Solyc07g052520	-44465	Unknown Protein
Solyc07g052530	-47682	Peroxidase

Example 3 sequence analysis of ICL Gene

From the above examples, it can be seen that the tomato ICL gene is located on chromosome 7, the NCBI sequence of the gene is NM-001246949.2, and the Soxhlet number of the SGN gene is Solyc07g052480.2. The sequence was analyzed using the SGN network, which is located on the chromosome at SL2.50ch07:60973492.. 60970926. The full-length gDNA of ICL has 2022bp, and comprises 3 exons and 2 introns, which code for 575 amino acids. The amino acid sequence of ICL was analyzed and the region between 27 and 575 amino acids was found to be a conserved ICL domain.

Further analysis revealed that the ICL encoded protein contained Leu-169, Lys-170, Pro-171(LKP) and Thr-210, Lys-211, Lys-212(TKK) motifs, with reported substrate binding domain function, and a putative Peroxisome Targeting Signal (PTS) Ala-Arg-Met (ARM) tripeptide. The LKP and TKK motifs constitute a putative substrate binding domain and ARM is a putative peroxisome targeting signal (figure 3).

According to the tomato core germplasm resource re-sequencing data and the first generation sequencing result, a plurality of SNP sites (detailed genome sequence information is shown in https:// solgenomics. net/search/locus) exist in ICL in different materials, and the ICL can be divided into 4 haplotypes (hapI, hapII, hapIII and hapIV) according to the SNP sites.

The results are shown in FIG. 4, where there is a T/C mutation in the coding region, this mutation site is located on the second exon, 1299bp from the start codon, this SNP difference causes the CTG-TTG codon difference, resulting in a synonymous mutation of leucine, indicating that the exon SNP difference does not cause amino acid changes, and this SNP difference is distributed among three varieties and the difference is not significant (FIG. 4 a). Thus, this example excludes the differences in coding region SNPs that cause differences in ascorbic acid content; further sequence analysis of the 2kb promoter fragment of ICL revealed 4 distinct SNPs located 286bp (SNP ch07_60973535), 847bp (SNP ch07_60974096), 1193bp (SNP ch07_60974395) and 1981bp (SNP ch07_60975230) upstream of the start codon (FIG. 4 a).

In this example, the distribution of the 4 haplotypes in the natural population of tomato is further counted, and according to the SNP site variation and the distribution characteristics thereof, after excluding the difference of coding regions, HapIII and HapIV promoters are found to be the same, PIM (black currant tomato) is mainly concentrated into HapI haplotypes, CER (cherry tomato) and BIG (tomato BIG fruit) are mainly concentrated into HapIII and HapIV haplotypes, so that it can be speculated that the promoter difference between HapI and HapIII and HapIV haplotypes may be the main reason for causing the ascorbic acid content to change in the natural population.

To further verify the effect of ICL on ascorbic acid content, this example also performed statistics on the relative ascorbic acid content of different haplotypes in the natural population, and the results show (FIG. 4b) that haplotype Hap I ascorbic acid content is higher than other phenotypes. These results all indicate that the different haplotypes of the promoter cause differences in ascorbic acid content.

The ICL gene promoter was further predicted using plantaCARE (see http:// bioinformatics. psb. agent. be/webtools/plantaCARE/html /) and planta 3.0 (plantan. itps. ncku. edu. tw), which showed that a difference of 286bp upstream of the promoter might cause a difference of a cis-element (CAATCA/CAAACA) which might produce a difference of HD-ZIP transcription factor (AT 3G60390: HAT3 in Arabidopsis); analysis of this HD-ZIP transcription factor of Arabidopsis thaliana revealed that the gene with the highest homology in tomato is Solyc08g078300.2 (homeobox-leucoine zipper protein HB 2-like). HAT3 can control apical embryonic development and meristem function in arabidopsis thaliana (Turchi et al, 2013); arabidopsis leaf development was controlled (Bou-Torrent et al, 2012). The ICL regulation of the HD-ZIP transcription factor needs to be further verified. Further statistics on the ascorbic acid content of the SNP site at 286bp show that the ascorbic acid content of the base A (containing complete cis-elements) is higher than that of the base G (without cis-elements), which indicates that the difference of the SNP at 286bp may cause the difference of ascorbic acid (FIG. 4 c).

