WO2023207932A1 - 控制玉米蛋白含量和氮高效的关键基因 - Google Patents

控制玉米蛋白含量和氮高效的关键基因 Download PDF

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WO2023207932A1
WO2023207932A1 PCT/CN2023/090463 CN2023090463W WO2023207932A1 WO 2023207932 A1 WO2023207932 A1 WO 2023207932A1 CN 2023090463 W CN2023090463 W CN 2023090463W WO 2023207932 A1 WO2023207932 A1 WO 2023207932A1
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corn
gene
thp9
protein
seq
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巫永睿
黄永财
王海海
路小铎
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中国科学院分子植物科学卓越创新中心
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Definitions

  • the invention belongs to the field of agricultural genetic engineering and relates to the application of wild corn asparagine synthase 4 gene Thp9 in improving corn grain protein content, plant total nitrogen content and/or nitrogen efficiency.
  • the quality of corn directly affects the yield and quality of meat and milk, and is an important determinant of the development level of animal husbandry.
  • protein nutritional quality total protein
  • the common corn protein content in production is between 7% and 9%.
  • protein must be supplemented by additional soybean meal, etc., which greatly increases the feed cost. Therefore, increasing the protein content of corn kernels will reduce or even eliminate it in the feed. Adding soybean meal is an important way to promote the healthy development of my country's feed industry and animal husbandry.
  • the endosperm of corn kernels is the main storage organ of nutrients, among which starch and protein are the two most important storage substances.
  • the total protein content of common corn inbred lines is about 10% and starch is about 70% (Flint-Garcia et al., 2009). Proteins are divided into zein, albumin, globulin and glutenin according to their solubility (Wu and Messing, 2017). When corn is used as feed and grain, the abundance and amino acid composition of different types of proteins vary greatly, which determines the nutritional quality of corn.
  • the main storage protein of corn is gliadin, or zein, which accounts for more than 60% of the total protein.
  • ⁇ -zein has the highest abundance, accounting for more than 50% of the total zein content (Esen, 1987; Thompson and Larkins, 1994). However, almost all zeins do not contain the essential amino acids lysine and tryptophan, which results in total corn endosperm protein being extremely deficient in these two amino acids (Mertz et al., 1964).
  • Opaque2 O2 is an important transcription factor in corn endosperm.
  • the expression of gliadin zein decreases by more than 60%.
  • the expression of non-gliadin proteins is compensatoryly upregulated, and the final total protein content is only slightly decline.
  • Non-gliadin is rich in lysine, so the lysine content in the O2 mutant is about twice that of ordinary corn; mice fed with O2 corn grew significantly faster than the control (with ordinary corn) (Mertz et al. ,1965).
  • O2 is a powdery endosperm, and the kernels are easily broken and susceptible to mildew and diseases.
  • the total protein content is low (about 8-9%) and the yield is low, so it cannot be directly industrialized and planted for utilization. Creating new high-quality, high-protein corn will have great significance in terms of food production and safety.
  • wild corn is a new method for creating high-protein corn.
  • Gene donor resources with excellent germplasm introducing wild corn as a donor into cultivated corn is a method to increase the protein content of corn.
  • Ames21814 hereinafter represented by Ames21814 or Teosinte (Teo)
  • Teo Teosinte
  • the protein content of wild corn Ames21814 reaches 30%, and both ⁇ -zein and non-zein parts rich in nutritional lysine content are significantly increased, which is a good choice for improving One of the representatives of natural donors with high zein content.
  • This gene can not only significantly increase corn protein content and biomass, but also increase the nitrogen utilization efficiency of corn and reduce the amount of nitrogen fertilizer used.
  • a molecular marker for this gene introduced the wild corn high-protein gene Thp9 into cultivated corn, and created new high-protein corn germplasm resources. Accordingly, the present invention includes the following technical solutions.
  • the first aspect of the present invention is to provide the application of wild corn asparagine synthase 4 genes such as Thp9 in improving corn grain protein content, plant total nitrogen content and/or nitrogen efficiency.
  • the application of the wild corn asparagine synthase 4 gene such as Thp9 can be selected from the following methods: introducing the wild corn asparagine synthase 4 encoding gene such as Thp9 into the common corn chromosome; overexpressing wild corn in corn Asparagine synthase 4 genes such as Thp9; make corn overexpress the original asparagine synthase 4 gene ZmASN4 of ordinary corn; introduce the regulatory region that controls the expression of wild corn asparagine synthase 4 gene into corn to increase the expression of this gene Increase, thereby increasing the activity of asparagine synthase 4 or enhancing the expression of asparagine synthase 4, thereby increasing the asparagine content in corn.
  • the above wild maize asparagine synthase 4 encoding gene is a mutant of the original asparagine synthase 4 of common maize.
  • the above-mentioned original asparagine synthase 4 gene ZmASN4 of common corn refers to Zm00001d047736 for common cultivated corn such as inbred corn B73.
  • nucleotide sequence of the above-mentioned wild maize asparagine synthase 4 gene, such as Thp9 is selected from the following group:
  • SEQ ID NO:1 The polynucleotide shown in SEQ ID NO:1 is derived from wild corn teosinte (Zea mays ssp. Parviglumis, Ames21814), named Thp9 (Teosinte high protein locus in 9th chromosome), and the gene number is Teo09G002926, NCBI Genome submission:SUB11272093;
  • (B) A polynucleotide with a homology of ⁇ 80%, ⁇ 85%, ⁇ 90%, preferably ⁇ 95%, and more preferably ⁇ 98% with the nucleotide sequence shown in SEQ ID NO:1.
  • the wild corn asparagine synthase 4 is a polypeptide selected from the following group:
  • polypeptide derived from (a) that is formed by substituting, deleting or adding one or more amino acid residues to the SEQ ID NO:2 amino acid sequence and having the function of (a) polypeptide;
  • SEQ ID NO:2 has the following amino acid sequence:
  • amino acid sequence of asparagine synthase 4 expressed in the inbred maize line B73 is:
  • the method of introducing wild maize asparagine synthase 4 encoding genes such as Thp9 into the maize chromosome includes the following steps:
  • the expression vector of the gene is transformed into young corn embryos using Agrobacterium-mediated method to obtain transgenic corn that overexpresses the gene.
  • step (2) can be to transform the expression vector of the gene into Agrobacterium competent cells after sequencing verification.
  • Cells use transformants to transform young corn embryos; after corn culture and growth, positive plants are obtained through identification at the genome level and transcription level.
  • the pCAMBIA vector may be a pCAMBIA3300 vector driven by the maize Ubiquitin promoter.
  • the ZmASN4 gene can be loaded downstream of the Ubiquitin promoter.
  • the second aspect of the present invention is to provide a kit for implementing the above application, which includes: the Thp9 gene fragment of SEQ ID NO: 1 or its CDS sequence SEQ ID NO: 3, and is used to combine the gene fragment or its CDS sequence SEQ ID NO: 3. PCR primers required for cloning CDS sequences into plant expression vectors;
  • the above-mentioned gene expression vector contains: the above-mentioned gene expression vector, a reagent for transferring the gene expression vector into Agrobacterium;
  • Agrobacterium transformed into the above gene expression vector may include: Agrobacterium transformed into the above gene expression vector, and a reagent for transforming Agrobacterium into plants.
  • a third aspect of the present invention provides a method for detecting the above-mentioned genes in the corn genome, including the following steps:
  • the PCR product is 198 bp SEQ ID NO: 4, which is Thp9 high protein Molecular markers of sites:
  • PCR product is 176bp SEQ ID NO:5, which is also a molecular marker of the Thp9 high protein site:
  • the PCR product is 151 bp SEQ ID NO: 6, which is the molecular marker of the maize B73 gene Zm00001d047736:
  • the forward primer asn4-is-F CCGTTCCTCGACAAGGAGTT
  • the reverse primer asn4-is-R ATCAGAGCTGAAAGTGGGGC
  • the PCR product is 455 bp SEQ ID NO :7, which is wild corn Molecular markers inserted into the Ames21814 genotype:
  • PCR detection can be carried out using ordinary PCR MIX and programs. If a band can be amplified, it means that the wild maize Ames21814 genotype is inserted, and it cannot be amplified. If it is out, it means it does not carry the high protein site.
  • the fourth aspect of the present invention is to provide a kit for implementing the above method, which includes corresponding primers for detecting SEQ ID NOs: 4-7, or DNA/RNA probes, or a microarray of DNA/RNA probes chip.
  • the present invention can not only increase the protein content of corn, but also promote the nitrogen efficiency of corn to improve the utilization rate of nitrogen fertilizer, which is suitable for new high-protein corn germplasm.
  • Resource creation, agricultural production and environmental protection are all of great economic significance and have broad application prospects.
  • Figure 1 shows the phenomenon of strong selection of protein content during the process of domestication of wild corn to cultivated corn.
  • a a schematic diagram of the selection of cultivated corn inbred lines from wild corn with tillers after about 9000 years of domestication
  • b the analysis and determination of more than 30 samples of two types of wild corn, Parviglumis and Mexicoana, and the grain protein content is about 28.6% ⁇ 1.0 %, while the protein content of 405 cultivated corn inbred lines is 6.5%-16%, with an average value of 11.52%
  • c representative wild corns (registration numbers: Ames and PI series) are selected for alcohol-soluble and non-gliadin proteins Analysis showed that the gliadin and non-gliadin contents of wild corn were significantly higher than those of the control B73 inbred line
  • d the free amino acid asparagine content in the roots, stems and leaves of wild corn Ames21814 was significantly higher than that of the control B73.
  • Figure 2 shows the results of gliadin analysis of 500 inbred lines from the natural population and GWAS analysis of ⁇ -zein population variation.
  • a analysis of 500 inbred line gliadins from natural populations
  • b grading of ⁇ -zein content with the largest change in zein content of 500 inbred lines, and the difference in ⁇ -zein content between 19 and 22-kD.
  • Genome-wide association analysis GWAS was performed after three levels (19-kD content higher than, equal to, and lower than 22-kD ⁇ -zein).
  • GWAS results show The main site of difference in the content of 19-kD ⁇ -zein and 22-kD ⁇ -zein is located on the short arm of chromosome 4.
  • Figure 3 shows the results of third-generation sequencing assembly of wild maize genome and gliadin copy number analysis.
  • a wild corn Z. mays ssp. Parviglumis Ames21814, F 1 of B73 x Ames21814 and plants of common cultivated corn B73;
  • b wild corn genome assembly flow chart;
  • c high-quality wild corn genome, from outer circle to inner circle Gene density, repeat sequence density, TIR density, Indel number, SNP number, Copia density, Gypsy density, Knob density and GC content are displayed respectively;
  • d gliadin tandem repeat copy number analysis, respectively counting ⁇ -zein different tandem gene repeats Sequence copy numbers of different gene clusters in inbred line B73, wild maize Teosinte and inbred line W22.
  • Figure 4 shows the genetic basis analysis of high protein and the protein detection results during the population construction process.
  • a inbred line B73, wild corn Teo, and seeds of B73 x Teo and B73 x Teo F 2 , the seed protein measurement data are marked above, n is the number of assays;
  • b inbred line B73, wild corn Teo , and SDS-PAGE gel analysis of B73 x Teo gliadin ;
  • c seed gliadin analysis of B73
  • the picture shows the gliadin analysis of 12 separate fruit ears, B73 is the control; e, F 1 BC 2 population, 12 grains were taken from the same high-protein fruit ear for separate protein content analysis, B73 is the control; F, F 1 BC 3 population
  • the protein content of 30 ears was measured, and the total protein content of the grains showed a separation of 10% and 15%;
  • g 8 high-protein ears of the F 1 BC 3 population, 7 grains were taken from each for protein measurement, and the protein content of each grain on
  • FIG. 5 shows the results of localization and expression analysis of the Thp9 gene.
  • a G'value analysis of BSA mapping of F 1 BC 4 population
  • b analysis of three BSA sequencing infiltrated genes of F 1 BC 4 , F 1 BC 6 and F 1 BC 8
  • c Thp9 map cloning and Thp9 mapping
  • Thp9 Teosinte high protein locus in 9th chromosome, gene number is Teo09G002926, NCBI Genome submission: SUB11272093
  • d Schematic diagram of ASN4 transcripts in B73 and wild maize
  • e Statistics of ASN4 transcripts in B73 and wild maize in root and leaf RNA-Seq sequencing
  • f Near et al.
