CN1970744A - Corn embryosperm ADP- glucose pyrophosphorylase mutant and its screening method and application - Google Patents

Abstract

The invention discloses a corn endosperm ADP-glucose pyrophosphorylase mutant, a screening method and application thereof. The screening method comprises the following steps: 1) using hydroxylamine hydrochloride mutagen to randomly mutate the large and small subunit cDNA of corn endosperm AGPase under the condition of 70-80°C warm bath; 2) taking the mutated corn endosperm AGPase large and small subunit cDNA and the The large and small subunit cDNA of wild-type corn endosperm AGPase was transformed into Escherichia coli glgC mutant strains, and positive single clones were screened; 3) The positive clones were streaked on Conberg enriched solid culture plates and cultured at 35-39°C; 4) The single colony grown on the Conberg enriched solid culture plate was stained with iodine-potassium iodide solution, and compared with the color of the control colony, the mutant strain was darker than the control colony, and then verified by sequencing, the corn endosperm ADP- Glucose pyrophosphorylase mutants. The maize endosperm ADP-glucose pyrophosphorylase mutant and its screening method of the invention will play an important role in the improvement of maize varieties.

Description

Maize endosperm ADP-glucose pyrophosphorylase mutant and its screening method and application

technical field

The present invention relates to plant ADP-glucose pyrophosphorylase mutants and their screening methods and applications, in particular to ADP-glucose pyrophosphorylase mutants derived from corn endosperm and their screening methods and the mutants and their coding genes Application in corn variety improvement.

Background technique

Starch plays an important role in human life, so researchers have not stopped studying starch synthesis. In the fields of agriculture and forestry, people have long been committed to increasing the yield of crops (such as corn, etc.) to provide enough food and industrial raw materials for the continuously growing world population. At the same time, from the perspective of sustainable development and environmental protection, the demand for the yield and quality of corn production will increase and improve day by day. In addition, because plant cells or organs with a high starch content absorb less fat or frying oil, they can be used to produce "healthier" products that are lower in calories. According to statistics, more than 80% of the starch in the world comes from corn, and the proportion in the United States is as high as 95%. There are still many defects in the research in this area in our country. Therefore, it is necessary to accelerate the cultivation and improvement of high-starch corn varieties for industrial use. It is imperative.

A series of enzymes are involved in the process of starch synthesis: ADP-Glucose pyrophosphorylase (AGPase for short), starch synthase (SS for short), starch branching enzyme (starch branching enzyme, Abbreviated as SBE), starch debranching enzyme (starch debranching enzyme, abbreviated as DBE) and so on. Among them, ADP-glucose pyrophosphorylase is the key enzyme for the synthesis of bacterial glycogen and plant starch. ADPG, which catalyzes the generation of 1-P-Glucose and ATP, participates in the synthesis of amylose and amylopectin as the substrate of starch synthase. The activity level is the key factor to determine the rate of starch accumulation. In higher plants, the enzyme has a complex heterotetrameric structure. So far, AGPase has been isolated and purified from various plants such as corn, wheat, spinach, sugar beet, Arabidopsis, barley and potato.

The level of AGPase activity is regulated by many factors, among which allosteric regulation is an important regulation method, which achieves the purpose of controlling enzyme activity by adjusting the ratio of activator 3-PGA and inhibitor inorganic phosphorus. 1969, Cattaneo etc. (Cattaneo J, O.M., Sigal N, Sanchez-Medina G, Puig J.Geniticstudies of E.coliK12 mutants with alterations in glycogenesis and properties of an altered AGPase. Biochem Biophys Res Commun.1964-371:69 .) found a mutant strain in the Escherichia coli (E.coli) K12 strain, and its glycogen content was increased by more than 33% compared with the wild-type strain. Further studies confirmed that the increase in glycogen in mutants was due to the mutation of AGPase, a key enzyme controlling glycogen synthesis, which changed the enzyme's response to allosteric regulators, that is, it was insensitive to allosteric regulation (LeeY M, K.A., Preiss J. Amino acid sequence of an E. coli ADPglucose synthetase allostefric mutants as deduced from the DNA sequence of the glgCgene. Nucleic Acids Res, 1987, 15: 10603.). In 1992, Stark et al. (David M.Stark, Kurt P.Timmerman et al.Regulation of the amount of starch in planttissues by AGPase.Science, 258:287-291.) of Monsanto Company transferred the mutated AGPase gene into potato , Obtained transgenic plants with increased starch content, the average content increased by 35% compared with the control, and some could reach 60%. This is the first successful example of using genetic engineering to increase the starch content of plants.

In order to study the catalytic and regulatory properties of AGPase in depth, so as to achieve the purpose of regulating starch synthesis, researchers used chemical modification and mutation methods to study the substrate and allosteric agent binding sites of the enzyme. The results confirmed that the enzyme contained multiple ligand binding sites, and besides the substrate ATP and G-1-P catalytic sites, there were also activator 3-PGA binding sites and inorganic phosphorus inhibitory binding sites. At the same time, some corresponding mutants have also been obtained, such as mutants sensitive to the activator 3-PGA (Greene, T.W., I.H.Kavakli, et al. Generation of up-regulated allosteric variants of potato ADP-glucose pyrophosphorylase byreversion genetics." Proc Natl Acad Sci USA.1998, 95 (17): 10322-10327.) etc. But more studies are AGPase derived from potato tubers and AGPase of spinach leaves, and there are relatively few mutants of corn endosperm AGPase. The method of transposon-induced mutation induces the large subunit gene (Shrunken-2) of AGPase in the maize endosperm to produce mutations, and the seed weight of the mutant strain increases by 11-18% compared with the wild type. Further analysis shows that the increase of the seed weight is As mutated AGPase reduces sensitivity to inhibitor inorganic phosphorus (Giroux, M.J., J.Shaw, et al. A single mutation that increases maizeseed weight. Proc Natl Acad Sci USA. 1996, 93(12): 5824-5829 .). In addition, the AGPase of maize endosperm is a thermolabile enzyme, Greene et al. Acad SciUSA.1998, 95(22): 13342-13347.) A thermally stable mutant was obtained by using mutation technology, and the analysis results showed that the thermal stability was due to the enhanced interaction between the large and small subunits.

