The hexose transport mutant gene ZmSWEET4c in maize 446th SNP tagging and its application

Abstract

This application discloses a maize hexose transport mutant gene. ZmSWEET4c 446th This study focuses on SNP markers and their applications, aiming to address the current technical problems of poor nutritional quality and low lysine content in maize kernels. The SNP markers used are from the maize reference genome B73 RefGen_v4 version. Zm00001d015912 A C→T mutation exists at nucleotide 446 of the gene's CDS sequence. The primer set for the dCAPS marker includes the upstream primer dCAPS-F: 5'-TTCGGCACCGGCATGTACGCAGCGC-3' and the downstream primer dCAPS-R: 5'-TGTAGTAGTAGCTACAGCGGCT-3'. This application provides a maize hexose transport mutant gene. ZmSWEET4c 446th It is a new one. SWEET4c The types of gene mutations provide a material basis for breeding new maize varieties with high lysine content. On the other hand, it also provides a method for detecting hexose transport mutations in maize. ZmSWEET4c 446th The dCAPS marker can be applied to marker-assisted breeding of maize with high lysine content, enabling seedling selection with high lysine content and greatly shortening the breeding cycle.

Description

Technical Field

[0001] This invention application relates to the field of bioengineering technology, specifically to a hexose transport mutant gene in maize. ZmSWEET4c 446th SNP tags and their applications. Background Technology

[0002] Corn is my country's largest grain crop and an important food, feed, and energy crop. Ordinary corn kernels contain only 1.5-2% lysine, while the optimal lysine content for the human diet is 5% (Young et al., 1998). The nutritional quality of ordinary corn kernels falls far short of human needs. Storage proteins in corn endosperm are an important source of nutritionally valuable nitrogen for humans and animals. Storage proteins can be divided into prolamins and non-prolamins. Prolamins are the most abundant storage proteins in corn endosperm, accounting for 50-70% of the total protein (Azevedo et al., 2003); non-prolamins include albumin, globulin, and glutenin, accounting for approximately 30-50%. The essential amino acid lysine for humans mainly comes from non-prolamins. Discovering and cloning genetic loci that control the lysine content of corn kernels to create superior germplasm resources is one of the important ways to cultivate high-quality protein corn.

[0003] In recent years, researchers at home and abroad have made significant progress in elucidating the genetic mechanism of lysine content in maize endosperm. o2 It is a starchy endosperm mutant. The mutant grains have a decreased proportion of alcohol-soluble proteins and a significantly increased content of lysine-rich non-alcohol-soluble proteins, resulting in a lysine content twice that of wild-type grains (Mertz and Nelson, 1964). o2 Molecular mechanism studies of the upregulated expression of genes encoding lysine-rich proteins in mutants have shown that the expression of elongation transcription factor eEF1A is upregulated, and the content of this transcription factor is significantly correlated with the endosperm lysine content (Habbenet et al., 1995). o7 and o2 It is also one of the mutants with the highest lysine nutritional value. o7 This is a natural mutation in the W22 background (Tsai and Dalby, 1974). In the mutant endosperm, the decreased content of α-prolyzin, resulting in fewer protein bodies and smaller size, leads to an opaque phenotype and an increase in lysine content in the grain. Analysis of amino acids and metabolites reveals... o7 It may affect amino acid biosynthesis by regulating α-ketoglutarate and oxaloacetate. Map-based cloning was used to isolate the mutant gene, which encoded an acyl activator highly similar to AAE3. o7The cloning suggests a new regulatory mechanism for endosperm storage proteins and provides an effective target for genetic alterations in cereal storage protein content (Wang et al., 2011).

[0004] Although these mutants have high lysine content and high nutritional value, these mealy endosperm mutants are all accompanied by a chain of unfavorable agronomical traits, such as low grain bulk density and soft texture, resulting in easily broken grains, susceptibility to pests, and poor storage, which seriously restricts their commercial application (Crow and Kermicle, 2002). Therefore, foreign breeders are trying to find the modifying genes of these mutant genes in order to breed superior varieties that can maintain high nutritional value while improving the undesirable characteristics of mealy and soft endosperm. o2 Shortly after the discovery of genes, foreign corn breeders began searching for ways to maintain... o2 High lysine content, and can change o2 Genes that modify endosperm hardness and texture ( mo2 Several have already been identified. mo2 Modification sites (Holding et al., 2008). South African and CIMMYT breeders use these... mo2 Gene modification introduction o2 Among the germplasm, "high-quality protein maize" was obtained (Prasanna et al., 2001; Vasal et al., 1980). This maintained both the phenotype and yield of ordinary maize and its high lysine content. However, several... mo2 The import process is quite complex, requiring maintenance during gene aggregation. o2 Besides requiring homozygosity at specific loci, the need for regular monitoring of amino acid composition changes severely limits the breeding and widespread application of QPM germplasm. Therefore, it is urgent to identify and explore more genetic loci controlling lysine content in maize kernels to provide an important theoretical basis for creating high-quality protein maize, and to offer new genetic resources and selection targets for high-quality protein maize breeding. Summary of the Invention