  • ZmAsn4 is significantly highly expressed in the roots and leaves of NILTHP9; g, ZmASN4 protein analysis of the roots and leaves of the near-isogenic line NILTHP9 and the control NILB73.
  • Figure 6 shows the results of linked marker development and phenotypic analysis of Thp9 wild maize high protein variation sites.
  • a F 2 BC 7 population ear
  • Asn4-B73 represents Thp9 as B73 genotype
  • Asn4-H represents Thp9 as heterozygous genotype
  • Asn4-Teo represents Thp9 as homozygous wild maize genotype
  • b F 2 BC Grain protein of different Thp9 genotypes in 7 populations of fruit ears Content determination
  • Asn4-B73 represents Thp9 as B73 genotype
  • Asn4-H represents Thp9 as heterozygous genotype
  • Asn4-Teo represents Thp9 as homozygous wild maize genotype
  • c F 2 BC 7 population ears with different Thp9 genotype roots
  • Asn4-B73 represents the B73 genotype of Thp9
  • Asn4-H represents the heterozygous genotype of Thp9
  • Asn4-Teo represents the homozygous wild maize
  • Figure 7 shows the results of phenotypic analysis of the near-isogenic line NILTHP9.
  • a the grain protein content of the near-isogenic line NILTHP9 and the control NILB73 in different ecological zones of Shanghai, Sanya and Northeast China
  • b the total nitrogen content in the roots, stems and leaves of the near-isogenic line NILTHP9 and the control NILB73
  • c the near-isogenic line NILTHP9 and the control NILB73 grain protein content in different ecological zones in Shanghai, Sanya and Northeast China Determination of free amino acid asparagine content in roots of isogenic line NILTHP9 and control NILB73
  • d near-isogenic line NILTHP9 and control NILB73 plants
  • e determination of plant height of near-isogenic line NILTHP9 and control NILB73, plants planted in Sanya in 2021
  • f Fresh weight of leaves, stems and whole plants of the near-isogenic line NILTHP9 and the control
  • Figure 8 shows the results of genetic validation of Thp9.
  • a the relative expression of Thp9 in the roots of two independent transgenic events that overexpress Thp9, OE-1 (Overexpression-1) and OE-2 (Overexpression-2);
  • b two independent transgenic events that overexpress OE-1 and OE-2 leaves;
  • c overexpression of two independent transgenic events OE-1 and OE-2 THP9 immunoblot in roots;
  • d overexpression of two independent transgenic events OE-1 and OE- 2 Determination of grain protein content;
  • e GWAS analysis of grain protein content of 405 and 438 inbred lines in 2019 and 2020, respectively, showing a significant signal at ASN4 on chromosome 9;
  • f ASN4 in natural populations Schematic diagram of the gene structure of three haplotypes, among which HAP1 is the Thp9 haplotype of wild maize, HAP3 is the B73 haplotype and is 47bp less than HAP1, and HAP2 is 22bp less than HAP
  • Figure 9 shows the results of the nitrogen efficiency test of the near-isogenic line NILTHP9 at the Shanghai Experimental Base in 2020.
  • a the plants of NILB73 and NILTHP9 under normal nitrogen application and no nitrogen application
  • b the roots of NILB73 and NILTHP9 plants under normal nitrogen application and no nitrogen application
  • c the expression of ASN4 gene is induced by nitrogen application.
  • ASN4 in the near-isogenic line of NILTHP9 under no nitrogen fertilization reaches the expression of ASN4 in NILB73 under normal nitrogen fertilization; d, biomass measurement of aboveground plants of NILB73 and NILTHP9 under normal nitrogen fertilization and no nitrogen fertilization; e, Determination of the biomass of underground roots of NILB73 and NILTHP9 under normal nitrogen fertilization and no nitrogen fertilization; f, Determination of total biomass of NILB73 and NILTHP9 under normal nitrogen fertilization and no nitrogen fertilization; g, Determination of grain protein content.
  • Figure 10 shows the results of the nitrogen efficiency test of the near-isogenic line NILTHP9 at the Sanya Experimental Base in 2020.
  • a 0%, 25%, 50% and 100% (the level of 100% is, fertilizer is applied once in the seedling stage and once in the jointing stage, a total of two times, each time 0-4-8-16g/plant , the nitrogen content is 17%, and other levels are reduced in sequence, with a planting density of 0.6m Determination of plant height of NILB73 and NILTHP9; c, determination of aboveground biomass of NILB73 and NILTHP9 under four different nitrogen application levels; d, determination of total nitrogen content of roots of NILB73 and NILTHP9 under four different nitrogen application levels; e, four types of Determination of total nitrogen content in leaves of NILB73 and NILTHP9 under different nitrogen application levels; f, determination of total nitrogen content in stems of NILB73 and NILTHP9 under four different nitrogen application levels; g, protein content in grains
  • Figure 11 shows the test results of Thp9 hybrids and improved new varieties to create high-protein corn.
  • a the phenotype of F 2 hybrids created with NILB73 and NILTHP9 and Mo17
  • b the comparison of 100-kernel weight and protein content determination of hybrids carrying Thp9
  • c the creation of high-protein Zhengdan 958 by improving Thp9 Dan 958THP9 plant
  • d Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 hybrid fruit ear
  • e Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 and control Zhengdan 958 fresh weight determination of the shoot
  • f Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 hybrid fruit ear
  • f Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 and control Zhengdan 958 fresh weight determination
  • Determination
  • High protein content corn is an important germplasm resource for modern hybrid corn breeding and is also an important agronomic trait. Since high protein in maize is controlled by small-effect polygenes and the genetic mechanism is complex, the quantitative trait loci QTL that control its formation in natural populations are difficult to clone and have not been reported so far. At the same time, the formation mechanism of high protein maize is also unclear, so high The genetic improvement of protein corn and the expansion of genetic germplasm are very slow and difficult. Not only is it time-consuming, labor-intensive, inefficient, but the progress is very slow and the direction is unclear. Each generation also requires the huge and tedious work of measuring protein content.
  • the inventor cloned the maize high-protein gene Thp9 from wild maize (Ames21814) for the first time.
  • the allele of this gene in the B73 genome is ZmASN4, and the gene number is Zm00001d047736.
  • Wild corn Thp9 can significantly increase the protein content of corn kernels and plants, increase the biomass of corn, and improve corn quality. Nitrogen fertilizer use efficiency of rice.
  • the asparagine synthase 4 gene ASN4 studied in this article includes both the asparagine synthase 4 gene derived from common cultivated corns, such as Zm00001d047736 derived from inbred corn B73, and the nucleotide sequence derived from wild corn teosinte. Thp9 of SEQ ID NO:1.
  • the present invention also developed molecular markers to identify Thp9 high-protein sites including 198 bp SEQ ID NO: 4 and 176 bp SEQ ID NO: 5, which facilitates the use of this high-protein site. Genetic improvement of common corn.
  • the present invention in order to detect whether the Thp9 gene is inserted into the maize genome, the present invention further developed the molecular marker SEQ ID NO: 7 to identify the high protein site of Thp9 in natural populations, which is used to screen excellent allelic variations in maize natural populations.
  • molecular markers are very convenient to use and can be completed by those of ordinary skill in the field through simple molecular biology experiments. For example, by extracting genomic DNA from corn leaves, performing a PCR reaction with the above-mentioned mutation site-specific primers we developed, and then using the provided primers for sequencing, the above-mentioned mutation sites can be detected.
  • the ASN4 gene application and detection objects of the present invention are applicable to all natural populations of corn, including but not limited to existing wild species, inbred lines, farm varieties and hybrids.
  • the application method and the target gene detection method of the present invention can be carried out using a kit, and the required materials are concentrated in one kit.
  • the above kit may also include respectively At least one of the following: a carrying tool divided into defined spaces that can accommodate one or more containers, 96-well plates or strips, such as test kits, vials, test tubes, and the like , each container containing a separate component for use in the method of the invention; instructions, which may be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of the container, For example, it is paper with an operation demonstration video APP download window such as a QR code.
  • the instructions can also be in the form of multimedia, such as CD, U disk, network disk, IC card, etc.
  • ASN4 genes including Zm00001d047736 and Thp9
  • over-expression for example, through natural variation and artificial mutagenesis (all mutagen mutagenesis and genetic engineering methods) leading to variation in the promoter region of wild corn or common corn ASN4 gene, and the distal regulatory region of the gene causing gene
  • the expression level is increased; by screening the upstream regulatory factors of maize ASN4 and their mutations, the expression level of the ASN4 gene is increased.
  • the key gene Thp9 that controls high protein and nitrogen efficiency in corn that we cloned from wild corn Ames21814 includes at least the following advantages:
  • Thp9 has very strong function and good genetic stability. Introducing Thp9 hybridization of wild corn into different corn inbred lines can increase the grain protein content, which is theoretically effective for most inbred lines, which will greatly expand high-protein corn germplasm resources. Moreover, the genetic stability of Thp9 is very good, and the phenotype is very stable in Northeast, Shanghai and Sanya.
  • Thp9 The time required to improve high-protein corn using Thp9 is short. Since the genes and mechanisms that control the formation of high-protein corn are unclear, conventional genetic improvement of high-protein corn requires large-scale field investigations at multiple sites over many years to obtain stable materials. Now when improving high-protein corn, as long as it is crossed with other inbred lines and identified through the molecular markers SEQ ID NOs: 4-5 and 7 we developed, plants carrying excellent Thp9 loci can be obtained; Crossing or crossing other corn pollens will produce seeds that are all high-protein corn. Moreover, during the import process, at least one ear only needs to be retained for hybridization and selfing to obtain stable hard grain material, which can greatly save workload.
  • Thp9 to improve high-protein corn is simple and suitable for large-scale operations.
  • the technology of corn hybridization and selfing is relatively simple and can be mastered by ordinary workers.
  • the corn leaf DNA extraction, PCR reaction and sequencing involved are all routine molecular experiments and can be completed in ordinary laboratories and sequencing companies.
  • Thp9 to improve high-protein corn has low cost. Compared with traditional corn genetic improvement, using Thp9 to improve high-protein corn has strong effects, good stability, shortens time, and reduces workload, thus greatly saving costs. In addition, molecular experiments for Thp9 genotype identification are also routine and low-cost. Traditional identification of grain protein content requires the use of a nitrogen analyzer to measure the protein content of each ear. The nitrogen determiner is expensive and has low throughput. It also costs more than 500,000 yuan to purchase a Rapid N nitrogen analyzer, and maintenance during the period is complicated. Consumables are also expensive.
  • Thp9 can be used to create high-protein hybrid corn.
  • Thp9 is a natural main locus that increases protein content. Introducing the wild corn Thp9 locus into corn inbred lines and hybrid parents can significantly increase the protein content and biomass of inbred lines and hybrids. Our study found that the nitrogen content in the grains, stems and roots of the near-isogenic line NILThp9, which contains the high-protein site Thp9, is significantly higher than that of the near-isogenic line NILB73, which does not contain the high-protein site. At the same time, it is excessive in B73 Expression of Thp9 can significantly increase the total nitrogen content of grains, stems and roots.
  • Thp9 has high nitrogen efficiency and can significantly improve the nitrogen use efficiency of corn and reduce the use of nitrogen fertilizer. It is an important gene that starts a new green revolution in corn and other crops.
  • Molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly refer to "Molecular Cloning Experiment Guide” (Third Edition), J. Sambrook, Edited by D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, specific experimental conditions can be determined through simple experiments.
  • PCR amplification experiments were performed according to the reaction conditions provided by the reagent supplier or the kit instructions. If necessary, it can be adjusted through simple experiments.
  • Example 1 Measuring corn protein content and amino acid content, mining high-protein wild corn donor materials
  • corn kernels that need to be measured for total protein content are dried in an oven at 60°C, and then ground into powder through a crushing instrument.
  • the dry 50-70 mg corn powder is weighed to make a test sample, which is passed through Dumas Rapid of German Elementar Company.
  • Total protein was measured using a rapid nitrogen analyzer (rapid N exceed).