Although AGPase from maize endosperm has high amino acid sequence homology with AGPases from potato tubers and spinach leaves, in terms of subcellular localization, regulatory properties, and thermal stability, AGPase from maize endosperm and AGPase from both sources exist. There is a big difference, for example, the AGPase of potato tubers is located in the starch body of the storage organ, while the AGPase of the corn endosperm is mainly located in the cytoplasm outside the starch; the potato tuber AGPase is quite sensitive to activators and inhibitors, and in the absence of activators However, the activity of corn endosperm AGPase is negligible (it is difficult to detect the activity), and the enzyme activity is still high in the absence of activators, and the presence of activators can partially compensate for the inhibitor's effect on the activity. In addition, potato AGPase can be regulated by different levels, such as transcriptional level, post-transcriptional level and post-translational reductive modification regulation. In maize kernels, the changes in the content of the large and small subunits of AGPase are consistent with the transcription levels of its coding genes Shrunken-2 and Brittle-2, and no other level of regulation has been found so far; in terms of thermal stability, due to the two There is cysteine at the N-terminal of each small subunit, so a relatively stable disulfide bond structure is formed, while the large and small subunits of maize AGPase do not have this structure. Therefore, an in-depth study of corn endosperm AGPase will not only help to reveal the similarities and differences between monocots and dicots in the starch synthesis pathway, but also help to further increase the corn starch content.

Contents of the invention

The purpose of the present invention is to provide a simple and feasible method for screening corn endosperm ADP-glucose pyrophosphorylase mutants with improved enzyme activity.

The method for screening corn endosperm ADP-glucose pyrophosphorylase mutant provided by the present invention comprises the following steps:

1) Use hydroxylamine hydrochloride mutagen to randomly mutate the cDNA of maize endosperm AGPase large and small subunit genes (Sh2 and Bt2) at 70-80°C in a warm bath; the formulation of the hydroxylamine hydrochloride mutagen is: 0.15-0.25g hydroxylamine hydrochloride, 125mM pH7.0 sodium pyrophosphate 1.0-1.6mL, 1M NaCl0.2-0.4mL, 500mM pH8.0 EDTA10-14μl, dilute to 3mL with deionized water;

2) Transform the Escherichia coli glgC mutant strains with the mutated maize endosperm AGPase subunit cDNA and the wild-type maize endosperm AGPase subunit cDNA as a control in step 1), and screen for transformation with the mutated maize endosperm AGPase subunits respectively. Positive single clones of the base cDNA and wild-type maize endosperm AGPase large and small subunit cDNA;

3) Inoculate the positive clones screened in step 2) on a Conberg-enriched solid culture plate and culture at 35-39°C for 12-24 hours; the formula of the Conberg-enriched solid medium is: potassium dihydrogen phosphate 8-9g , dipotassium hydrogen phosphate 10-12g, yeast extract 5-7g, glucose 8-12g, agar 15-25g, dilute to 1L with water, final pH7.0;

4) Use iodine-potassium iodide (I ₂ -KI) solution to stain the single colony grown on the Conberg enriched solid culture plate in step 3), and compare it with the color of the control colony, and the color darker than the control is a mutant The bacterial strain is sequenced to obtain a corn endosperm ADP-glucose pyrophosphorylase mutant; the iodine-potassium iodide solution is an aqueous solution containing 0.08-0.12mol/L iodine and 0.25-0.35mol/L potassium iodide.

In the above screening method, the warm bath temperature in step 1) is preferably 75°C, and the formula of the hydroxylamine hydrochloride mutagen is preferably: 0.2084g hydroxylamine hydrochloride, 1.2mL of sodium pyrophosphate (pH7.0) of 125mM, 0.3mL of 1M NaCl , 500mM EDTA (pH8.0) 12μl, deionized water 1.488mL.

In step 2), the mutated maize endosperm AGPase large and small subunit cDNA and the wild-type maize endosperm AGPase large and small subunit cDNA as a control can be obtained by containing the mutated maize endosperm AGPase large and small subunit cDNA or the wild-type maize endosperm AGPase large and small subunit cDNA Escherichia coli glgC mutant strain transformed with the prokaryotic expression vector, the prokaryotic expression vector can be any prokaryotic expression vector that can express foreign genes in Escherichia coli, such as pGEX-KG, pET28a, pET30a, pET-11c, pET-22b, etc. .

The inoculation method in step 3) is preferably streak inoculation, and the culture temperature is preferably 37° C.; the formula of Conberg enriched solid medium is preferably: 8.5 g of potassium dihydrogen phosphate, 11 g of dipotassium hydrogen phosphate, 6 g of yeast extract, glucose 10g, 20g agar, dilute to 11 with water, final pH7.0.

The formula of the iodine-potassium iodide solution in step 4) is preferably: an aqueous solution containing 0.1mol/L iodine and 0.3mol/L potassium iodide.

In addition, in order to screen out a corn endosperm ADP-glucose pyrophosphorylase mutant with higher activity, steps 1)-4) in the above screening method can be repeated 1-3 times.

The corn endosperm ADP-glucose pyrophosphorylase mutant obtained by screening with the above method also belongs to the protection scope of the present invention.

The ADP-glucose pyrophosphorylase mutant named AGPase-10-3-53 may have one of the following amino acid residue sequences:

1) SEQ ID NO: 1 in the sequence listing;

2) The amino acid residue sequence of SEQ ID NO: 1 in the sequence listing is subjected to substitution, deletion or addition of one to ten amino acid residues, or a protein with AGPase activity.