In order to further analyze the relationship between the haplotype distribution of ICL in the natural population and PIM, CER and BIG, the proportion of PIM, CER and BIG in different ICL haplotypes in the natural population is counted. The results are shown in FIG. 5, in Hap I, PIM, CER, BIG are distributed, wherein PIM accounts for 48%, CER accounts for 25%, BIG accounts for 28%; only PIM and CER in Hap II account for 38% and 62% respectively; both HapIII and HapIV have only CER and BIG, and HapIII has 82% of BIG, 18% of CER, 39% of BIG and 61% of CER in haplotype IV.

Therefore, by analyzing the ICL gene haplotype in the natural population, whether the ICL gene is a germplasm resource with high content of ascorbic acid can be known, and the ICL gene germplasm resource with high content of ascorbic acid can be helped for tomatoes.

Example 4 expression analysis of ICL in high and Low ascorbic acid Material

To further verify that SNP difference at 286bp upstream of ICL gene initiation codon causes difference of phenotype ascorbic acid content, this example measured ICL expression amount in ascorbic acid extreme material. 6 parts of high-ascorbic acid content and low-extreme material are selected respectively, and the ascorbic acid content is determined, and the result is shown in figure 6, wherein the ascorbic acid content of the extremely high material is higher than that of the extremely low material (figure 6 a); the expression level of ICL in the extreme material was determined and the results showed that the expression level of ICL in the extreme high material was significantly higher than that of ICL in the extreme low material (fig. 6b), indicating that the difference in ascorbic acid content in the natural population was caused by the difference in promoters of ICL.

Example 5 differential promoter of ICL GUS staining analysis

To further explore the function of the ICL promoter, this example used Primer premier 5 to design primers to amplify the ICL 2K promoter fragment, with the Primer sequences Fw (shown in SEQ ID NO. 1) and Rv (shown in SEQ ID NO. 2).

Taking gDNA of a high ascorbic acid material TS158 and a low ascorbic acid material TS9(AC) as templates, amplifying to obtain an ICL 2kb promoter fragment, connecting to a pMV2 (containing a GUS reporter gene) expression vector through homologous recombination, and transferring into Escherichia coli Trans T1; obtaining two expression vectors of proICL (TS158), GUS and proICL (AC), GUS, and sending the positive detected single clone of colibacillus to Tianyihui Yuan company for sequencing. Wherein the TS158 promoter type belongs to haplotype I, and the AC (TS9) promoter type belongs to haplotype IV. The results of the electrophoretic detection of the carrier are shown in FIG. 7.

The plasmid proICL (TS158) with correct sequencing results, GUS and proICL (AC), GUS vector plasmid, is transferred into tobacco leaves for transient expression by an agrobacterium-mediated method, and the agrobacterium-mediated and transient expression methods are referred to (Song Jian, Ph university in Huazhong, academic thesis, 2019). The tobacco leaf after injection contains proICL (TS158), GUS and proICL (AC), GUS expression vector, and is stained in GUS staining solution for 12h (the GUS staining solution contains 0.1mol phosphate buffer, pH7.0, 0.5mmol K3[ Fe (CN 6) ]]，0.5mmol K₄[Fe(CN)₆]·3H₂O，10mmol EDTA-Na₂2H₂O, 0.1% Triton X-100, 2% X-Gluc, which can be dyed blue to different degrees, removing chlorophyll by 95% (V/V) ethanol, and observing whether the tobacco leaves are dyed blue or not and observing the range and the shade of the color; sampling the tobacco leaves mediated by agrobacterium, extracting RNA (Invitrogen Ambion RNA kit, Saimerfei), carrying out qPCR detection after reverse transcription into cDNA, determining the expression quantity of GUS genes in the leaves, and carrying out RNA extraction, reverse transcription of cDNA and qPCR detection according to a reference method (Liugen loyalty, Phd academic thesis of university of agriculture in Huazhong, 2020).

The results are shown in FIG. 8, which shows that the blue color of the tobacco leaves after the GUS expression vector is injected is significantly darker than that of the tobacco leaves after the GUS expression vector is injected (AC) (FIG. 8 a); the analysis result of the tobacco leaf expression level also shows that the expression level of the GUS gene in the GUS is obviously higher than that of the proICL (AC) and the GUS (figure 8 b).