  • Sample pretreatment dry the sample at 65°C, grind it, and pass it through a 100-mesh screen. Weigh an appropriate amount of sample, add distilled water and shake for 1 minute, soak at 4°C for 8 hours, then add steel balls and homogenize.
  • Example 2 Analysis and determination of protein from 500 inbred lines in natural population
  • 500 corn inbred line seeds and corresponding genotype data were provided by the laboratory of Professor Lai Jinsheng of China Agricultural University.
  • the gliadin content of 500 corn inbred lines was analyzed by SDS-PAGE gel electrophoresis.
  • the ⁇ -zein content with the largest change in zein content was classified into three grades.
  • the difference in 19 and 22-kD ⁇ -zein content was divided into three levels.
  • a genome-wide association analysis GWAS was performed. The method of genome-wide association analysis was based on the method published in our laboratory (Liu et al., PNAs, 2015).
  • Example 3 3rd generation sequencing and assembly of high-quality genome of wild corn Ames21814
  • the final chromosome mounting rate was 91.30%, the genome size was 2460Mb, and the contig N50 was 62.29Mb, scaffoldN50 reaches the gold level of 243.71Mb wild corn genome.
  • BUSCO estimates that the wild maize genome is 96.8% complete.
  • the wild maize genome contains 80.80% repetitive sequences, of which LTR transposons are the main transposable elements, accounting for approximately 61.48% of the genome.
  • the wild maize genome has annotated 58,092 genes, encoding 108,712 transcripts.
  • the sequence of the wild maize genome assembly has been uploaded to NCBI (Genome submission: SUB11272093).
  • Example 5 BSA sequencing and map-based cloning of the key gene Thp9 that controls the formation of high-protein corn
  • CTAB extraction buffer Chloroform: isoamyl alcohol (24:1): 500mL chloroform, add 20.8mL isoamyl alcohol, and mix well.
  • the filtered high-quality sequencing data was compared to the B73 reference genome, and GATK software was used for SNP detection.
  • the F 1 BC 6 population was used for high-throughput testing of 150 samples (mixed pool 1) of high-protein type (content about 15%) and 150 samples (mixed pool 2) of B73 type (content about 10%) selected from the isolated population. Sequencing (X-Ten100X, total data volume 500G) sequencing, the analysis method is the same as above.
  • F 1 BC 8 population was used for high-throughput testing of 50 samples (mixed pool 1) of high protein type (content about 15%) and 50 samples (mixed pool 2) of B73 type (content about 10%) selected from the isolated population. Sequencing (X-Ten 50X, total data volume 250G) sequencing. The analysis method is the same as above.
  • Align the reference genome Align the B73 sample, high-protein HP sample and low-protein LP sample data of F 1 BC 4 (the same for F 1 BC 6 and F 1 BC 8 ) to the merged genome of B73 and Teo ;
  • Statistical comparison of the coverage depth on the Teo gene compare the bam files of each sample, use 50kb as the window and 25kb as the step size sliding window to count the coverage depth of each window area;
  • Data Standardization In order to make the coverage depth of different samples comparable to each other, we standardized the coverage depth of each sample (formula: coverage depth of each sample/50kb-B73 sample coverage depth/50kb);
  • Obtain drawing data Subtract the coverage depth of the low-protein LP sample (same for BC4, BC6, and BC8) from the normalized coverage depth of the high-protein HP sample to obtain the difference (Delta), which is used for drawing.
  • Drawing Use the ggplot2 package of R language for drawing.
  • Genome-wide polymorphic markers were designed and developed based on the wild corn genome and the B73 reference genome, and then the developed molecular markers were selected for verification in the high-protein populations of wild corn, B73, and F 1 .
  • the protein content of 2,000 populations was measured, and the genotype genetic exchange information and the grain protein content corresponding to the exchanged individual plants were analyzed.
  • PCR used 2 ⁇ HieffTM PCR Master Mix (Shanghai Yisheng Biotechnology Co., Ltd., 10102ES03) and standard procedures for amplification, and 3% agarose gel for identification.
  • Map-based cloning molecular marker primer information Map-based cloning molecular marker primer information:
  • This 147kb interval was found to contain only The expression level of one gene, teo09G002926, changed significantly (c in Figure 5). Its corresponding B73 version gene is ZmAsn4, gene number Zm00001d047736, encoding asparagine synthase 4, ASN4.
  • Asn4 of wild maize has a 47bp insertion in the 10th exon relative to B73, and this insertion causes the Asn4 gene transcript of wild maize to be different from B73.
  • Example 6 Thp9, a key gene in the formation of high-protein corn, and verification of linked markers
  • the leaf DNA extraction and PCR identification methods are the same as step 5.4 above.
  • the identification primers are:
  • ZmAsn4 is the genotype of B73
  • SEQ ID NO:4 a molecular marker that analyzed 200 individuals in the F 3 BC 7 population, and found that the protein content of ZmAsn4-B73 (ZmAsn4 is the genotype of B73) in the population was significantly low.
  • ZmAsn4-H is the heterozygous genotype
  • wild maize genotype Asn4-Teo is the genotype of wild maize
  • the content of ZmAsn4-H type and Asn4-Teo type was significantly higher than that of ZmAsn4-B73 type (c in Figure 6), indicating that the level of THP9 protein content is linked to this marker.
  • the results further prove that high expression of ZmAsn4 is an important factor in increasing the protein content of corn.
  • the molecular marker SEQ ID NO:4 we developed can be used to identify the imported wild corn high protein site and the variation of this site at the population level.
  • the methods for protein content determination and amino acid content analysis are the same as steps 1.1 and 1.3 above.
  • the protein content of NILTHP9 seeds in Sanya was about 15.39 ⁇ 0.95%, and the control NILB73 was about 11.17 ⁇ 0.95%;
  • the protein content of NILTHP9 seeds in the Northeast is also about 11.96 ⁇ 0.65%, and the control NILB73 is about 9.16 ⁇ 0.52% in the Northeast, which increased by 35.19%, 47.78 and 30.57% in Shanghai, Sanya and Northeast respectively (a in Figure 7 ).
  • NILTHP9 in addition to increasing grain protein content, NILTHP9 also increased the total nitrogen content in plant stems and roots (b in Figure 7).
  • the determination of free amino acid content found that asparagine in NILTHP9 was significantly higher than that in NILB73 (c in Figure 7).
  • the plant height and aboveground biomass of NILTHP9 were significantly increased, the plant height was also relatively increased by 10% (d and e in Figure 7), and the aboveground biomass was increased by 20% (f in Figure 7), indicating that THP9 is excessively accumulated in Asparagus Amides are beneficial to plant growth.
  • THP9 has great potential in increasing corn grain protein content, increasing total nitrogen in silage corn stalks, and increasing plant biomass and plant height.
  • Example 8 Genetic verification shows that Thp9 is a key gene controlling the formation of high-protein corn
  • Thp9 vector was constructed by using wild maize Thp9 cDNA as a template for amplification.
  • the amplification primers were: forward primer ASN4-3300-FlgF:
  • Reverse primer ASN4-3300-R3 gggaaattcgagctcTTACACCGCGATGGCGACAGC.
  • PCR conditions Use Toyobo's KOD-FX-NEO enzyme for PCR amplification.
  • the system follows the KOD enzyme standard mixing system, pre-denaturation at 94°C for 2 minutes, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 68°C for 2 minutes, 35 cycles.
  • the PCR amplified fragment was cloned into the pCAMBIA3300 vector using homologous recombination (ClonExpress II One Step Cloning Kit, C112-02, Novozymes) and placed downstream of the corn Ubiquitin (UBI) promoter to construct an overexpression Thp9 vector. .
  • Agrobacterium tumefaciens EHA105 competent cells were prepared.
  • the overexpression Thp9 vector was transformed into maize B73 immature embryos using Agrobacterium-mediated method to obtain overexpression transgenic maize.
  • the genetic transformation was performed at Weimi Biotechnology (Jiangsu) Co., Ltd.
  • Nitrogen analyzer analyzes the protein content of 500 corn inbred lines in corn grains
  • Thp9 of the wild maize haplotype through the UBI promoter in the B73 background.
  • the expression of Thp9 gene in the roots and leaves of the over-expression transgenic event was significantly increased (a and b in Figure 8); Western blot analysis showed that Thp9 significantly accumulated in the roots of the over-expression transgenic event (c in Figure 8), and the grain protein content increased from the control. 12.08 ⁇ 0.88% increased to 15.18 ⁇ 1.03% for Asn4-OE-1 and 15.81 ⁇ 1.13% for Asn4-OE-2, respectively (d in Figure 8).
  • Thp9 also has great potential in increasing the protein content of corn grains, increasing the total nitrogen of silage corn stalks, and increasing plant height, once again proving the important value of Thp9.
  • the protein content of 405 and 438 inbred lines planted at the Damao Base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan in 2019 and 2020 was determined. It was found that the variation in protein content of the inbred lines in 2019 was 6.5%-16%. , the average value is 11.52%; while the variation of protein content in 2020 is 7.7%-16.8%, and the average value is 12.3%.
  • the materials were uniformly planted in the Damao Base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan.
  • the protein content of the inbred lines was analyzed by GWAS of the protein content of the natural population. It was found that the main locus controlling the protein content of the natural population is still the Thp9 gene located on chromosome 9. site (e in Figure 8).
  • the protein content determination method is the same as step 1.1 above.
  • NILTHP9 near-isogenic line NILTHP9 carrying Thp9 high protein
  • control NILB73 not carrying Thp9 in soil under different nitrogen levels
  • the expression of the ZmAsn4 gene increased significantly with the increase of nitrogen levels; and in low nitrogen
  • the plant height, aboveground and underground biomass and grain protein content of NILTHP9 under the level of no artificial nitrogen fertilizer all reached the normal nitrogen application rate of NILB73 (applied in 2 times, 10g each time at the seedling stage and jointing stage). ) biomass and plant height (a-g in Figure 9). This shows that the high expression of wild maize ZmASN4 gene has the potential for high nitrogen efficiency.
  • Thp9 locus The PCR identification method of Thp9 locus is the same as step 5.3.
  • the primers are thp9-F/R and asn4-is-F/R. See Example 6 for amplification information.
  • Thp9 In order to test the application potential of Thp9, first the Thp9 near-isogenic line (B73 background) and the control NILB73 Hybrids were created with Mo17 respectively, and it was found that the protein content of the hybrids carrying the Thp9 locus was significantly increased (a and b in Figure 11). Furthermore, we backcrossed Thp9 into Zheng 58 and Chang 7-2, the two parents of the main corn hybrid Zhengdan 958.
  • Peng,B.,H.Kong,Y.Li,L.Wang,M.Zhong et al., 2014 OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice. Nat Commun 5:4847.