The SEQ ID NO in the sequence listing: 1 is made up of 475 amino acid residues, compares with unmutated maize endosperm ADP-glucose pyrophosphorylase, from amino terminal (N terminal) 323rd by Ile mutation to Met, since The amino terminal position 499 was mutated from Met to Arg.

The gene (AGPase-10-3-53) encoding the above-mentioned ADP-glucose pyrophosphorylase mutant is one of the following nucleotide sequences:

1) The DNA sequence of SEQ ID NO: 2 in the sequence listing;

2) The DNA sequence of SEQ ID NO: 1 in the coding sequence list;

3) A nucleotide sequence having more than 90% homology with the DNA sequence defined by SEQ ID NO: 2 in the sequence listing and having AGPase activity;

4) A nucleotide sequence that can hybridize with the DNA sequence defined by SEQ ID NO: 2 in the sequence listing under high stringency conditions.

The high stringency conditions are: use 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridize at 65° C. and wash the membrane.

SEQ ID NO in the sequence listing: 2 consists of 1428 bases, its coding sequence is the 1-1428th base from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID NO: 1 in the sequence listing, Bases 967-969 from the 5' end encode the mutated amino acid residue Met, and bases 1345-1347 from the 5' end encode the mutated amino acid residue Arg.

Expression vectors, transgenic cell lines and host bacteria containing the ADP-glucose pyrophosphorylase mutant gene AGPase-10-3-53, and primers for amplifying any fragment of AGPase-10-3-53 also belong to this invention. protection scope of the invention.

Another object of the present invention is to provide a method for increasing the starch content of corn seeds.

The method for increasing the starch content of corn seeds provided by the invention is to introduce the ADP-glucose pyrophosphorylase mutant gene AGPase-10-3-53 into corn tissues or cells, so that the starch content of corn seeds is increased.

The ADP-glucose pyrophosphorylase mutant gene AGPase-10-3-53 can be introduced into maize tissue or cells through a plant expression vector containing the ADP-glucose pyrophosphorylase mutant gene AGPase-10-3-53 .

The starting vectors used to construct the plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage The untranslated region transcribed at the 3' end of the protein gene) has similar functions.

When using AGPase-10-3-53 to construct a plant expression vector, any enhanced promoter or inducible promoter can be added before its transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter , root-specific expression promoters, etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

Specifically, the starting vector for constructing the plant expression vector may be pCAMBIA2301, pBI121, pCAMBIA1301 or pCAMBIA1300, etc.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The plant expression vector carrying AGPase-10-3-53 of the present invention can transform corn tissue or cells, and growing the transformed maize tissue or cells into plants.

The invention provides a corn endosperm ADP-glucose pyrophosphorylase mutant and a screening method thereof. The screening method is to randomly mutate the wild-type corn endosperm AGPase gene, transform Escherichia coli glgC mutant strain, inoculate the recombinant on Conberg enriched solid medium, and finally screen the grown single clone by iodine staining method. Obtain corn endosperm ADP-glucose pyrophosphorylase mutant. The screening method has the advantages of simplicity, stability and reliability. In addition, the test results show that the enzymatic activity of the corn endosperm AGPase mutant obtained by the screening method of the present invention is 134% of that of wild-type AGPase, and the AGPase mutant is in Escherichia coli Compared with the wild-type AGPase, the ability of accumulating glycogen in AGPase was also significantly improved; the analysis of kinetics and regulatory properties showed that the affinity of the mutant to the substrate (ATP and G-1-P) was enhanced, but the affinity to the activator The sensitivity to inhibitors was the same as that of the wild-type AGPase, indicating that the enhanced affinity of the mutant to the substrate was the reason for the increased enzyme activity and glycogen accumulation ability of the mutant. The maize endosperm ADP-glucose pyrophosphorylase mutant and the screening method thereof of the invention will play an important role in the improvement of maize varieties, and have broad application prospects.

The present invention will be described in further detail below in conjunction with specific embodiments.

Description of drawings

Figure 1 shows the results of agarose gel electrophoresis detection of RT-PCR amplified maize endosperm AGPase large and small subunit gene cDNA

Figure 2 is a schematic diagram of the partial structure of the prokaryotic expression vector pGEX-KG-Sh2-Bt2 of the maize endosperm AGPase subunit gene

Figure 3 is the SDS-PAGE detection results of the expression of the corn endosperm AGPase subunit gene in the E. coli mutant strain without glgC activity and the functional complementation experiment results with glgC

Figure 4 shows the results of the first round of screening of the large and small subunits of maize endosperm AGPase after random mutation

Figure 5 is the comparison result of the iodine staining depth of mutants 10-3 and 10-3-53 and the control

Fig. 6 is the amino acid residue sequence alignment result of the corn endosperm AGPase mutant and AGPase small subunit parts from other sources

Figure 7 is the SDS-PAGE detection result of the purified corn endosperm AGPase mutant AGPase-10-3-53

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified. The primer synthesis and sequencing work were completed by Shanghai Sangong.