[0005] The purpose of this application is to provide a maize hexose transport mutant gene. ZmSWEET4c 446th SNP markers and their applications are used to address the current technical problems of poor nutritional quality and low lysine content in corn kernels.

[0006] To solve the above-mentioned technical problems, this application proposes to adopt the following technical solution:

[0007] The first aspect of this application provides a maize hexose transport mutant gene. ZmSWEET4c 446thIts CDS sequence is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

[0008] A second aspect of this application provides an SNP marker, which is based on the maize reference genome B73 RefGen_v4 version. Zm00001d015912 A C→T mutation exists at nucleotide 446 in the gene's CDS sequence.

[0009] A third aspect of this application provides a method for detecting... ZmSWEET4c 446th The dCAPS molecular marker for the mutant gene, based on the corresponding SNP site, uses the following primers:

[0010] dCAPS-F:5'-TTCGGCACCGGCATGTACGCAGCGC-3'

[0011] dCAPS-R:5'-TGTAGTAGTAGCTACAGCGGCT-3';

[0012] The SNP sites are reference genome sites for maize. Zm00001d015912 A C→T mutation exists at nucleotide 446 in the gene's CDS sequence.

[0013] Furthermore, the restriction endonuclease used for the dCAPS molecular marker at the SNP site is... Eco47 III.

[0014] In a fourth aspect of this application, a kit is proposed, comprising the aforementioned primers and restriction endonucleases. Eco47 III.

[0015] In another aspect, this application relates to the aforementioned hexose transport mutant gene in maize. ZmSWEET4c 446th Protein encoding, SNP markers, or kits can be used to increase the lysine content of corn kernels.

[0016] In another aspect of this application, the aforementioned maize hexose transport mutant gene... ZmSWEET4c 446th Protein encoding, SNP markers, or kits are used in the preparation of formulations that increase the lysine content of maize kernels.

[0017] In another aspect of this application, the aforementioned hexose transport mutant gene in maize is described. ZmSWEET4c 446th Encoded proteins, SNP markers, or kits are used in breeding programs to identify and / or assist in screening for characteristics of lysine content in maize endosperm.

[0018] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages:

[0019] 1. This invention provides a mutant gene for hexose transport in maize. ZmSWEET4c 446th Compared to the wild-type gene CDS sequence, the 446th base C is replaced with a base T, resulting in changes to the nucleotide and amino acid sequences, which is a novel change. SWEET4c Gene mutation types provide a material basis for the breeding of new maize varieties with high lysine content.

[0020] 2. This invention also provides a method for detecting hexose transport mutant genes in maize. ZmSWEET4c 446th The dCAPS marker can be applied to marker-assisted breeding of maize with high lysine content, enabling seedling selection with high lysine content and greatly shortening the breeding cycle. Attached Figure Description

[0021] Figure 1 As shown in one embodiment of this application ZmSWEET4c 446th Endosperm ultrastructure observation; in the figure, WT represents... ZmSWEET4c 446th Wild-type kernels from F2 ears obtained by self-pollination after hybridization with Mo17; smk11 is mutant kernels from the same F2 ear.

[0022] Figure 2 As shown in one embodiment of this application ZmSWEET4c 446th The total amino acid content and the content of various amino acids were determined in comparison with the wild type; ** in the figure represent statistically significant differences in amino acid content between this strain and the control.

[0023] Figure 3 For fine positioning of smk11 in another embodiment of this application.

[0024] Figure 4 Mutant gene in another embodiment of this application ZmSWEET4c 446th The structure and location of point mutations. Figure 5 This is a mutant allelism test in one embodiment of this application.

[0025] Figure 6 As shown in one embodiment of this application ZmSWEET4c 446th Functional marker identification electrophoresis diagram. Detailed Implementation

[0026] To better understand the technical solution of this application, the above technical solution will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the test materials and reagents involved are all commercially available; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0028] Example 1 smk11 Cultivation of mutants

[0029] The mutant used in this experiment was a small-grain mutant that was isolated from the F2 ears after crossbreeding with Mo17 following EMS-induced mutation of B73 pollen and self-pollination. It was named... smk11 The number of mutant and normal kernels on three F2 single ears was counted. The results showed that there were an average of 244 kernels per ear, of which 189 were wild-type and 55 were mutant. Fitness tests showed that χ²... 2 =0.66<χ 2 (0.05，1) =3.84, the difference is not significant, which is consistent with Mendelian inheritance laws of a 3:1 segregation ratio, indicating that... smk11 It is a recessive mutant controlled by a single gene.