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Abstract

本发明公开了野生玉米天冬酰胺合成酶4基因Thp9在提高玉米籽粒蛋白含量、植株总氮含量和/或氮高效中的应用，通过提高天冬酰胺合成酶4的活性或者增强天冬酰胺合成酶4的表达，能够提高玉米的蛋白含量和对氮肥的利用率，对于高蛋白玉米新种质资源创制和农业生产及保护环境都具有重大经济意义。

Description

控制玉米蛋白含量和氮高效的关键基因 技术领域
本发明属于农业基因工程领域，涉及野生玉米天冬酰胺合成酶4基因Thp9在提高玉米籽粒蛋白含量、植株总氮含量和/或氮高效中的应用。
背景技术
玉米(Zea mays L.)的品质直接影响肉奶的产量和质量，是影响畜牧业发展水平的重要决定因素。同时，随着人民生活水平的提高，消费者对玉米品质的关注度越来越高，而蛋白营养品质(总蛋白总量)是广泛关注的重要指标。然而生产中普遍的玉米蛋白含量约在7％-9％之间，作为饲料须通过额外添加豆粕等进行蛋白补充，大大提高了饲料成本，因此，提高玉米籽粒蛋白含量，在饲料中降低甚至零添加豆粕，是促进我国饲料工业和畜牧业的健康发展的重要途径。同时，作为青储玉米秸秆总氮的提高，游离氨基酸含量的提高，也将对畜牧业产生重大意义。因此，克隆控制玉米总蛋白含量的基因，解析高蛋白形成的机理，创制高蛋白含量新种质资源是保障粮食安全的重要策略。已报道在水稻上克隆到一个水稻的数量性状基因座(QTL)qPC1，编码的氨基酸转运蛋白OsAAP6与水稻蛋白含量相关，OsAAP6的高表达与高籽粒蛋白含量相关(Peng et al.,2014)。此外，通过测定400多份水稻种质资源的总蛋白以及贮藏蛋白含量，并通过图位克隆与功能研究明确了qGPC-10(OsGluA2)负责编码稻米贮藏蛋白中的谷蛋白前体，能够显著影响稻米蛋白质含量并最终影响稻米的营养品质(Yang et al.,2019)。科学家通过近红外分析测定了2009年和2010年961份群体材料，发现玉米籽粒蛋白含量存在7.32％-15.20％的变异(Karn et al.,2017)，然而至今还未克隆到控制其蛋白含量的基因位点。
玉米籽粒胚乳是营养物质的主要储存器官，其中淀粉和蛋白质是最主要的两种储藏物质。常见玉米自交系的总蛋白含量约为10％，淀粉约为70％(Flint-Garcia et al.,2009)。蛋白质根据溶解性分为醇溶蛋白(zein)、清蛋白(albumin)、球蛋白(globulin)和谷蛋白(glutelin)(Wu and Messing,2017)。玉米作为饲料和粮食时，不同种类蛋白质的丰度和氨基酸构成差异极大，决定了玉米的营养品质。玉米主要储存蛋白是醇溶蛋白，即zein，占总蛋白60％以上。Zein根据氨基酸同源性分为α(19和22-kD)、β(15-kD)、γ(50，27和16-kD) 和δ(18和10-kD)四个亚家族。α-zein丰度最高，占总zein含量50％以上(Esen,1987；Thompson and Larkins,1994)。然而，几乎所有的zein都不含必需氨基酸赖氨酸和色氨酸，这导致玉米胚乳总蛋白极度缺乏这两种氨基酸(Mertz et al.,1964)。Opaque2(O2)是玉米胚乳重要转录因子，在O2突变体中，醇溶蛋白zein表达下降60％以上，然而由于蛋白质的平衡机制使非醇溶蛋白表达补偿性地上调，最终总蛋白含量只有略微下降。非醇溶蛋白中赖氨酸含量丰富，因此O2突变体中赖氨酸含量是普通玉米的两倍左右；用O2玉米饲养的小白鼠明显比对照(用普通玉米)生长快(Mertz et al.,1965)。然而O2是粉质胚乳，籽粒易破碎，且易发霉感病，总蛋白含量偏低(约8-9％)，产量低，因此不能直接进行产业化和种植利用。创制新型优质高蛋白玉米在粮食生产和安全性上都将产生重大意义。
已报道在水稻氮素高效利用研究中克隆出几个影响水稻氮高效的基因，硝酸盐转运蛋白基因NRT1.1B的自然变异是介导籼稻和粳稻氮素利用效率不同的关键因子，籼稻型NRT1.1B等位基因具有氮高效的特性(Hu et al.,2015)。此外，利用全基因组关联分析(genome-wide association study，GWAS)，发现水稻TCP转录因子家族成员基因OsTCP19是控制水稻适应不同土壤氮素高低环境的关键因子(Liu et al.,2021)。同时，研究揭示GA信号通路协同调控水稻生长与氮代谢，水稻转录因子GROWTH-REGULATING FACTOR 4(GRF4)(Li et al.,2018)和APETALA2结构域的转录因子NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5(NGR5)是介导氮素调控分蘖形成的关键因子(Wu et al.,2020)。这些基因的优异等位变异实现了低氮生长条件下的增产、稳产，为水稻氮素高效利用提供了重要资源。然而，如何提高氮肥的利用率或者提高玉米植株的氮素高效感知吸收同化转运，挖掘玉米氮高效基因的挖掘和分子模块是现阶段农业生产上亟待解决的重大科学难题。
发明内容
提高玉米蛋白含量需要具有高蛋白含量的材料作为供体，我们对30余份不同野生玉米进行蛋白含量测定和分析，发现野生玉米蛋白含量约为30％，因此，野生玉米是创制高蛋白玉米新种质优良的基因供体资源，将野生玉米作为供体导入到栽培玉米是一种提高玉米蛋白含量的方法。为了深入解析野生玉米中控制蛋白含量的主效QTL基因位点，从2012年起，我们选用野生玉米大刍草(Zea mays ssp.Parviglumis,Ames21814，下文中用Ames21814或者Teosinte(Teo)表示)作为我们构建群体的导入供体。野生玉米Ames21814蛋白含量达到30％，并且α-zein和富含营养赖氨酸含量的non-zein部分均显著增加，是提 高玉米蛋白含量的天然供体代表之一。历经10年的努力，大量遗传群体的分析，连续10代近等基因系群体的创建，上万份蛋白含量的测定，我们克隆到玉米首个控制总蛋白含量的主效QTL基因位点，并通过三代测序，解析组装了高质量的野生玉米Ames21814基因组序列。本发明挖掘野生玉米中控制高蛋白玉米形成关键基因Thp9，该基因不仅可以显著增加玉米蛋白含量和生物量，同时可以增加玉米的氮素利用效率，减少氮肥使用量。此外，我们还开发出该基因的分子标记，将野生玉米高蛋白基因Thp9导入栽培玉米，培育创制出高蛋白玉米新种质资源。据此，本发明包括如下所述的技术方案。
本发明的第一个方面在于提供野生玉米天冬酰胺合成酶4基因比如Thp9在提高玉米籽粒蛋白含量、植株总氮含量和/或氮高效中的应用。
具体地讲，所述野生玉米天冬酰胺合成酶4基因比如Thp9应用可以选自下组方式：将野生玉米天冬酰胺合成酶4编码基因比如Thp9导入普通玉米染色体中；使玉米过表达野生玉米天冬酰胺合成酶4基因比如Thp9；使玉米过表达普通玉米原有天冬酰胺合成酶4基因ZmASN4；将控制野生玉米天冬酰胺合成酶4基因表达量的调控区域导入玉米使该基因表达量提高，从而提高天冬酰胺合成酶4的活性或者增强天冬酰胺合成酶4的表达，进而增加玉米中天冬酰胺的含量。
上述野生玉米天冬酰胺合成酶4编码基因是普通玉米原有天冬酰胺合成酶4的突变体。
上述普通玉米原有天冬酰胺合成酶4基因ZmASN4对于普通栽培玉米比如自交系玉米B73而言是指Zm00001d047736。
在一种实施方式中，上述野生玉米天冬酰胺合成酶4基因比如Thp9的核苷酸序列选自下组：
(A)如SEQ ID NO:1所示的多核苷酸，其来源于野生玉米大刍草(Zea mays ssp.Parviglumis，Ames21814)，命名为Thp9(Teosinte high protein locus in 9th chromosome)，基因编号为Teo09G002926，NCBI Genome submission:SUB11272093；
(B)与SEQ ID NO:1所示核苷酸序列的同源性≥80％、≥85％、≥90％、优选≥95％、更优选≥98％的多核苷酸。
所述野生玉米天冬酰胺合成酶4是选自下组的多肽：
(a)具有SEQ ID NO:2氨基酸序列的多肽；
(b)将SEQ ID NO:2氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；
(c)与(a)限定的多肽序列有95％以上同源性，优选地98％以上同源性，更优地99％以上同源性，且具有(a)多肽功能的由(a)衍生的多肽；或
(d)序列中含有(a)或(b)或(c)中所述多肽序列的衍生多肽。
其中，SEQ ID NO:2具有如下氨基酸序列：
而自交系玉米B73中表达的天冬酰胺合成酶4的氨基酸序列为：
作为上述应用的一种方式，将野生玉米天冬酰胺合成酶4编码基因比如Thp9导入玉米染色体中的方法包括如下步骤：
(1)将所述野生玉米天冬酰胺合成酶4编码基因比如Thp9克隆到适合于在农杆菌中表达的植物表达载体中，得到该基因的表达载体；
(2)载体经过测序验证后，将该基因的表达载体用农杆菌介导法转化玉米幼胚，获得过表达该基因的转基因玉米。
优选地，步骤(2)可以是将该基因的表达载体经过测序验证后，转化农杆菌感受态 细胞；用转化子转化玉米幼胚；玉米培养生长后经过基因组水平和转录水平鉴定获得阳性植株。
例如，所述pCAMBIA载体可以是以玉米Ubiquitin启动子驱动的pCAMBIA3300载体。ZmASN4基因可加载于Ubiquitin启动子下游。
本发明的第二个方面在于提供一种用于实施上述应用的试剂盒，其包含：SEQ ID NO:1的Thp9基因片段或者其CDS序列SEQ ID NO:3、用于将该基因片段或者其CDS序列克隆入植物表达载体所需的PCR引物；
或者包含：上述的基因表达载体，用于将基因表达载体转入农杆菌中的试剂；
或者包含：转入了上述的基因表达载体的农杆菌，用于将农杆菌转化植株的试剂。
本发明第三个方面提供了一种检测玉米基因组中上述基因的方法，包括下述步骤：
检测核苷酸序列为SEQ ID NO:1的Thp9基因时，正向引物thp9-F：CTCTGTGCCATGCATCCTCC，反向引物thp9-R：CGTCAGCGCTGGTTAGC，PCR产物为198bp的SEQ ID NO:4，其为Thp9高蛋白位点的分子标记：
或者PCR产物为176bp的SEQ ID NO:5，其也为Thp9高蛋白位点的分子标记：
而PCR产物为151bp的SEQ ID NO:6，其为玉米B73基因Zm00001d047736的分子标记：
检测核苷酸序列为SEQ ID NO:1的Thp9基因是否插入玉米基因组时，正向引物asn4-is-F：CCGTTCCTCGACAAGGAGTT，反向引物asn4-is-R：ATCAGAGCTGAAAGTGGGGC，PCR产物为455bp的SEQ ID NO:7，其为野生玉米 Ames21814基因型插入的分子标记：
在检测核苷酸序列为SEQ ID NO:1的Thp9基因是否插入玉米基因组时，PCR检测可以用普通PCR MIX及程序进行，能扩增出条带则为有野生玉米Ames21814基因型插入，不能扩出则为不携带该高蛋白位点。
本发明的第四个方面在于提供一种实施上述方法的试剂盒，其包括用于检测SEQ ID NOs:4-7的相应引物、或者DNA/RNA探针、或者DNA/RNA探针的微阵列芯片。
本发明通过将野生玉米天冬酰胺合成酶4基因Thp9应用于栽培玉米，不仅能够提高玉米的蛋白含量，还能够促进玉米的氮高效从而提高对氮肥的利用率，这对于高蛋白玉米新种质资源创制、农业生产和环境保护都具有重大经济意义，应用前景广阔。
附图说明
图1显示了野生玉米驯化到栽培玉米过程中蛋白含量受到强烈选择现象。其中，a，有分蘖的野生玉米经过约9000年的驯化选择到栽培玉米自交系示意图；b，分析测定30余份两个种类型的野生玉米Parviglumis和Mexiana籽粒蛋白含量约为28.6％±1.0％，而405份栽培玉米自交系蛋白含量为6.5％-16％，平均值为11.52％；c，选取代表性的野生玉米(登记号为Ames和PI系列)进行醇溶和非醇溶蛋白分析，发现野生玉米醇溶蛋白和非醇溶蛋白含量都显著高于作为对照的B73自交系；d，野生玉米Ames21814根、茎和叶中游离氨基酸天冬酰胺含量都显著高于对照B73。