Subsequently, the two expression vectors were genetically transformed with AC as a background material to obtain proICL (TS158) containing GUS and proICL (AC) containing GUS transgenic material. Extracting gDNA of the transgenic tomato material, carrying out positive detection, carrying out GUS staining on positive plant seedlings for 12h, removing chlorophyll through 95% (V/V) ethanol, and observing whether the tobacco leaves are stained with blue color or not, and the range and the depth of the color. The results showed that both expression vectors were expressed in leaves and stems, but proICL (TS158): GUS transgenic seedlings were significantly more blue than proICL (AC): GUS transgenic seedlings, and also expressed in the heels (FIG. 8 c). These results all indicate that the activity of the ICL promoter in TS158 is significantly higher than that of the AC material, which has a regulatory effect of promoting the anabolism of ascorbic acid.

Example 6 ICL subcellular localization

In order to investigate the relationship between ICL and ascorbic acid content, the amino acid sequence of ICL was analyzed for its subcellular localization via the PSI website (http:// bis. zju. edu. cn/PSI /), indicating that ICL may be localized to chloroplasts and mitochondria. In order to verify the location of ICL, Primer premier 5 is used to design a Primer for removing the stop codon of ICL by amplification, a recombination sequence (shown as SEQ ID NO. 6) is added in front of a forward Primer and a recombination sequence (shown as SEQ ID NO. 7) is added in front of a reverse Primer, AC material cDNA is used as a template amplification fragment, and the amplification fragment is connected to a 101-YFP vector (North Nuo Life technology) through homologous recombination. The homologous recombination fragment is transferred into an escherichia coli Trans T1 competent cell, colony PCR is carried out on a single clone to identify a positive clone, and the colony with positive detection is directly sent to Tianyihui company for sequencing. Activating the colony with correct sequencing, extracting plasmid, transferring the plasmid into agrobacterium strain GV2260 through electric shock transformation, and performing PCR positive detection on the single clone to obtain 35S, wherein the detection of ICL-YFP fusion expression vector is shown in figure 9.

The CaMV35S promoter is used for driving and constructing 35S, namely ICL-YFP fusion expression vector, and transient expression is carried out in tobacco leaves. Plant group DNA was extracted using leaves of transgenic material. For the excess of CaMV35S strong promoter, a CaMV35S primer and a target gene reverse primer are used; for TS158 and AC self-promoter overages, gene self-promoter overage forward primer and pHelles gate8 reverse primer were used; the CPISPR knockout material is detected by using a PTX (PTX vector) primer (presented by the institute of genetic development of Chinese academy of sciences) in the T0 generation, a plant without vector insertion is removed, a fragment detection primer is designed by using a fragment with a total fragment length of 600-800 bp and containing two target points, the fragment detection primer is used for detection, then a PCR product amplified by the detection primer is sent to a Tianyihui-Chiang company for sequencing, the target point knockout condition is analyzed, and a plant with large fragment deletion or insertion is reserved for subsequent research.

Subcellular localization observations by confocal microscopy with mitochondrial Marker gene MTBR as localization control (chloroplast autofluorescence) showed that ICL was localized in mitochondria (figure 10), while the key gene for the ascorbic acid biosynthetic pathway, L-galactose-1, 4-lactone dehydrogenase, was present in mitochondria (Bartoli et al 2000), indicating that ICL and ascorbic acid were spatially consistent.

Example 7 ICL transgenic tomato Gene expression and ascorbic acid content

Construction of overexpression vector:

to further verify the relationship between ICL and ascorbic acid content, this example utilizes Primer premier 5 design to amplify the full-length open reading frame of the candidate gene, adding recombination sequences (as shown in SEQ ID NO. 3) and XhoI cleavage sites before the forward Primer, and recombination sequences (as shown in SEQ ID NO. 4) and XbaI cleavage sites before the reverse Primer. The gDNA of the AC material was used as a template to amplify the fragment, and the fragment was ligated to pHels gate8 vector by homologous recombination to construct CaMV35S promoter overexpression vector ICL-pro35S-OE, and the vector detection results are shown in FIG. 1.