Example 1. Obtaining of Maize Endosperm ADP-Glucose Pyrophosphorylase Mutant and Its Encoding Gene

1. Obtaining the large and small subunit cDNA of maize endosperm AGPase

1. Primer design

Two pairs of RT-PCR primers (Sh and Bt) were designed according to the cDNA sequences of the corn endosperm AGPase large subunit gene Shrunken-2 and small subunit gene Brittle-2 (GenBank numbers: M81603, AF334959), and for the convenience of vector construction , when designing the primers, a special design was made for the following two points: a. The recognition site of the restriction endonuclease NdeI was added at the 5' end of Shrunken-2, and the recognition site of the restriction endonuclease SmaI and SacI was added at the 3' end of Shrunken-2 site; b. Because there is a restriction endonuclease NcoI recognition site at the ATG position at the 5' end of Brittle-2, the A at position 39 at the 5' end of Brittle-2 is mutated to C, and the presence at this site is deleted The second NcoI recognition site, and a restriction endonuclease SacI recognition site is added at the 3' end of Brittle-2, the primer sequence is as follows:

Primer pair Sh for amplifying maize endosperm AGPase large subunit gene Shrunken-2 cDNA:

Sh2F: 5'-CCCATATGCAGTTTGCACTTGCATTGGACACG-3'

Sh2R: 5'-GAGAGCTCCCCGGGCTATATGACAGACCCATCGTTGATG-3';

Primer pair Bt for amplifying the small subunit Brittle-2 cDNA of maize endosperm AGPase:

Bt2F: 5'-CAAACCATGGACATGGCTTTGGCGTCTAAAGCCTCCCCTCCGCCCTGGAATGCCACCGCCGCCG-3'

Bt2R: 5'-GAGCTCTCATATAACTGTTCCACTAGGGAGTAAAGC-3'.

2. Synthesis of first-strand cDNA

Use the Trizol RNA extraction kit (purchased from Tianwei Times Company) and refer to the kit instructions to extract the total RNA of corn seeds 18 days after pollination, and use this as a template to reverse transcribe and synthesize the first-strand eDNA: take 1 μL of ligo(dT) ₁₈ was added to 10 μL of total RNA solution with a concentration of 200 ng/μL, reacted at 70°C for 5 minutes, then cooled on ice for 5 minutes, and then used Promega’s AMV Reverse Transcriptase and added the following components according to the instructions: 5× reverse transcription buffer Solution (included in the kit) 5 μL, dNTPs (10 mM each) 2.5 μL, RNasin ribonuclease inhibitor (concentration: 50 U/μL) 1 μL, AMV reverse transcriptase (30 U/μL) 3 μL, dilute with DEPC-treated water Make up to 25 μL. The reaction conditions were: room temperature for 10 minutes, 42°C for 60 minutes, 70°C for 10 minutes, and ice bath for 2 minutes.

3. PCR amplification

Using the first-strand cDNA synthesized in step 2 as a template, under the guidance of the primer pair Sh and the primer pair Bt, respectively, PCR amplified Shrunken-2 and Brittle-2 cDNA, the reaction system was: 10 × buffer 5 μL, dNTPs (per Seed 10mM) 1μL, primer Sh2F (Bt2F) (concentration is 10μM 1μL, primer Sh2R (Bt2R) (concentration is 10μM) 1μL, Ex-Taq (5U/μL, TaKaRa company) 1μL, first-strand cDNA (concentration is 10ng/uL ) 5 μL, dilute to 50 μL with sterilized water. The PCR reaction conditions are: first 94°C for 5 minutes; then 94°C for 1 minute, 62°C for 45 seconds, 72°C for 2 minutes, 10 cycles; then 94°C for 1 minute, 65°C for 45 seconds, and 72°C 2min, 25 cycles; last 10min at 72°C, stored at 4°C. After the reaction, the PCR amplified product was detected by 0.7% agarose gel electrophoresis, and the detection result is shown in Figure 1 (swimming lane Sh2: PCR amplified Shrunken -2, lane Bt2: Brittle-2 amplified by PCR, a Shrunken-2 cDNA fragment of about 1.5kbp and a Brittle-2 cDNA fragment of 1.4kbp were amplified by PCR, consistent with the expected results. Recover and purify these two target fragments , which were respectively cloned into the vector pGEM ^(R) -T Easy (purchased from Promega Company), the cloning vector containing the Shrunken-2 cDNA fragment was identified by double enzyme digestion with restriction endonucleases BamHI and SmaI, and obtained The DNA fragments of 1.5kbp and 3.0kbp were in line with the expected results; the cloning vector containing the Brittle-2 cDNA fragment was identified by single enzyme digestion with the restriction endonuclease EcoRI, and the DNA fragment of 1.4kbp was obtained after digestion, which was in line with the expected The results are consistent: the above-mentioned cloned plasmid was sequenced again, and the sequencing results showed that, except for the expected mutation of Brittle-2 from the 39th base A at the 5' end to C, the other sequences were consistent with the sequence registered on GenBank, indicating that the The cDNAs of large subunit gene Shrunken-2 and small subunit gene Brittle-2 of maize endosperm AGPase were amplified by RT-PCR.

2. Heterologous expression of large and small subunit genes of maize endosperm AGPase in E. coli mutant strains without glgC activity

1. Transformation of multiple cloning sites of prokaryotic expression vector pGEX-KG

Because the N-terminus and the C-terminus of the large and small subunits of potato tuber AGPase play an important role in the regulatory properties of this enzyme, so in order to make the large and small subunit gene of corn endosperm AGPase use vector pGEX-KG (purchased from Invitrogen Company, the physical See literature for map and construction method, Guan KL, Dixon JE. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991, 192(2): 262-7) for prokaryotic No fusion protein is produced during expression. The carrier is now modified so that the expression of the GST protein on the carrier can be terminated early, no fusion protein is produced, and the expression of the target protein is not affected. The designed multiple cloning site (MCS) is as follows:

CTA GGA TCC TAA TAA CCG GGC AGG TCT AGA CCA TGG GAG CTC

BamHI stop codon Xbal Ncol Sacl

CAC CAC CAC CAC CAC CAC AAG CTT ACC

His Tag Hindllll

The multiple cloning site fragment was synthesized, after it was double-digested with restriction endonucleases BamHI and HindIII, it was connected with the prokaryotic expression vector pGEX-KG that had been double-digested with the same enzymes with T4 DNA ligase, and the ligation system and reaction The conditions are as follows: 10× ligase buffer 2ul, pGEX-KG BamHI and HindIII double-digested fragment 2ul, MCSBamHI and HindIII double-digested fragment 10ul, T4 DNA ligase (TaKaRa company) 1ul, sterilized distilled water 5ul, ligation at 16°C 12-24 hours. After the reaction, the ligation product was transformed into Escherichia coli DH5a competent cells, and the transformed cells were cultured at 37°C for 12-24 hours, and the single clone grown on the LB resistance plate (containing 100 mg/L ampicillin) was picked, The plasmids were extracted, and the restriction endonucleases BamHI, EcoRI, XhoI and XbaI were used for single-enzyme digestion and identification respectively. The results were in line with the expected results, and then one of the single clones was picked for sequencing. The sequencing results showed that the newly synthesized multiple cloning site fragments Instead of the multiple cloning sites in the original vector, the transformed vector pGEX-KG was named pGEX-KG1.