图2显示了自然群体500份自交系醇溶蛋白分析及α-zein群体变异GWAS分析结果。其中，a，自然群体500份自交系醇溶蛋白分析；b，对500份自交系玉米醇溶蛋白含量变化最大的α-zein含量高低划分等级，19和22-kDα-zein含量差异分成三个等级(19-kD含量高于、等于和低于22-kDα-zein)后进行全基因组关联分析GWAS。GWAS结果显示 19和22-kDα-zein含量差异的主效位点在4号染色体短臂。
图3显示了野生玉米基因组3代测序组装及醇溶蛋白拷贝数分析结果。其中，a,野生玉米Z.mays ssp.Parviglumis Ames21814，B73 x Ames21814的F1以及普通栽培玉米B73的植株；b，野生玉米基因组组装流程图；c，野生玉米高质量基因组，从外圈到内分别显示基因密度，重复序列密度，TIR密度，Indel数量，SNP数量，Copia密度，Gypsy密度，Knob密度以及GC含量；d，醇溶蛋白串联重复拷贝数分析，分别统计α-zein不同串联基因重复序列自交系B73，野生玉米Teosinte和自交系W22不同基因簇的拷贝数。
图4显示了高蛋白的遗传基础分析和群体构建过程中蛋白检测结果。其中，a，自交系B73，野生玉米Teo，以及B73 x Teo和B73 x Teo F2的种子，种子蛋白测定数据标注在上方，n为测定份数；b，自交系B73，野生玉米Teo，以及B73 x Teo醇溶蛋白SDS-PAGE胶分析；c，B73 x Teo F2的种子醇溶蛋白分析，F2均为高蛋白，B73为对照；d，F1BC2群体不同果穗蛋白分析图为12个单独的果穗醇溶蛋白分析，B73为对照；e，F1BC2群体同一个高蛋白果穗上取12粒籽粒单独进行蛋白含量分析，B73为对照；F，F1BC3群体30个果穗蛋白测定，籽粒总蛋白含量呈现10％和15％的分离；g，F1BC3群体8个高蛋白果穗，每个取7粒进行蛋白测定，高蛋白果穗上每粒籽粒蛋白含量均～15％，B73作为对照；h，F1BC4群体30个果穗蛋白测定，籽粒总蛋白含量呈现10％和～15％的分离；i，F1BC4群体8个高蛋白果穗，每个取7粒进行蛋白测定，高蛋白果穗上每粒籽粒蛋白含量均～15％，B73作为对照。
图5显示了Thp9基因的定位和表达分析结果。其中，a，F1BC4群体BSA定位G’value分析；b，F1BC4，F1BC6，F1BC8这三次BSA测序渗入基因的分析；c，Thp9图位克隆，Thp9定位在标记143.7与143.8这147kb的区间中，仅包含一个基因表达量发生显著变化，命名为Thp9(Teosinte high protein locus in 9th chromosome，基因编号为Teo09G002926，NCBI Genome submission:SUB11272093)，对应B73中的ZmASN4，Zm00001d047736；d，ASN4在B73和野生玉米中转录本的示意图；e，ASN4在B73和野生玉米中转录本在根(root)和叶(leaf)RNA-Seq测序中统计数量；f，近等基因系NILTHP9和对照NILB73根和叶的转录组分析，ZmAsn4在NILTHP9的根和叶中显著高表达；g，近等基因系NILTHP9和对照NILB73根和叶的ZmASN4蛋白分析。
图6显示了Thp9野生玉米高蛋白变异位点连锁标记开发和表型分析结果。其中，a，F2BC7群体果穗，Asn4-B73代表Thp9为B73基因型，Asn4-H代表Thp9为杂合基因型，Asn4-Teo代表Thp9为纯合野生玉米基因型；b，F2BC7群体果穗不同Thp9基因型籽粒蛋白 含量测定，Asn4-B73代表Thp9为B73基因型，Asn4-H代表Thp9为杂合基因型，Asn4-Teo代表Thp9为纯合野生玉米基因型；c，F2BC7群体果穗不同Thp9基因型根的游离氨基酸天冬酰胺含量测定，Asn4-B73代表Thp9为B73基因型，Asn4-H代表Thp9为杂合基因型，Asn4-Teo代表Thp9为纯合野生玉米基因型。
图7显示了近等基因系NILTHP9表型分析结果。其中，a，近等基因系NILTHP9和对照NILB73籽粒在不同生态区上海、三亚和东北籽粒蛋白含量；b，近等基因系NILTHP9和对照NILB73根、茎和叶叶中总氮含量；c，近等基因系NILTHP9和对照NILB73根中游离氨基酸天冬酰胺含量测定；d，近等基因系NILTHP9和对照NILB73植株；e，近等基因系NILTHP9和对照NILB73的株高测定，植株2021年种植于三亚；f，近等基因系NILTHP9和对照NILB73叶片、茎秆和整株植株的鲜重。
图8显示了Thp9的遗传验证结果。其中，a，过表达Thp9两个独立的转基因事件OE-1(Overexpression-1)和OE-2(Overexpression-2)根中Thp9相对表达量；b，过表达两个独立的转基因事件OE-1和OE-2叶中Thp9相对表达量；c，过表达两个独立的转基因事件OE-1和OE-2根中THP9免疫印迹；d，过表达两个独立的转基因事件OE-1和OE-2籽粒蛋白含量测定；e，2019年和2020年两年分别405份和438份自交系籽粒蛋白含量GWAS分析，显示在9号染色体ASN4处有一个显著的信号；f，自然群体中ASN4的3种单倍型基因结构示意图，其中HAP1是野生玉米Thp9单倍型，HAP3是B73单倍型比HAP1缺失47bp，HAP2比HAP1缺失22bp；g，自然群体中ASN4的3种单倍型蛋白含量分析。
图9显示了近等基因系NILTHP9于2020年上海实验基地的氮高效试验结果。其中，a，正常施氮和不施氮情况下NILB73和NILTHP9的植株；b，正常施氮和不施氮情况下NILB73和NILTHP9的植株的根；c，ASN4基因的表达受到施氮的诱导，且不施氮水平下NILTHP9近等基因系ASN4的表达水平达到正常施氮情况下NILB73中ASN4的表达；d，正常施氮和不施氮情况下NILB73和NILTHP9的地上部分植株的生物量测定；e，正常施氮和不施氮情况下NILB73和NILTHP9的地下部分根的生物量测定；f，正常施氮和不施氮情况下NILB73和NILTHP9的总生物量测定；g，籽粒蛋白含量测定。
图10显示了近等基因系NILTHP9于2020年三亚实验基地的氮高效试验结果。其中，a，0％、25％、50％以及100％(100％的水平为，苗期施了一次，拔节期施了一次，共施肥两次，每次0-4-8-16g/株，含氮量17％，其它水平的依次减少，种植密度0.6m x 0.25m)4个梯度的施氮田间试验，左边均为NILB73，右边为NILTHP9；b，4种不同施氮水平下 NILB73和NILTHP9株高的测定；c，4种不同施氮水平下NILB73和NILTHP9地上部分生物量的测定；d，4种不同施氮水平下NILB73和NILTHP9根的总氮含量测定；e，4种不同施氮水平下NILB73和NILTHP9叶的总氮含量测定；f，4种不同施氮水平下NILB73和NILTHP9茎的总氮含量测定；g，4种不同施氮水平下NILB73和NILTHP9籽粒的蛋白含量测定。
图11显示了Thp9杂交种测试及改良新品种创制高蛋白玉米的测试结果。其中，a，用NILB73和NILTHP9与Mo17创造的杂交种F2果穗的表型；b，携带Thp9杂交种百粒重比较和蛋白含量测定；c，Thp9改良郑单(Zhengdan)958创制高蛋白郑单958THP9植株；d，Thp9改良郑单958创制高蛋白郑单958THP9杂交种果穗；e，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958地上部鲜重测定；f，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958株高测定；e，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958地上部鲜重测定；g，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958籽粒蛋白含量测定；h，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958根总氮测定；i，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958茎总氮测定；e，Thp9改良郑单958创制高蛋白郑单958THP9和对照郑单958叶总氮测定。
具体实施方式
高蛋白含量玉米是现代杂交玉米育种的重要种质资源，同时也是重要的农艺性状。由于玉米高蛋白受微效多基因控制，而且遗传机制复杂，自然群体中控制其形成的数量性状基因座QTL很难克隆，迄今还没有报道，同时高蛋白玉米的形成机制也不清楚，因此高蛋白玉米的遗传改良及其遗传种质的拓展都非常缓慢而且艰难，不仅费时费力、效率低、而且进展非常缓慢方向不明确，每一代还需要测定蛋白含量这项巨大又繁琐的工作。我们通过多年玉米高蛋白遗传规律分析、基因组测序和组装、艰难的基因克隆、以及确凿严谨的遗传验证和田间试验，经过10年的坚持和努力，终于首次从野生玉米中首次克隆到控制玉米高蛋白形成的关键QTL-Thp9，并将其应用于玉米杂交种的创制，积极的实验结果预示该关键基因的开发利用将会大大促进高蛋白玉米的遗传改良及种质资源创新，具有非常广泛的应用前景和经济价值。
发明人首次从野生玉米(Ames21814)中克隆到控制玉米高蛋白基因Thp9，该基因在B73基因组中的等位基因为ZmASN4，基因号为Zm00001d047736。研究表明，野生玉米Thp9可以显著提高玉米籽粒和植株的蛋白含量，可以增加玉米的生物量，同时可以提高玉 米的氮肥利用效率。
本文中研究的天冬酰胺合成酶4基因ASN4既包含普通栽培玉米来源的天冬酰胺合成酶4基因比如自交系玉米B73来源的Zm00001d047736，也包含野生玉米大刍草来源的核苷酸序列为SEQ ID NO:1的Thp9。
在本文中，为了描述简便，有时会将某种蛋白比如天冬酰胺合成酶4与其编码基因ASN4(或Asn4)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如，对于ASN4，用于描述天冬酰胺合成酶功能或类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该酶的基因。
为了将Thp9基因应用于玉米种质改良，本发明还开发了鉴定Thp9高蛋白位点的分子标记包括198bp的SEQ ID NO:4和176bp的SEQ ID NO:5，便于将该高蛋白位点用于普通玉米的遗传改良。
另一方面，为了检测Thp9基因是否插入玉米基因组，本发明进一步开发了鉴定自然群体中Thp9高蛋白位点的分子标记SEQ ID NO:7，用于筛选玉米自然群体中的优良等位变异。
这些分子标记使用很方便，本领域的普通技术人员通过简单的分子生物学实验即可完成。例如提取玉米叶片基因组DNA，用我们开发的上述变异位点特异引物进行PCR反应，然后用提供的引物进行测序，即可检测上述突变位点。
本发明的ASN4基因应用和检测对象适用于所有玉米自然群体，包括但不限于现有的野生种、自交系、农家种和杂交种。
考虑到操作方便目的，作为一种优选方式，本发明的应用方式和目的基因检测方式可以采用试剂盒进行，将所需的材料集中在一个试剂盒中。在优选的实施方式中，上述试剂盒除了包含：ASN4基因片段、ASN4基因扩增PCR引物、植物双元表达载体、限制性内切酶、农杆菌和必要的试剂等之外，还可分别包括下述物品中的至少之一：携带工具，其空间划分为可以收容一种或多种容器、96孔板或板条的限定空间，该容器例如是试剂盒、药瓶、试管、和类似物，每样容器都含有一个单独的用于本发明方法的组分；说明书，其可以写在瓶子、试管和类似物上，或者写在一张单独的纸上，或者在容器的外部或内部，例如是带有操作演示视频APP下载窗口比如二维码的纸件，说明书也可以是多媒体的形式，比如CD、U盘、网盘、IC卡等。
本领域技术人员容易理解，促进ASN4基因(包括Zm00001d047736和Thp9)在玉米 中过表达的方式有多种，例如，通过自然变异和人工诱变(所有诱变剂诱变和基因工程方法)导致野生玉米或普通玉米ASN4基因启动子区变异，基因远端调控区域导致基因表达量上调；通过筛选玉米ASN4上游调控因子及其变异等导致ASN4基因表达量上调。