[0030] Example 2 smk11 Grain ultramicro observation

[0031] Select immature normal kernels with 16 DAP from the same ear and smk11 After cutting a portion of the endosperm from the same location in the mutant grain, the endosperm was immediately fixed in 2.5% glutaraldehyde electron microscopy fixative at 4℃ for 12 h, followed by embedding with ethanol and propylene oxide. The samples were then cut into 70 nm ultrathin sections using a Reichert MILtracutE diamond scalpel microtome. Endosperm proteins were observed using a JEM-1400Plus transmission electron microscope. The results showed… smk11 The number of protein bodies in the endosperm increased significantly, and their diameter increased ( Figure 1 Select mature, normal kernels from the same ear of grain. smk11 The seeds were clamped on both sides with pliers and then longitudinally split to form a natural fracture surface. They were then dried for 15 minutes, and the fracture surface was sputtered with gold using an E-100 ion sputtering system. Finally, the fracture surface was observed using a HITACHI S-3000N scanning electron microscope. The results showed... smk11 In the endosperm, starch granules are densely packed, their number increases, and their size decreases; the protein matrix in the endosperm tightly coats the surface of the starch granules, and its number increases. Figure 1 ).

[0032] Example 3 smk11 Determination of total amino acid content and content of various amino acids

[0033] After drying, the seeds were pulverized using a high-speed grinder and sieved through a 60-mesh sieve. 100 mg of sample was accurately weighed and placed in a capped glass test tube. Using the acid hydrolysis method, HCl was added, and the mixture was heated to 110°C for 24 h for complete hydrolysis. The mixture was then cooled to room temperature, and the test tube was rinsed several times with distilled water. The washings were poured into a volumetric flask. The pH was adjusted with NaOH, and the solution was brought to volume and thoroughly mixed. The mixture was allowed to stand in a glass beaker for 30 minutes, then filtered through a 0.45 μm filter membrane. The sample solution was transferred to a liquid chromatography vial and finally analyzed by the HPLC system. The results showed that the mutant material… smk11 The total amino acid content was significantly increased compared with the wild type, and the content of essential amino acids such as lysine and methionine was significantly increased. Figure 2 ).

[0034] Example 4 smk11 Fine mapping and candidate gene structure analysis

[0035] DNA was extracted from 10 mutant and 10 wild-type plants from the F2 segregating population, and the mixtures were used to construct [specific structures]. smk11 A pool of mutant and wild-type plant DNA was used for linkage marker screening using 432 SSR markers covering the entire maize genome and exhibiting polymorphism between B73 and Mo17. The molecular marker umc1692 was found to be linked at the 5.03 bin position. smk11 Linkage was found at the loci, and subsequent verification of the linkage marker was performed on 22 mutant plants. A few plants showed heterozygous banding due to chromosomal recombination, while the rest showed B73 banding, further confirming the linkage between the molecular marker umc1692 and... smk11 They are linked.

[0036] InDel markers were developed near the umc1692 marker, and 1924 markers were analyzed in the F2 segregating population. smk11 The genotypes of the mutant plants were identified, recombinant single plants were screened, and finally... smk11 Located within an interval of approximately 170kb between markers LM4 and RM5. Figure 3 Referring to the annotation results of B73 RefGen_v4genome on the Gramene website (http: / / www.gramene.org), it was found that there is only one coding gene within the localized region. Zm00001d015912 This gene encodes a hexose transporter, ZmSWEET4C, with a CDS length of 783 bp, encoding 261 amino acids. Primers were designed to amplify the target fragment, and the PCR product size was approximately 783 bp. The primer sequences are as follows:

[0037] SWEET4C-F: 5'-TGTTGTTTGGTGTTACTTGGATCCATGGTCTCGGCGGATACCATCC-3' (SEQ IDNo. ​​3);

[0038] SWEET4C-R: 5'-AATGAGCTTTTGCTCCATGAGCTCGTAGCCGTTGCCGCCGCCGCCG-3' (SEQ ID No. 4).