2. Construction of the prokaryotic expression vector of maize endosperm AGPase large and small subunit genes

After the cDNA of the corn endosperm AGPase large subunit gene Shrunken-2 amplified in step 1 was double-digested with restriction endonucleases NdeI and SacI, it was combined with the vector pET30a(+) (purchased from Novagen company) connection to obtain the recombinant vector containing Shrunken-2 cDNA, which is named as pET30Sh2; after the cDNA of the corn endosperm AGPase small subunit gene Brittle-2 amplified in step 1 is cut with restriction endonuclease NcoI and SacI after double enzyme digestion , and the carrier pET28a(+) (purchased from Novagen) through the double digestion with the same enzyme, to obtain a recombinant vector containing Brittle-2 cDNA, named pET28Bt2; Under the guidance of 3' and Bt2R, the eDNA of Brittle-2 and its upstream ribosome binding site were amplified by PCR. After the amplification was completed, the target fragment of about 3.0kbp was recovered, and it was subjected to a single step with the restriction endonuclease SacI. After digestion, it was connected with the recombinant vector pET30Sh2 digested with the same enzyme, and after identification, the recombinant vector connected with the large and small subunit genes of corn endosperm AGPase and their respective ribosome binding sites was obtained, which was named pET30Sh2-Bt2; The endonucleases XbaI and SacI double-digested pET30Sh2-Bt2, recovered and purified about 3.0kbp of the maize endosperm AGPase subunit gene with their respective ribosome binding sites, and combined it with the vector that had been double-digested with the same enzymes pGEX-KG1 was connected to obtain the prokaryotic expression vector of the maize endosperm AGPase subunit gene, which was named pGEX-KG-Sh2-Bt2, and its partial structure is shown in FIG. 2 .

3. Heterologous expression and functional complementation of maize endosperm AGPase subunit gene in E. coli mutant strain without glgC activity

1) Heterologous expression of large and small subunit genes of maize endosperm AGPase in E. coli mutant strains without glgC activity

The maize endosperm AGPase large and small subunit gene prokaryotic expression vector pGEX-KG-Sh2-Bt2 constructed in step 2 was transformed into Escherichia coli mutant strain BL21-dglgC without ADP-glucose pyrophosphorylase activity (the bacterial strain was purchased from Beijing Tianwei Times Company, See the literature for the screening method, Govons S, Vinopal R, Ingraham J, Preiss J. Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycol. J Bacteriol. 1969 Feb; 97(2): 970-972), screening positive clones , pick positive single clones and inoculate them in 2mL LB liquid medium (containing 100 μg/mL ampicillin), shake culture at 37°C for 12-24 hours, and then transfer to 50mL LB liquid medium (containing 100 μg/mL ampicillin), shake culture at 37°C until OD ₆₀₀ is 0.3-0.5, add different concentrations of IPTG (final concentrations are 0, 0.005, 0.01, 0.1 mM), and continue shaking culture at 37°C for 3-4 hours Induce the expression of the target protein, and end the culture when the OD ₆₀₀ of the bacterial solution is 1.0. After the incubation, centrifuge at 10,000rpm for 5 minutes, discard the supernatant, and add an equal volume of 1× loading buffer (250mM Tris HCl (pH6.8), 500mM mercaptoethanol, 10% SDS, 0.5% bromophenol) to the pellet Blue, 50% glycerol, stored at 4°C, diluted 10 times when used), fully suspended the bacteria, boiled in boiling water for 5 minutes, then ice-bathed for 2 minutes, then centrifuged at 4°C, 12000rpm for 1 minute, and loaded for SDS-PAGE detection. With the BSA standard as a control, the test results are shown in Figure 3 in Figure A (swimming lane 1 is pGEX-KG1 induced by 0.1mmol/L IPTG at a final concentration, and pGEX-KG1 induced by 0.1mmol/L IPTG at a final concentration of swimming lane 2 , lane 3 is pGEX-KG-Sh2-Bt2 induced without IPTG, lane 4 is pGEX-KG-Sh2-Bt2 induced by a final concentration of 0.005mmol/L IPTG, lane 5 is induced by a final concentration of 0.01mmol/L IPTG pGEX-KG-Sh2-Bt2, lane 6 is the pGEX-KG-Sh2-Bt2 induced by IPTG with a final concentration of 0.1mmol/L), induced by IPTG, the large subunit of 57kDa and the small subunit protein of 52kDa of maize endosperm AGPase All were expressed, and there was no significant difference in the expression of the maize endosperm AGPase subunits induced by different concentrations of IPTG (0, 0.005, 0.01, 0.1mM). Further experimental results showed that the higher the added IPTG concentration (such as 1mM) , the expression level of maize endosperm AGPase subunits was lower.