在现有技术的ASN4基因(包括Zm00001d047736和Thp9)基础上，进一步提高天冬酰胺合成酶4基因的活性也是本技术领域所期盼的，例如，通过自然变异和人工诱变(所有诱变剂诱变和基因工程方法)导致ASN4基因功能获得或改变；通过筛选玉米ASN4的互作蛋白改变ASN4的功能；通过筛选变异导致玉米ASN4的表达量发生改变，或基因功能发生改变。
我们从野生玉米Ames21814中克隆的控制玉米高蛋白和氮高效的关键基因Thp9至少包括如下优点：
1.Thp9的功能非常强并且遗传稳定性好。将野生玉米的Thp9杂交导入不同玉米自交系中，可以将其籽粒蛋白含量提高，理论上对绝大部分自交系都有效，这将大大拓宽高蛋白玉米种质资源。而且Thp9的遗传稳定性非常好，在东北、上海和三亚表型都非常稳定。
2.用Thp9进行高蛋白玉米改良的时间短。由于控制高蛋白玉米形成的基因和机制不清楚，常规的高蛋白玉米遗传改良需要经过多年多点的大规模田间考察才能获得稳定的材料。而现在进行高蛋白玉米改良时，只要和其它自交系杂交，通过我们开发的分子标记SEQ ID NOs:4-5、7鉴定获得携带优良Thp9位点的植株；其长出的果穗不论是自交还是杂交其它玉米花粉，产生的种子全为高蛋白玉米。而且导入过程中，最少只要保留一个穗子进行杂交、自交就可以获得稳定的硬粒材料，可以大大节约工作量。
3.用Thp9进行高蛋白玉米改良的操作简单，适合进行规模化作业。玉米的杂交和自交技术相对简单，普通工人就可以掌握。我们开发了Thp9的分子标记引物SEQ ID NOs:4-5、7，可以便捷地对优良变异的Thp9基因型进行鉴定。而所涉及的玉米叶片DNA提取、PCR反应和测序这些都是常规的分子实验，在普通实验室和测序公司就可以完成。
4.用Thp9进行高蛋白玉米改良成本低。相比传统的玉米遗传改良，用Thp9进行高蛋白玉米改良的效果强、稳定性好、缩短时间、减少工作量，从而大大节约成本。除此之外，Thp9基因型鉴定的分子实验也很常规，成本较低。传统的鉴定籽粒蛋白含量需要用定氮仪对每个果穗进行蛋白含量测定，定氮仪测定的成本昂贵，而且通量低，购买Rapid N定氮仪器也耗费50万余元，期间维护复杂，耗材同样花费大。
5.用Thp9可以用于创制高蛋白杂交种玉米。我们将Thp9基因型导入主栽玉米品种的亲本中，在保持原有杂种优势的基础上可以迅速获得高蛋白玉米的杂交种，进行推广可以 创造巨大的经济和社会效益。我们已将Thp9导入郑单958的2个亲本郑58和昌7-2中，创制了高蛋白玉米杂交品种。
6.Thp9是一个天然的提高蛋白含量的主效位点，将野生玉米Thp9位点导入玉米自交系和杂交种亲本中可以显著提高自交系和杂交种的蛋白含量和生物量。我们的研究发现，含高蛋白位点Thp9的近等基因系NILThp9籽粒、茎秆和根中的氮含量都明显比不含高蛋白位点的近等基因NILB73中的高，同时在B73中过量表达Thp9能显著提高籽粒、茎秆和根的总氮含量。
7.Thp9具有氮高效，可以显著提高玉米的氮素利用效率而降低氮肥的使用量，是开启玉米及其他作物新绿色革命的重要基因。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于举例说明目的，而不是对本发明的限制。此外应理解，在阅读了本发明的构思之后，本领域技术人员对其做出的各种改变或调整，均应落入本发明的保护范围内，这些等价形式同样属于本专利所附权利要求书限定的范围。
实施例
实施例中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。
材料和方法
玉米的自交、杂交、转基因操作、大田育种等按照常规的育种方式进行。
实施例中的引物合成及基因测序皆由上海博尚生物技术有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等，主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据试剂供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
实施例1：测量玉米蛋白含量和氨基酸含量，挖掘高蛋白野生玉米供体材料
1.1定氮仪分析玉米籽粒蛋白含量
将需要测定总蛋白含量的玉米籽粒放于60℃烘箱烘干，然后通过粉碎仪器打磨成粉末，称取干燥的50-70mg玉米粉末制成测试样品，通过德国elementar公司的杜马斯快速 定氮分析仪(rapid N exceed)进行总蛋白的测定。
1.2 SDS-PAGE胶分析醇溶蛋白和非醇溶蛋白
(1)将胚乳在37℃烘箱干燥，研磨机60Hz，60s打磨成粉末，称取100mg研磨干燥好的粉于2mL管中，加入醇溶蛋白提取液1mL，充分混匀，室温放置2h以上或过夜。(2)向2mL管中放入钢珠，研磨机震荡1min，放于桌面20min后，15871g转速下离心15min。(3)取上清100mL于新的1.5mL管中，再分别加入10μL 10％(g/mL)的SDS，真空45℃抽气(选择旋转、抽乙醇溶液)70min。加入100μL ddH2O，放于4℃冰箱过夜，即醇溶蛋白Zein抽提完成。(4)将上述(2)中的剩余液体倒出，再加入1mL醇溶蛋白提取液，震荡，放置2h以上，15871g转速下离心15min，去上清。重复此过程3次，即用醇溶蛋白提取液抽提醇溶蛋白。(5)重复3次后，去除上层液体，将沉淀放于真空45℃抽气(选择旋转、抽乙醇溶液)40min。(6)加入非醇溶提取液1mL，震荡涡旋，放置2h，15871g转速下离心15min，取上层液体100mL于新的管子中，即完成非醇溶蛋白提取。(7)制作15％的SDS-PAGE胶进行蛋白分析。醇溶蛋白上样量为3μL样品加8μL 2 x loading buffer混合，95℃变性5min后上样；非醇溶蛋白上样量为4μL样品加8μL x loading buffer混合，95℃变性5min后上样。(8)电泳180V，70min，样品蓝色平行线刚跑出即可。用考马斯亮蓝染色2h，再用脱色液脱色3次，每45min换一次脱色液。
醇溶蛋白提取溶液
非醇溶蛋白提取溶液

1.3玉米不同组织游离氨基酸含量测定
(1)样本前处理，样本65℃烘干，研磨，过100目筛网。称取适量样本，加蒸馏水震荡1min，4℃浸泡8小时后加钢珠，匀浆。匀浆液4500g离心5min，取上清待用；(2)衍生化过程：取标准品混标(混标浓度见原始数据中S1-S5，氨基酸试剂盒：北京质谱医学研究有限公司，MSLAB50AA，批号：MSLAB50AA211201#；天冬酰胺Asn标准曲线：y＝0.00147x+0.00104(r＝0.9984)；y指标名称Asn，x分析值，r相关系数；混标浓度S1-S5分别为1.25μmol/L,6.25μmol/L,12.5μmol/L,50μmol/L和100μmol/L)、待测样本50μl加50μl蛋白沉淀剂(含NVL)，混匀后13200转冷冻离心4分钟。取上清10μl，加50μl标记缓冲液混匀，瞬离。再加20μl衍生液混匀、瞬离后置55℃恒温衍生15min。衍生后样本置冰箱冷却后混匀瞬离，取50μl上机检测。仪器型号：HPLC-MS/MS；LC液相：戴安公司Ultimate3000；MS质谱仪：美国AB公司：API 3200 Q TRAP；氨基酸试剂盒：北京质谱医学研究有限公司MSLAB50AA，批号：MSLAB50AA170601#；甲醇、已腈等均购自Fisher。
从2012年，我们就开始进行玉米高蛋白供体材料的寻找、分析以及群体构建。为了寻找高蛋白的供体材料，我们分别测定了30余份野生玉米的种子和常见栽培玉米自交系的蛋白含量，发现野生玉米蛋白含量为28.6％±1.0％，而普通玉米自交系蛋白含量为10％(B73作为代表)(图1中a和b)。我们通过SDS-PAGE胶进一步分析了野生玉米和自交系B73的醇溶蛋白和非醇溶蛋白，发现野生玉米醇溶蛋白和非醇溶蛋白相对于B73都显著提高(图1中c)。通过测定野生玉米和B73植株的根、茎和叶的游离氨基酸含量，发现游离氨基酸组分中天冬酰胺含量显著下降(根：野生玉米16611μg/g下降到栽培玉米的4120μg/g；茎：野生玉米13668μg/g下降到栽培玉米的2529μg/g；叶：野生玉米14689μg/g下降到栽培玉米的2946μg/g)(图1中d)。这些证据表明，野生玉米是天然存在的优异的高蛋白供体材料。
实施例2：自然群体500份自交系蛋白分析和测定
2.1 SDS-PAGE凝胶电泳分析500份玉米自交系的醇溶蛋白含量
我们于2014年在哈尔滨种植500份自交系，成熟收获干燥后，每份自交系取3个果穗中部的籽粒，去胚，混合磨样。醇溶蛋白提取和SDS-PAGE胶分析方法如上步骤1.2的 醇溶蛋白提取。
2.2 GWAS关联分析
500份玉米自交系种子和对应的基因型数据由中国农大赖锦盛教授实验室提供。通过SDS-PAGE凝胶电泳分析了500份玉米自交系的醇溶蛋白含量，对玉米醇溶蛋白含量变化最大的α-zein含量高低划分等级，19和22-kDα-zein含量差异分成三个等级(19-kD含量高于、等于和低于22-kDα-zein)后，进行全基因组关联分析GWAS。全基因组关联分析的方法参照本实验室发表文献中的方法(Liu et al.,PNAs,2015)。
通过分析自然群体中自交系的蛋白含量，发现自然群体中也存在高蛋白的主效因子。玉米的主要贮藏蛋白是醇溶蛋白，占总蛋白的60％以上。我们进一步通过SAD-PAGE胶分析500份自然群体的醇溶蛋白(图2中a)，发现不同自交系之间差别很大，并对玉米醇溶蛋白含量变化最大的α-zein含量高低划分等级，进行全基因组关联分析GWAS。α-zein含量差异的主效位点在4号染色体短臂，即19和22-kDα-zein基因拷贝成簇分布的区域，暗示自然群体自交系中醇溶蛋白含量的变化与醇溶蛋白拷贝数有关(图2中b)。那么野生玉米蛋白含量高是因为醇溶蛋白串联重复的拷贝数决定的吗？
实施例3：野生玉米Ames21814高质量基因组3代测序和组装
为了解析复杂的醇溶蛋白串联重复的拷贝数和为下游的基因克隆提供参考序列，我们通过3代测序完成了野生玉米Ames21814高质量基因组的组装和注释(图3中a-c)。我们的测序材料是野生玉米(Ames21814)和B73杂交后的F1，并采用最新的HiFi模式测序后，我们得到6752166条序列，共约104Gb的高质量的CCS序列，读长N50达到了15.4Mb。按照B73基因组大小2.2Gb计算，大约47X的数据量。我们的组装是基于Trio-binning方法，通过借用二代亲本序列提取野生玉米CCS序列后，利用Hifisam和Yak程序组装成contig级别。同时使用375.56Gb的Hi-C数据，通过juicer和bwa mem默认参数，R1和R2分别比对PacBio组装的基因组，根据HiC数据提供的互作信息进行染色体的聚类，排序和两轮错误纠正，最后再将HiC互作矩阵导入到juicebox中进行可视化和手动检查，确定没有异常后导出，在每个contig之间添加500个N，最终染色体挂载率为91.30％，基因组大小2460Mb，contig N50为62.29Mb，scaffoldN50达到243.71Mb黄金级别的野生玉米基因组。BUSCO评估得到野生玉米基因组的完整比例为96.8％。野生玉米基因组中含有80.80％的重复序列，其中LTR转座子是最主要的转座元件，约占基因组的61.48％。野生玉米基因组注释到了58,092个基因，编码108,712个转录本。野生玉米基因组组装完成的序列已经上传至NCBI(Genome submission:SUB11272093)。在获得的高质量基因组的基 础上，我们通过序列比对继续分析和注释了野生玉米所有醇溶蛋白的拷贝，发现野生Ames21814所有醇溶蛋白的拷贝数和B73相比没有明显的变化，说明野生玉米高蛋白形成不是因为醇溶蛋白拷贝数引起的(图3中d)。
实施例4：高蛋白的遗传基础分析和群体构建