Primer premier 5 is used for designing primers to amplify the full-length ORF and the 2kb promoter fragment of the candidate gene, a recombination sequence (shown as SEQ ID NO. 5) is added in front of a forward Primer, and SacI is addedEnzyme cleavage site of (2)Adding a recombination sequence (shown as SEQ ID NO. 4) in front of the reverse primer, adding an XbaI restriction enzyme cutting site, respectively taking TS158 and AC material gDNA as templates, carrying out fragment amplification, respectively constructing TS158 and AC (TS9) self-starting overexpression vectors ICL-pro158-OE and ICL-proAC-OE, and detecting the vectors as shown in FIG. 12.

Genetic transformation: the vector is subjected to genetic transformation by utilizing an agrobacterium-mediated method, and the specific transformation steps are shown in Europe Bobo, 2003, doctor academic thesis of university of agriculture in Huazhong. The genetic transformation receptor materials used were TS158 and AC (introduced from the American tomato genetic resource center and preserved by the tomato theme group of university of agriculture, Huazhong); and (4) analyzing the sequences of the candidate genes, and analyzing the gene sequence structure by using an SGN website.

Construction of a CRISPR knockout vector: analyzing the sequence of the candidate gene, analyzing the gene sequence structure by using an SGN website, designing a primer by using a CRISPR knockout primer design website (http:// CRISPR. dbcls. jp/. The vector plasmid with the correct sequencing result is transferred into agrobacterium strain C58.

The expression levels of ICL in CaMV35S promoter-containing transgenic material and TS158 and AC (TS9) self-promoter-containing transgenic material were further determined, and the results are shown in FIG. 14, wherein the expression levels of ICL-pro35S-OE-11-2, ICL-pro35S-OE-18-7, and ICL-pro35S-OE-21-3 transgenic lines are 26.6, 23.2, and 39.3, respectively; the expression levels of the ICL-pro158-OE-2-2, ICL-pro158-OE-3-7 and ICL-pro158-OE-5-7 transgenic lines are respectively 10.1, 6.3 and 5.2; the expression levels of ICL-proAC-OE-1-4, ICL-proAC-OE-3-2 and ICL-proAC-OE-11-9 transgenic lines are 6.2, 7.3 and 14.5 respectively.

Subsequently, the ascorbic acid content of the transgenic tomato leaves and red ripe fruits is also tested in the embodiment, and the results are shown in fig. 15, wherein the ascorbic acid content of the excessive transgenic tomato leaves and red ripe fruits is obviously increased. In the leaf, the ascorbic acid content of three transgenic strains of ICL-pro35S-OE is respectively increased by 36%, 17% and 44% (FIG. 15a), and the ascorbic acid content of three transgenic strains of ICL-pro158-OE is respectively increased by 38%, 16% and 22% (FIG. 15 b); in the fruit, the ascorbic acid content of the two transgenic lines ICL-pro35S-OE was increased by 32%, 24% respectively (FIG. 15c), and the ascorbic acid content of the three transgenic lines ICL-pro158-OE was increased by 35%, 30% respectively (FIG. 15 d).

The content of the ascorbic acid in the leaves and the fruits of the ICL knocked-out transgenic material is measured, and the result shows that the content of the ascorbic acid in the CRISPR knocked-out transgenic material with TS158 and AC as background materials is reduced. Transgenic knockout lines ICL-CR-10-2, ICL-CR-14-6 and ICL-CR-16-5 with AC material as background were reduced in ascorbic acid content by 31%, 24% and 9%, respectively, in the leaf (FIG. 16a) and in the fruit by 35%, 21% and 32%, respectively (FIG. 16 c); the transgenic knockout lines ICL-CR-6-1, ICL-CR-20-4 and ICL-CR-50-6 with TS158 as background had a 25%, 38% and 24% decrease in ascorbic acid content in leaves, respectively (FIG. 16b), and a 17%, 26% and 27% decrease in fruits, respectively (FIG. 16 d).

In this example, it is found that the relative expression amount of ICL in the transgenic material with excess CaMV35S promoter is greater than 23, and the relative expression amount of ICL in the transgenic material with excess TS158 from promoter is only 5 times or more, but the percentage increase of ascorbic acid content in the transgenic material with excess TS158 from promoter is slightly higher than that of the transgenic material with excess CaMV35S promoter. Although the expression level of the ICL promoter in the TS158 is only 5 times, the content of the ascorbic acid is higher than that under the drive of the 35S promoter, which indicates that the driving efficiency of the TS158 promoter is higher. Meanwhile, the percent reduction of the ascorbic acid content in the knockout transgenic line with the TS158 as the background is higher than that in the knockout transgenic line with the AC as the background material. Therefore, the haplotype of the TS158 promoter of the high ascorbic acid material represents high ascorbic acid content in natural populations, and the difference of the haplotype can be used for designing a marker and applying the marker to molecular breeding and screening of the high ascorbic acid material.