2) Functional complementation experiment with glgC (ADP-glucose pyrophosphorylase gene, GenBank number: NC_000913)

Pick the single colony of the maize endosperm AGPase large and small subunit gene expression strain obtained in step 1), inoculate it in 2mL LB liquid medium, shake and culture it at 37°C for 12-24 hours, and then enrich it in Conberg enriched medium (potassium dihydrogen phosphate 8.5 g, dipotassium hydrogen phosphate 11g, yeast extract 6g, glucose 10g, agar 15g, dilute to 1L with water, final pH7) for streaking, culture at 37°C for 12-24 hours, use iodine-potassium iodide (I ₂ -KI ) solution (0.1mol/L iodine and 0.3mol/L potassium iodide) for staining, the results are shown in Figure B in Figure 3 (1 is the control: Escherichia coli glgC mutant strain, 2 is transformed with the corn endosperm AGPase subunit gene coli glgC mutant strain), the recombinant Escherichia coli glgC mutant strain transformed with the maize endosperm AGPase subunit gene grows well, indicating that the corn endosperm AGPase subunit co-expression in the E. coli glgC mutant strain can make the glgC mutation The strain synthesizes glycogen on Conberg's enriched medium, which complements the missing function of glgC.

3. Random mutation of large and small subunits of maize endosperm AGPase

Hydroxylamine hydrochloride mutagen: 0.2084g hydroxylamine hydrochloride, 1.488mL ddH ₂ O, 1.2mL 125mM sodium pyrophosphate (pH7.0), 0.3mL 1M NaCl, 12μl 500mM EDTA (pH8.0).

Digest pGEX-KG-Sh2-Bt2 with restriction endonucleases NdeI and SacI, recover and purify about 3.0kbp of maize endosperm AGPase large and small subunit cDNA fragments, then take 1-3mL of hydroxylamine hydrochloride mutagen, and purify 20-30 μg of maize endosperm AGPase large and small subunit cDNA fragments to be mutated were added to 1 mL mutagen solution, and random mutations were carried out under 75°C warm bath conditions, respectively at 10, 20, 30, 45, 50, 55, 60, 65, Take out 50ul each at 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, and 130 minutes, and place them on ice immediately. Connected into the prokaryotic expression vector pGEX-KG, electrotransformed Escherichia coli glgC mutant strain, and screened the mutant strain with the same iodine staining method as in step 2.

In the first round of screening, most of the mutant strains were light yellow after staining with iodine; the color of some mutant strains after dyeing was similar to that of the E. coli glgC mutant strain (control) transformed with the cDNA of the wild-type corn endosperm AGPase large and small subunits. Brown in color, only a small number of mutant strains become dark brown after staining. A total of about 12,000 single clones were selected in this round of screening, seeded in 96-well plates, cultured with shaking at 37°C for 12-24 hours, then streaked on Conberg enriched medium, and cultured at 37°C for 12-24 hours. Iodine staining method was used for screening. After 3 rounds of screening, two mutant strains with iodine staining color darker than the control were obtained, named 8-2 and 10-3 respectively. The final results of the first round of screening are shown in Figure 4 (CK: wild-type strain).

The plasmids of the two mutant strains screened were extracted and sequenced, and the sequencing results showed that both 10-3 and 8-2 were the small subunit gene Brittle-2 of the maize endosperm AGPase from the 5' end of the 1345-1347th position encoded by Met (M ) is mutated into a base encoding Arg (R).

Using the mutant strain 10-3 as the original material, the same method as above was used for the second random mutation to obtain a mutant strain with higher activity. Results Four good mutant strains were obtained, named 10-3-14, 10-3-24, 10-3-33 and 10-3-53 respectively. After repeated experiments, the mutant strain 10-3-53 had better reproducibility. Among them, the iodine staining conditions of the mutant strain 10-3-53(53), the mutant strain 10-3 and the control strain (CK) are shown in FIG. 5 .

The plasmid of the mutant strain 10-3-53 was extracted and sequenced. As a result, the corn endosperm AGPase subunit gene was mutated on the basis of the corn endosperm AGPase subunit gene of the mutant strain 10-3, named AGPase-10 -3-53, having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, consisting of 1428 bases, its coding sequence is the 1-1428th base from the 5' end, and the encoding has the SEQ ID in the sequence listing The protein of the amino acid residue sequence of NO: 1, the protein is named AGPase-10-3-53, from the 5' end 967-969 base coding mutant amino acid residue Met (wild type is Ile), from 5' Bases 1345-1347 at the 'end encode the mutated amino acid residue Arg (the wild type is Met). The amino acid residue sequence alignment results of AGPase-10-3-53 and other sources of AGPase small subunits are shown in Figure 6, from the 323rd position of the amino terminal Ile in maize endosperm and leaves, barley endosperm and leaves, wheat seeds The source of AGPase is highly conserved, and the amino acid residue at this position is Val in the AGPase derived from all known dicotyledonous plants, corn embryos, rice endosperms, and leaves.

Example 2, Determination of Enzyme Activity and Glycogen Content of Corn Endosperm AGPase Mutant

1. Determination of AGPase activity in crude extract of corn endosperm AGPase mutant

1. Obtaining crude extract of corn endosperm AGPase mutant

Pick the Escherichia coli glgC mutant strain (control) containing the wild-type corn endosperm AGPase gene and the single colonies of the mutant strains 10-3 and 10-3-53 obtained through screening in Example 1, and inoculate them in LB liquid medium (containing 100 μg/mL ampicillin), cultured at 37°C for 12-24 hours, and then transferred the overnight cultured bacterial solution to fresh LB liquid medium (containing 100 μg/mL ampicillin) at a ratio of 1:100. Shake culture at 37°C and 200rpm for 3-4 hours until the OD ₆₀₀ is 0.6-0.8, then add a final concentration of 0.1mM IPTG and induce at room temperature for 12-24 hours. After the cultivation, the cells were collected by centrifugation at 6000rpm, weighed, placed at -20°C for 12-24 hours, and 5 mL of protein extraction buffer (50 mM Hepes (pH7.5), 5 mM MgCl ₂ , 1 mM EDTA ( pH8.0), 20% sucrose, 100 μM ATP, 30% ammonium sulfate, 10 mM Pi, 5 mM DTT, 500 μg/mL lysozyme (Lysozyme), 1 μg/mL pepsin inhibitor (Pepstatin), 1 μg/mL leupeptin ( Leupeptin), 1mM Benzamidine, 1mM PMSF, 10μg/mL serine protease inhibitor (Chymostatin)), resuspended bacteria, ice-bathed for 30min, ultrasonicated for 10-15min, and finally centrifuged at 30000rpm for 20min, and the supernatant was collected as a crude extract for enzyme activity determination liquid.