野生玉米控制高蛋白的性状是如何遗传的呢？为了解析这个问题，我们将野生玉米Ames21814与B73构建遗传群体来进行分析。首先，以B73当做母本，获得的F1籽粒蛋白含量为11.6±0.8％，同母本B73蛋白含量10.8±1％相似。我们继续将F1自交以及将其花粉再授到B73上，F1自交获得的F2种子蛋白含量为19.9±1.2％，而且F2不同种子间的蛋白含量没有变化，说明控制高蛋白形成的因子是由母体植株的基因型决定的(图4中a-c)。我们再将收获的F1BC1继续种植，接下来均用B73作为父本进行回交，对收获的F1BC2(图4中d和e)，F1BC3(图4中f和g)和F1BC4(图4中h和i)群体均进行蛋白分析，发现蛋白含量的高低均以果穗为单位进行分离，而回交群体中不同果穗之间蛋白含量发生10％至15％的变异，而同一个果穗上的不同籽粒蛋白含量是一样的(图4中d-i)。以上的遗传学群体构建和蛋白分析，进一步证明控制蛋白含量的因子是由母体植株的基因型决定的，并且野生玉米导入栽培玉米自交系能提高蛋白含量，成为创制高蛋白玉米重要的策略。基于此遗传学基础，我们测定每一代回交的遗传群体，选择高蛋白的果穗继续种植，用B73不断进行回交，创建高代的近等基因系用于后续高蛋白基因的定位。
实施例5：BSA测序和图位克隆控制高蛋白玉米形成的关键基因Thp9
5.1叶片DNA提取
(1)取玉米叶片放于2mL管中，加入钢柱，液氮处理后进行研磨(60Hz，60s)。(2)研磨后，加入0.6mL CTAB提取缓冲液，混匀。(3)放入65℃烘箱60min，期间每10-15min混匀一下。(4)取出置于室温5-10min，加入等体积的氯仿：异戊醇(24：1)至离心管，密封后摇晃5min。(5)室温15871g转速下离心15min，吸取上清至新1.5mL离心管中。(6)加等体积异丙醇，来回颠倒混匀，放置-20℃20min。(7)室温15871g转速下离心1min，倒掉上清。用1mL的75％乙醇洗DNA沉淀1至2次，每次15871g转速下离心1min后，将乙醇倒出。(8)短暂离心，吸出多于液体，在室温中晾干DNA沉淀。(9)加入0.3mL H2O溶解DNA沉淀。
CTAB提取缓冲液


氯仿：异戊醇(24:1)：500mL氯仿，加入20.8mL的异戊醇，混匀即可。
5.2 BSA测序和分析
对F1BC4、F1BC6和F1BC8群体进行使用定氮仪(方法同上述步骤1.1)进行蛋白含量测定，选取每套群体2个极端表型相同数量的个体进行DNA的提取，等量混池进行BSA测序。在F1BC4群体中，对分离群体中选出的高蛋白型(含量约15％)100个样本(混池1)，B73型(含量约10％)100个样本(混池2)进行高通量测序(X-Ten 100X，总数据量500G)，轮回亲本B73重测序(30X，数据量75G)。将过滤后的高质量测序数据比对到B73参考基因组，采用GATK软件进行SNP检测，通过计算两个混池间和与亲本间的SNP index差值，分析与高蛋白性状紧密连锁的区段(该方法可参考发明人之前发表的论文Huang et al.,Plant Cell，2019)。F1BC6群体对分离群体中选出的高蛋白型(含量约15％)150个样本(混池1)，B73型(含量约10％)150个样本(混池2)进行高通量测序(X-Ten100X，总数据量500G)测序，分析方法同上。F1BC8群体对分离群体中选出的高蛋白型(含量约15％)50个样本(混池1)，B73型(含量约10％)50个样本(混池2)进行高通量测序(X-Ten 50X，总数据量250G)测序。分析方法同上。
5.3 F1BC4、F1BC6和F1BC8绘图及数据分析
(1)比对参考基因组：将B73样本，F1BC4的高蛋白HP样本和低蛋白LP样本数据(F1BC6和F1BC8同)比对到B73和Teo合并后的基因组上；(2)统计比对到Teo基因上的覆盖深度：将每个样本比对后的bam文件，以50kb为窗口，25kb为步长滑窗统计每个窗口区域的覆盖深度；(3)数据标准化：为了使不同样本间的覆盖深度可以互相比较，我们将每个样本的覆盖深度进行了标准化(公式：每个样本覆盖深度/50kb-B73样本覆盖深度/50kb)；(4)获得绘图数据：用高蛋白HP样本标准化后的覆盖深度减去低蛋白LP样本的(BC4、BC6和BC8同)，获得差值(Delta)，用于绘图。(5)绘图：用R语言的ggplot2包进行绘图。
5.4分子标记PCR检测方法
基于野生玉米基因组和B73参考基因组设计开发基因组范围的多态性标记，然后将开发好的分子标记在高蛋白群体中分别选择野生玉米、B73和F1进行验证。筛选9号染色体140Mb-152Mb可用的分子标记，将2000份F1BC9群体用上述筛选的分子标记通过PCR后琼脂糖跑胶进行基因型判断。同时，测定2000份群体的蛋白含量，将基因型遗传交换信息与交换单株对应的籽粒蛋白含量进行分析。PCR采用2×HieffTM PCR Master Mix(上海翊圣生物科技有限公司，10102ES03)及标准程序进行扩增，3％的琼脂糖胶进行鉴定。
图位克隆分子标记引物信息：
5.5 RNA提取和RNA-seq分析
(1)取实验组和对照组同一时期的组织样品，如根和叶片，液氮速冻，并迅速转移至-80℃超低温冰箱保存。(2)通过研磨机器，在液氮冻存的条件下，进行充分研磨(60Hz， 70s)后再存放于液氮中。(3)将充分研磨的组织粉末分别加入1mL的Trizol提取液，充分震荡，放置5min；再向混合液中加200μL的氯仿，充分震荡，冰上放置5min，13523g转速下4℃离心10min，吸取上清液(切勿吸到中间层和下面有机相)500μL到新的离心管中。(4)向上清中加500μL的异丙醇，充分震荡，放置冰上10min，13523g转速下4℃离心10min后，弃上清。(5)加1mL的70％乙醇溶液，轻弹使沉淀漂浮，冰上放置1min后，13523g转速下4℃离心5min，弃上清。(6)离心机短暂离心，用小枪头吸取多余液体。室温晾干RNA沉淀2min左右，加100μL的ddH2O溶解沉淀。(7)利用Qiagen的RNeasy Plus Mini Kit试剂盒(Qiagen,catalog number:74,106)，按照试剂盒标准方法将上述初提RNA过柱进行纯化处理，其中涉及使用DNaseI(Qiagen,catalog number:79,254)进行DNA的去除，最后用30μL无RNA酶的H2O进行溶解。(8)采用Pomega公司的反转录试剂盒(ImProm-IITM Reverse Transcription System)进行RNA的反转录，方法按照试剂盒中标准方法进行。
5.6定量PCR检测
通过NCBI设计特异性引物，并进行特异性分析，引物Asn4-rt-1F/R用于RT-qPCR，以Actin为内参基因。按照Takara的SYBR Green试剂盒标准方法进行定量分析。每个样品进行三个技术重复，将上述反转录后的cDNA样品统一稀释8倍，用20μL的反应体系进行定量分析。用SYBR Green Mix，按照20μL的反应体系，加入mix 10μL，引物各1μL，稀释后的cDNA 2μL，ddH2O 6μL进行。通过BIO-RAD荧光定量分析仪CFX，采用两步PCR扩增法检测基因表达量，反应条件：预变性95℃，30s；扩增：95℃，5s；60℃，35s；40个循环；终止：95℃，15s；60℃，60s；95℃，15s。采用EXCELL 2010和△△CT法对定量数据进行分析，ZmAsn4定量引物如下。
5.7 Western Blotting检测
(1)总蛋白提取：称量新鲜根和叶磨好粉的样品各100mg于2mL离心管中。加入1mL非醇溶蛋白提取缓冲液，室温孵育2h。于15871g转速下离心15min，取上清到新的1.5mL离心管中，保存于4℃冰箱备用。
(2)Western免疫印迹：吸取抽提好的蛋白15μL，4x Protein Loading Buffer 5μL于 200μL离心管中并涡旋混匀，使用PCR仪95℃孵育5min进行变性。制备分离胶12％，浓缩胶为4％的PGAE凝胶备用。使用1x SDS-PAGE Buffer进行电泳，120V，60min。裁剪和凝胶大小相同的PVDF膜，并使用甲醇活化5s备用。电泳结束后使用BIO-RAD半干转膜仪进行转膜。将转好的膜放入干净的杂交盒中，使用1x TBST溶解的5％进口脱脂奶粉在4℃房间过夜封闭。第二天使用1x TBST按照1：1000的比例稀释一抗稀释液，在室温下杂交孵育1h。用1x TBST进行洗膜，每隔15min更换一次TBST，重复4次洗膜。用1x TBST按照1：5000的比例稀释二抗稀释液，在室温下孵育1h。用1x TBST进行洗膜，每隔15min更换一次TBST，重复4次洗膜。加入化学发光液进行显色反应和成像。
5.8基因克隆
为了确定控制高蛋白玉米主效的遗传因子，我们分别在F1BC4(n＝500)、F1BC6(n＝1650)以及F1BC8(n＝2000)进行3次BSA混池测序，并且通过对5个高蛋白和5个低蛋白稳定的F3BC6材料进行深度的重测序，发现控制高蛋白的因子位于9号染色体区间(图5中a和b)。进一步通过图位克隆进行基因的定位。通过对2000份用F1BC9群体的蛋白含量测定和基因多态性标记的开发筛选，我们将Thp9基因缩小到标记143.7和143.8之间，这147kb间隔根据野生玉米单倍型序列发现仅包含一个基因teo09G002926表达量发生显著变化(图5中c)，其对应的B73版本基因为ZmAsn4，基因号Zm00001d047736，编码天冬酰胺合成酶4，ASN4。通过分析野生玉米3代基因组序列，我们发现野生玉米的Asn4相对于B73在第10个外显子有一个47bp的插入，而这个插入导致野生玉米Asn4基因转录本和B73不一样，通过分析野生玉米和B73根和叶中Asn4的转录本，发现野生玉米中Asn4采用的转录本和B73中不同，并且野生玉米转录本显著高表达(图5中d)。进一步分析携带该高蛋白位点的NILTHP9近等基因系和对照NILB73根和叶片的RNA-Seq，发现近等基因系NILTHP9在根和叶中，Asn4都显著高表达，NILB73几乎不表达(图5中e)。免疫印迹分析同样证明ASN4在近等基因系的根和叶中高积累(图5中f)。通过一系列的定位测序，大规模群体的蛋白含量测定考察，分子标记筛选，我们最终定位到高蛋白主效基因是Asn4-Thp9。
实施例6：高蛋白玉米形成的关键基因Thp9及连锁标记验证
叶片DNA提取和PCR鉴定方法同上述步骤5.4，鉴定引物为：

基于该47bp插入，我们开发了一个分子标记SEQ ID NO:4，对F3BC7群体中的200个体进行分析，发现群体中ZmAsn4-B73(ZmAsn4为B73的基因型)的蛋白质含量显着低于杂合ZmAsn4-H(ZmAsn4为杂合的基因型)和野生玉米基因型Asn4-Teo(ZmAsn4为野生玉米的基因型)(图6中a和b)，根中游离氨基酸天冬酰胺含量同样是ZmAsn4-H型和Asn4-Teo型含量显著高于ZmAsn4-B73型(图6中c)，表明THP9蛋白含量的高低与该标记连锁。结果进一步证明，ZmAsn4高表达是提高玉米蛋白含量的重要因素，我们开发的这个分子标记SEQ ID NO:4可以用于鉴定导入的野生玉米高蛋白位点以及群体水平中该位点的变异。
实施例7：Thp9高蛋白玉米在不同生态条件下的表现
蛋白含量测定和氨基酸含量分析方法同上述步骤1.1和1.3。我们分别在不同地点进行蛋白含量性状稳定性的测试，选取了上海、三亚以及东北哈尔滨进行不同年份、不同地点的测试。我们分别发现近等基因系NILTHP9种子在上海蛋白含量约为13.1±0.38％，对照NILB73在上海约为9.69±0.43％；NILTHP9种子在三亚蛋白含量约为15.39±0.95％，对照NILB73约为11.17±0.95％；以及NILTHP9种子在东北蛋白含量同样约为11.96±0.65％，对照NILB73在东北约为9.16±0.52％，在上海、三亚和东北分别提高了35.19％，47.78以及30.57％(图7中a)。除此之外，我们还发现NILTHP9除了增加籽粒蛋白质含量外，还增加了植株茎秆中的总氮含量和根中的总氮含量(图7中b)。游离氨基酸含量测定发现NILTHP9中天冬酰胺显著高于NILB73(图7中c)。此外，NILTHP9的株高和地上生物量显着增加，植株高度也相对提高10％(图7中d和e)，地上部生物量提高20％(图7中f)，表明THP9过量积累天冬酰胺有利于植物生长。THP9在玉米籽粒蛋白含量提高、青贮玉米茎秆总氮提高、植株生物量和株高提高上都具有巨大的潜能。