Example 8 analysis of ascorbic acid pathway metabolism in ICL transgenic lines

Transcription factors can directly regulate genes of the biosynthetic pathway for the purpose of regulating the ascorbic acid content, whereas ICL is an isocitrate lyase, which may affect specific substrates or intermediates in the ascorbic acid biosynthetic pathway. To analyze whether ICL exerts an effect on the substrates of the ascorbic acid biosynthetic pathway, this example further performed GC-MS assay on transgenic red ripe tomato fruit (red ripe fruit of the second ear), analysis on ascorbic acid synthesis intermediate metabolites, GC-MS assay and metabolite analysis methods refer to example 1.

As shown in FIG. 17, in the two knockout transgenic red ripe fruits with TS158 as background, the contents of mannose, inositol and galacturonic acid related to ascorbic acid synthesis are respectively 15% -18%, 61% -69% and 90% -93% of the control, while the content of galactose is 1.4-2.1 times of the control, the contents of other substrates such as glucose, sucrose and citric acid are respectively 73% -95%, 52% -58% and 65% -75% of the control, and the content of malic acid is increased by 1.3-1.5 times.

As shown in FIG. 18, the opposite results occurred in the ICL hypertransgenic material, and the content of inositol and galacturonic acid related to ascorbic acid in the hypertransgenic red ripe fruit was increased, while the content of galactose was decreased, and the content of glucose, sucrose and citric acid was increased, and the content of malic acid was decreased. Thus, it is shown that SlICL has an effect on ascorbic acid biosynthesis substrates, may catalyze specific steps, affect intermediate product content, and thus affect ascorbic acid accumulation levels.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

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Claims

1.4 SNP loci causing the synthesis difference of tomato ascorbic acid are respectively positioned at 286bp, 847bp, 1193bp and 1981bp upstream of the initiation codon of an isocitrate lyase coding gene, the gene is positioned on chromosome 7 of tomato SL2.50ch07:60973492..60970926, the sequence of the gene is the NCBI serial number NM-001246949.2, and the Soxhlet number of the SGN gene is Solyc07g052480.2.

2. The SNP site according to claim 2, wherein the tomato is divided into four haplotypes, hapI, hapII, hapIII and hapIV; wherein the Hap I haplotype has higher ascorbic acid content than other genotypes;

the nucleotide bases of 4 SNP sites of the Hap I haplotype corresponding to the genotypes at 286bp, 847bp, 1193bp and 1981bp are A, T, G, C in sequence;

the nucleotide bases of 4 SNP sites of the corresponding genotype of Hap II haplotype at 286bp, 847bp, 1193bp and 1981bp are A, C, G, C in sequence;

the nucleotide bases of 4 SNP sites of the Hap III haplotype corresponding to the genotypes at 286bp, 847bp, 1193bp and 1981bp are G, C, A, T in sequence;

the nucleotide bases of 4 SNP sites of the corresponding genotype of Hap IV haplotype at 286bp, 847bp, 1193bp and 1981bp are G, C, A, T in sequence.

3. A method for screening SNP sites causing differences in tomato ascorbic acid synthesis comprises the following steps:

analyzing SNP sites of the candidate gene in different tomato materials according to the tomato core germplasm resource re-sequencing data and first-generation sequencing, and taking the SNP sites as candidate SNP sites;

eliminating SNP sites taken by the candidate gene codes to obtain the SNP sites causing the synthesis difference of the tomato ascorbic acid;

4. A method for identifying or assisting in identifying high-content ascorbic acid tomato germplasm resources comprises the following steps:

detecting SNP sites at 286bp, 847bp, 1193bp and 1981bp upstream of an initiation codon of an isocitrate lyase coding gene, wherein the gene is located on chromosome 7 of tomato SL2.50ch07:60973492..60970926, the sequence number of NCBI of the gene is NM-001246949.2, and the sequence number of SGN is Solyc07g052480.2;