2. Determination of AGPase activity

Take 10 μl of crude protein extract obtained in step 1, mix with 40 μl reaction buffer (80 mM pH8.0 Hepes, 5 mM MgCl ₂ , 0.5 mg/mL BSA, 1 mM ADPGlc, 5 mM 3-phosphoglycerate, 2 mM Dithiotreitol, 10 μM 1,6- 2 phosphate glucose) mixed, then add 2mM sodium pyrophosphate solution to start the reaction, react at 25°C for 10 minutes, boil for 5 minutes, centrifuge at 5000rpm for 5 minutes, absorb the supernatant, add 380μl ddH ₂ O to the supernatant, measure OD ₃₄₀ as A1, Then add 70 μl of reaction buffer (100 mM pH7.5 Hepes, 0.1 mg/mL BSA, 5 mM MgCl ₂ , 1 mM NADP, 0.8 U/mL phosphoglucomutase, 0.7 U/mL 6-phosphate glucose dehydrogenase), React at 25°C for 10 minutes, measure OD ₃₄₀ as A2, and calculate AGPase activity based on the change of OD ₃₄₀ .

The AGPase activity of contrast, 10-3 and 10-3-53 AGPase crude extract is respectively 0.058unit/mg, 0.066unit/mg and 0.078unit/mg, shows that the activity level of crude extract AGPase is the same as that of iodine in Example 1 The staining results were consistent, the AGPase activity of the mutant strain 10-3 AGPase crude extract was 113% of the wild-type corn endosperm AGPase, and the AGPase activity of the mutant strain 10-3-53 AGPase crude extract was 134% of the wild-type corn endosperm AGPase . From the consistency of the iodine staining result and the AGPase activity analysis result of the crude extract, the screening method of the present invention is simple and feasible, and can be used to screen corn endosperm AGPase mutants with improved AGPase activity.

2. Glycogen content analysis

Pick the Escherichia coli glgC mutant strain (control) containing the wild-type corn endosperm AGPase gene and the single colonies of the mutant strains 10-3 and 10-3-53 obtained through screening in Example 1, respectively, and inoculate them in Conberg enriched liquid medium (containing 100 μg/mL ampicillin), culture at 37°C for 12-24 hours, then centrifuge at 12000rpm for 10min, collect the cells, resuspend the cells with distilled water after weighing, and culture at 100°C for 15min, then add DMSO and Hydrochloric acid, incubate at 60°C for 30min, adjust the pH value of the culture medium to 4.5 with 0.5M NaOH solution, add 3-5U amyloglucosidase, and react in the culture buffer of sodium acetate (containing 50mM sodium acetate, pH5.2) at 55°C Glycogen was degraded for 40 minutes, and then the glucose content produced by glycogen degradation was measured according to the glucose determination method: add 10 μl culture to 250ul sugar reaction buffer (100mM pH6.9 Imidazao-HCl, 5mM MgCl ₂ , 2mM NAD, 1mM ATP) Add 1-2μl (1-2μ/μl) 6-phosphate glucose dehydrogenase, react at room temperature for 10min, measure absorbance at 340nm wavelength, add 1-2μl hexokinase (Hexokinase, HK), measure glucose content.

The glycogen content measurement results of the control, 10-3 and 10-3-53 were 32.6mg/g, 46.2mg/g and 71.9mg/g respectively, and the results were basically consistent with the iodine staining results of Example 1, that is, the iodine staining was light Its glycogen content is low, and its glycogen content of iodine staining deep is relatively high, and above-mentioned detection result shows, compares with wild-type corn endosperm AGPase, the AGPase activity of the corn endosperm AGPase mutant screened with the inventive method not only obtains and its ability to accumulate glycogen in Escherichia coli has also been correspondingly improved, further proving the reliability of the screening method.

Embodiment 3, the purification of corn endosperm AGPase mutant

1. Protein Solubility Analysis

First, the bacterium liquid of the corn endosperm AGPase mutant strain 10-3-53 obtained in Example 2 induced by IPTG for 12-24 hours was centrifuged at 4° C. and 5000 rpm for 5 minutes to collect the induced cells, discard the supernatant, and use 1/ 10 volumes of TE (50mM Tris-HCl pH 8.0, 0.2mM EDTA) to resuspend the cell pellet, then add 10mg/mL lysozyme (prepared with fresh TE buffer) to a final concentration of 100μg/mL, then add 1/10 volume of Triton-100, incubate at 30°C for 15 minutes, put the centrifuge tube in an ice-water bath, ultrasonicate, then centrifuge at 12,000rpm at 4°C for 15 minutes, take the supernatant and precipitate for SDS-PAGE electrophoresis: supernatant Contains soluble protein, you can add an equal volume of 2×SDS loading buffer, boil for 5 minutes, centrifuge at 12000rpm for 5 minutes at room temperature, take 30mL supernatant for SDS-PAGE electrophoresis analysis; the precipitate contains inclusion bodies, you can use 1× After resuspension in SDS loading buffer, boil for 5 minutes, centrifuge at 12000rpm for 5 minutes at room temperature, and take 30mL supernatant for SDS-PAGE electrophoresis analysis. The results of electrophoresis analysis showed that most of the corn endosperm AGPase mutant protein existed in the pellet (that is, intracellular expression), and only a small amount of protein existed in the supernatant.