实施例8：遗传验证表明Thp9是控制高蛋白玉米形成的关键基因
8.1过表达Thp9载体构建及引物
过表达Thp9载体的构建是以野生玉米Thp9的cDNA为模板进行扩增，扩增引物是：正向引物ASN4-3300-FlgF：
反向引物ASN4-3300-R3：ggggaaattcgagctcTTACACCGCGATGGCGACAGC。
PCR条件：用Toyobo的KOD-FX-NEO酶进行PCR扩增，体系按照KOD酶标准混合体系，94℃预变性2min，98℃变性10s，60℃退火30s，68℃延伸2min，35个循环。
采用同源重组的方法(ClonExpress II One Step Cloning Kit，C112-02，诺唯赞)将PCR扩增片段克隆到pCAMBIA3300载体上，置于玉米Ubiquitin(UBI)启动子下游，构建得到过表达Thp9载体。
8.2遗传转化
参照《分子克隆实验指南》(第三版)，制备农杆菌EHA105感受态细胞。将过表达Thp9载体用农杆菌介导法转化玉米B73幼胚，获得过表达转基因玉米，遗传转化在未米生物科技(江苏)有限公司进行。
8.3转基因玉米的定量PCR分析和Western blotting
具体分析方法同5.5和5.6，定量引物为：
8.4定氮仪分析500份玉米自交系玉米籽粒蛋白含量
500份自交系统一种植在海南三亚中国农科院棉花研究所大茅基地(2019年和2020年)，每份材料均自交3个果穗，成熟收获干燥后，每份自交系取3个果穗中部的籽粒6粒，混合磨样。蛋白含量测定方法见上述步骤1.1。
进一步，我们在B73背景中通过UBI启动子过量表达野生玉米单倍型的Thp9。过表达转基因事件根和叶片中Thp9基因的表达均显著提高(图8中a和b)；免疫印迹分析表明，过表达转基因事件根中Thp9显著积累(图8中c)，籽粒蛋白含量从对照12.08±0.88％分别提高到Asn4-OE-1的15.18±1.03％和Asn4-OE-2的15.81±1.13％(图8中d)。通过转基因遗传的验证，我们证明过量表达Thp9同样在玉米籽粒蛋白含量提高、青贮玉米茎秆总氮提高、株高提高上都具有巨大的潜能，再次证明Thp9重要的价值。另外，我们测定 了2019年和2020年两年分别405份和438份种植在海南三亚中国农科院棉花研究所大茅基地自交系的蛋白含量，发现自交系2019年蛋白含量的变异为6.5％-16％，平均值为11.52％；而2020年蛋白含量的变异为7.7％-16.8％，平均值为12.3％。材料统一种植在海南三亚中国农科院棉花研究所大茅基地自交系的蛋白含量进行自然群体蛋白含量的GWAS分析，发现控制自然群体蛋白含量的主效位点依然是位于9号染色体的Thp9基因位点(图8中e)。自然群体中ASN4基因在之前鉴定到的野生玉米和B73插入缺失位置处存在3种单倍型(图8中f)，通过分析表明，B73的ASN4基因(Zm00001d047736)属于一种单倍型HAP3，相对于野生玉米单倍型HAP1缺失47bp，但没有功能，几乎不表达，蛋白含量大约10％；野生玉米单倍型HAP1以及缺失了22bp的自然群体中的HAP2单倍型都显著提高了自然群体中的蛋白含量(图8中g)。从野生玉米到栽培玉米，ASN4在驯化的过程中受到了选择，在自然群体中出现了分化。从群体变异水平再次证明ASN4的变异与蛋白含量显著相关。
实施例9：Thp9氮高效试验和大田测试
蛋白含量测定方法同上述步骤1.1。通过在不同氮素含量水平下的土壤中种植携带Thp9高蛋白近等基因系NILTHP9以及不携带Thp9的对照NILB73，发现ZmAsn4基因的表达随着氮素水平的增加而显著提高；并且在低氮(不人工施加氮肥)水平下的NILTHP9株高、地上地下生物量和籽粒蛋白含量，都达到对照NILB73在正常施氮量(分2次施入，分别在苗期和拔节期施入，每次10g)的生物量和株高(图9中a-g)。说明野生玉米ZmASN4基因的高表达具有氮高效的潜能。
进一步，我们通过大田试验，分别设置0％、25％、50％以及100％(100％的水平为，苗期施了一次，拔节期施了一次，共施肥两次，每次0-4-8-16g/株，含氮量17％，其它水平的依次减少；种植密度0.6m x 0.25m)这4个梯度的施氮试验，每个群体种植300颗(图10中a)，发现NILTHP9在各个水平下株高(图10中b)、地上部分生物量(图10中c)以及根叶茎的总N含量(图10中d-f)，籽粒蛋白含量都相对于对照NILB73有提高(图10中g)，并且25％水平下NILTHP9就具有对照50％和100％水平下的株高、生物量以及植株氮含量水平。通过大田试验进一步证明说明了野生玉米ZmAsn4具有氮高效，Thp9基因的导入对于减少氮肥的施入、环保绿色有着重大的意义。
实施例10：Thp9的应用潜力和价值
Thp9位点PCR鉴定方法同步骤5.3，引物为thp9-F/R和asn4-is-F/R，扩增信息见实施例6。为了检测Thp9的应用潜力，首先将Thp9近等基因系(B73背景)和对照NILB73 分别与Mo17创制杂交种，发现携带Thp9位点的杂交种蛋白含量显著提高(图11中a和b)。进一步，我们将Thp9回交导入主栽玉米杂交种郑单958的两个亲本郑58和昌7-2中，通过回交3代，再自交，分别获得Thp9改良版本的郑58和昌7-2。将双亲改良版本进行杂交，获得改良版本的郑单958。通过在三亚种植进行测试，发现高蛋白基因改良版本郑单958材料植株增加，植株生物量增加(图11中c-f)，蛋白含量显著高于对照原始杂交种，改良版本的郑单958-THP9种子蛋白质含量为11.14±1.13％，而对照郑单958为9.88±0.58％，提高了12.75％(图11中g)。此外，改良版本的郑单958-THP9的根、茎、叶总氮含量增加(图11中h-j)。我国玉米年产量约2.7亿吨，蛋白每提高1个百分点，相当于多生产270万吨蛋白，这将在农业生产上产生巨大的潜能和价值。
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Claims (10)

  1. 野生玉米天冬酰胺合成酶4基因在提高玉米籽粒蛋白含量、植株总氮含量和/或氮高效中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述应用选自下组方式：将野生玉米天冬酰胺合成酶4编码基因导入普通玉米染色体中；使玉米过表达野生玉米天冬酰胺合成酶4基因；使玉米过表达普通玉米原有天冬酰胺合成酶4基因ZmASN4；将控制野生玉米天冬酰胺合成酶4基因表达量的调控区域导入玉米使该基因表达量提高。
  3. 如权利要求2所述的应用，其特征在于，所述普通玉米原有天冬酰胺合成酶4基因ZmASN4对于自交系玉米B73而言是指Zm00001d047736。
  4. 如权利要求1所述的应用，其特征在于，所述野生玉米天冬酰胺合成酶4基因的核苷酸序列选自下组：
    (A)如SEQ ID NO:1所示的多核苷酸，命名为Thp9，基因编号为Teo09G002926，NCBI Genome submission:SUB11272093；
    (B)与SEQ ID NO:1所示核苷酸序列的同源性≥80％、≥85％、≥90％、优选≥95％、更优选≥98％的多核苷酸。
  5. 如权利要求1所述的应用，其特征在于，所述野生玉米天冬酰胺合成酶4是选自下组的多肽：
    (a)具有SEQ ID NO:2氨基酸序列的多肽；
    (b)将SEQ ID NO:2氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的，且具有(a)多肽功能的由(a)衍生的多肽；
    (c)与(a)限定的多肽序列有95％以上同源性，优选地98％以上同源性，更优地99％以上同源性，且具有(a)多肽功能的由(a)衍生的多肽；或
    (d)序列中含有(a)或(b)或(c)中所述多肽序列的衍生多肽。
  6. 如权利要求2或4所述的应用，其特征在于，将野生玉米天冬酰胺合成酶4编码基因导入玉米染色体中的方法包括如下步骤：
    (1)将所述野生玉米天冬酰胺合成酶4编码基因克隆到适合于在农杆菌中表达的植物表达载体中，得到该基因的表达载体；
    (2)载体经过测序验证后，将该基因的表达载体用农杆菌介导法转化玉米幼胚，获得过表达该基因的转基因玉米。
  7. 如权利要求5所述的应用，其特征在于，所述pCAMBIA载体是以玉米Ubiquitin启动子驱动的pCAMBIA3300载体。
  8. 一种用于实施如权利要求4所述应用的试剂盒，其包含：SEQ ID NO:1的Thp9基因片段或者其CDS序列SEQ ID NO:3、用于将该基因片段或者其CDS序列克隆入植物表达载体所需的PCR引物；
    或者包含：如权利要求5或6中所述的基因表达载体，用于将基因表达载体转入农杆菌中的试剂；
    或者包含：转入了如权利要求5或6中所述的基因表达载体的农杆菌，用于将农杆菌转化植株的试剂。
  9. 一种检测玉米基因组中如权利要求3或4所述的基因的方法，包括下述步骤：
    检测核苷酸序列为SEQ ID NO:1的Thp9基因时，正向引物thp9-F：CTCTGTGCCATGCATCCTCC，反向引物thp9-R：CGTCAGCGCTGGTTAGC，PCR产物为198bp的SEQ ID NO:4，其为Thp9高蛋白位点的分子标记：
    CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGATCGCATCTCCTAGACATCGTACCGTACGTCGTCCAAGTTAGCTAACCAGCGCTGACG(SEQ ID NO:4)；
    或者PCR产物为176bp的SEQ ID NO:5，其也为Thp9高蛋白位点的分子标记：
    CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATTTATGGCCACGCATCTCCTAGACATCGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG(SEQ ID NO:5)；
    而PCR产物为151bp的SEQ ID NO:6，其为玉米B73基因Zm00001d047736的分子标记：
    CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATGGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG(SEQ ID NO:6)；
    检测核苷酸序列为SEQ ID NO:1的Thp9基因是否插入玉米基因组时，正向引物asn4-is-F：CCGTTCCTCGACAAGGAGTT，反向引物asn4-is-R：ATCAGAGCTGAAAGTGGGGC，PCR产物为455bp的SEQ ID NO:7，其为野生玉米 Ames21814基因型插入的分子标记：
    CCGTTCCTCGACAAGGAGTTCGTCGACGTCGCGATGGGCATGGACCCCGAGTGGAAAATGGTACTGACGCGGGCCTTTTTCGACACGGCCCGGCCCTGCCGCCGCACGTCGGGGTCTCGGTTCTACGTATGATGATGACGCCTTCTTCTCTTCTTTGCGCAGTACGACAAGAACCTGGGTCGCATCGAGAAGTGGGTCCTGAGGAAGGCGTTCGACGACGAGGAGCACCCTTACCTGCCCGAGGTAAGAACATCTTCAGAGAAGGCTGGTCGTTTACCTCTGTGTCTGTGTGATTTCAAGCCTGAACTGACGCCTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGAT(SEQ ID NO:7)。
  10. 一种实施如权利要求9所述方法的试剂盒，其特征在于，包括用于检测SEQ ID NOs:4-7的相应引物、或者DNA/RNA探针、或者DNA/RNA探针的微阵列芯片。
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