[0039] PCR amplification was performed using the designed primers. The PCR reaction system consisted of 12.5 μL of 2× KODFX Buffer, 0.5 μL of KODFX, 1 μL each of the forward and reverse primers, 1.5 μL of DNA template, and 0.5 μL of 2 mM dNTPs. The volume was adjusted to 25 μL with ddH2O.

[0040] The PCR amplification program was as follows: 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 58℃ annealing for 30 s, 68℃ extension for 1 min, for a total of 35 cycles, and finally 68℃ extension for 5 min.

[0041] Gene sequencing revealed that the 446th base in the CDS sequence of this gene was replaced by thymine (T), resulting in the 149th amino acid, proline (Pro), being replaced by leucine (Leu). Pan-genomic analysis using Maize GDB (https: / / www.maizegdb.org / ) showed that this site is highly conserved in maize inbred lines such as B73 and Mo17, always containing a C base, with only... smk11 The mutant has a T base at this site ( Figure 4 ).

[0042] Example 5: Mutant Allelism Test

[0043] ZmSWEET4c- ref yes ZmSWEET4c Allelic mutants, ZmSWEET4c - ref The mutation site is located on the 5th exon, where the 584th base undergoes a C-to-T conversion, resulting in the 195th amino acid, proline (Pro), being replaced by leucine (Leu). ZmSWEET4c 446th It is a homozygous mutant. ZmSWEET4c-ref It is a heterozygous mutant. (Through...) ZmSWEET4c - ref and ZmSWEET4c 446th In hybridization, the ear of grain exhibits a 1:1 segregation ratio. Figure 5 This confirms ZmSWEET4cAbnormal gene function leads to a mutant phenotype, i.e. smk11 The generation of small particle phenotypes.

[0044] Example 6 ZmSWEET4c 446th Development and application of functional tags

[0045] Primers were designed at both ends of the mutation site using the online dCAPS Finder 2.0 software (http: / / helix.wustl.edu / dcaps / dcaps.html). A mismatch exists in the mutant sequence, which can form a primer that can be bound to the mutation site by a restriction endonuclease. Eco47 The site of restriction enzyme III cleavage was thus identified, allowing for the differentiation between normal and mutated genes. ZmSWEET4c 446th The dCAPS markers are used. The upstream primer sequence dCAPS-F is shown in SEQ ID NO.5, and the downstream primer sequence dCAPS-R is shown in SEQ ID NO.6.

[0046] PCR amplification was performed using the designed primers. The PCR reaction system consisted of 2.5 μL of 10×HiFi Buffer I, 0.5 μL of HiFi, 1 μL each of the forward and reverse primers, 2 μL of DNA template, 0.5 μL of 2.5 mM dNTPs, and was brought to a final volume of 25 μL with ddH2O.

[0047] The PCR amplification program was as follows: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 10 s, 68℃ annealing for 30 s, 72℃ extension for 1 min, 5 cycles; 94℃ denaturation for 10 s, 66℃ annealing for 30 s, 72℃ extension for 1 min, 5 cycles; 94℃ denaturation for 10 s, 64℃ annealing for 30 s, 72℃ extension for 1 min, 25 cycles; for a total of 35 cycles, a final extension at 72℃ for 5 min.

[0048] Use restriction endonucleases Eco47 The PCR product was digested with restriction enzyme III (5'-AGC↓GCT-3'). Agarose gel electrophoresis results showed that after digestion with restriction endonucleases, the mutant DNA fragment was 25 bp smaller than the wild type, therefore the mutant band migrated faster. Figure 6 ).

[0049] The above results demonstrate that the functional markers used in this application for identification of the materials... ZmSWEET4c 446th The gene is accurate and reliable, which can promote the application of molecular marker-assisted breeding of maize with high lysine content, and can quickly achieve seedling selection of materials with high lysine content, thereby shortening the breeding cycle.

[0050] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0051] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of its inventive concept. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A maize hexose transport mutant gene ZmSWEET4c 446th The CDS sequence of which is shown as SEQ ID NO.

1.

2. A maize hexose transport mutant gene ZmSWEERT4c 446th the amino acid sequence of which is shown as SEQ ID NO.

2.

3. A dCAPS primer for identifying a mutant gene of the group consisting of ZmSWEET4c 446th characterized in that, The primer sequences thereof are as follows: dCAPS-F: 5'-TTCGGCACCGGCATGTACGCAGCGC-3', dCAPS-R: 5'-TGTAGTAGTAGCTACAGCGGCT-3'.

4. A kit comprising the dCAPS primers and restriction endonuclease of claim 3. Eco47 III.

5. Use of the dCAPS primer of claim 3 or the kit of claim 4 in identifying and / or aiding in the screening of breeding for lysine content trait in maize kernels.

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