2. Mass induction of protein and preparation of crude extract

In the same method as in step 1 of Example 2, single bacterium colonies of Escherichia coli glgC mutant strain (control) and mutant strain 10-3-53 containing the wild-type corn endosperm AGPase gene were inoculated in LB liquid medium (containing 100 μg/mL Ampicillin), cultured at 37°C for 12-24 hours, then transferred the overnight cultured bacterial solution to fresh LB liquid medium (containing 100 μg/mL ampicillin) at a ratio of 1:100, at 37°C, Shake culture at 200 rpm for 3-4 hours until the OD ₆₀₀ is 0.6-0.8, then add a final concentration of 0.1 mM IPTG and induce at room temperature for 12-24 hours. After the cultivation, the cells were collected by centrifugation at 6000rpm, weighed, and placed at -20°C for 12-24 hours. Add 5mL of protein extraction buffer to each gram of cells, resuspend the cells, and ultrasonicate for 10-15 minutes after ice bathing for 30 minutes. Centrifuge at 30000rpm for 20min, collect the supernatant, rewash the precipitate once with 1/2 volume of protein extraction buffer, combine the supernatants to obtain corn endosperm AGPase protein bold solution, and set aside.

3. Fractional precipitation of ammonium sulfate of soluble protein

Respectively with 25%, 35%, 45%, 55%, 65%, 75% saturated ammonium sulfate solution, the AGPase protein crude liquid obtained in step 2 is carried out fractional precipitation, and the precipitation of each concentration section uses dialysis buffer (50mMHepes (pH8 .0), 5mM MgCl ₂ , 1mM EDTA (pH8.0), 20% Sucrose) suspension, dialyzed at 4°C for 12-24 hours, and then take a small amount of protein for SDS-PAGE and AGPase activity determination to determine the most suitable ammonium sulfate precipitation concentration. The results showed that most of the AGPase protein was precipitated at 45%-55% ammonium sulfate concentration, and the protein precipitate in this concentration range was collected, resuspended in 3 mL of dialysis buffer, and dialyzed at 4°C for 12-24 hours.

4. Ion exchange chromatography

Use fast protein liquid chromatography (FPLC for short) to pass the protein sample dialyzed in step 3 through the ion-exchange chromatography column first, and before the sample passes through the column, first use ion-exchange chromatography buffer A (50mM Hepes (pH8. MgCl ₂ , 5mM EDTA (pH8.0), 20% Sucrose, 5mM Pi, 0.5mM DTT) to equilibrate the Mono Q5/50 column (purchased from Pharmacia Company), and load the sample after the equilibrium is stable. Before loading the sample, the dialyzed Adjust the pH value of the protein sample to 8.0, and then use 5 column bed volumes of ion exchange chromatography buffer A to continue rinsing the column until the UV absorption value of the effluent reaches a stable value, and then use a 0-50% gradient for elution BufferB (50mM Hepes (pH7.0), 1mM MgCl ₂ , 5mM EDTA (pH8.0), 20% Sucrose, 5mM Pi, 0.5mM DTT, 1M NaCl) eluted the column at a flow rate of 8.0mL/min, and collected the effluent, Quickly put on ice for temporary storage. A small amount of samples with different absorption peaks were taken for SDS-PAGE, and the result was that the AGPase protein was eluted at about 400mM NaCl. Collect the sample effluent of this peak, measure AGPase enzyme activity, the result shows that the AGPase activity of the eluent under this peak is higher, merge the collected liquid of this peak, add solid ammonium sulfate to the final concentration of 75% and carry out the concentration of sample, use The pellet was resuspended in dialysis buffer, dialyzed at 4°C for 12-24 hours, and stored at -80°C for later use.

5. Gel filtration chromatography

Pass the dialyzed protein sample stored at -80°C in Step 4 through the Hiload 16/60 superdex75 column of FPLC (purchased from Pharmacia). Bubbles in water, and then use it to wash the column until it is stable, and then use gel filtration chromatography bufferC (50mM Hepes (pH7.5), 10mM MgCl ₂ , 5mM EDTA (pH8.0), 5% Sucrose, 200mM KCl, 1mM DTT) Equilibrate the column, load the sample after it is stable, load 4mL of the sample (the maximum volume is 5mL), and use the same buffer C for elution, collect the effluent at 0.2mL/tube, and perform SDS-PAGE detection to determine the elution of the target protein. The SDS-PAGE detection results of the off-peak and eluted proteins are shown in Figure 7 (swimming lane M is Marker, and swimming lanes 1-3 are purified target protein AGPase-10-3-53), indicating that the target protein with higher purity has been obtained. At the same time, the AGPase activity of the peak collection liquid was measured, and finally the active fraction was combined and stored at -80°C after aliquoting.

Example 4. Kinetic and Regulatory Analysis of Maize Endosperm AGPase Mutant AGPase-10-3-53

The purified maize endosperm AGPase mutant AGPase-10-3-53 and wild type maize endosperm AGPase (contrast) screened by the method of the present invention were analyzed for kinetics and regulatory properties as follows:

1) Prepare two substrates (ATP and 1-P-G, purchased from Sigma Company) with different concentrations, namely 0, 500nM, 1μM, 10μm, 50μM, 100μm, 500μm, 1mM and 5mM, respectively in the above-mentioned different concentrations of substrates The enzyme activity of AGPase-10-3-53 and wild-type corn endosperm AGPase was measured in the medium, and the reaction velocity v was calculated, and a straight line was obtained by plotting 1/v against 1/[S], and the intercept of the horizontal axis was -1/Km , the intercept of the vertical axis is 1/Vmax.

The enzymatic kinetic constants of maize endosperm AGPase mutant AGPase-10-3-53 and wild-type maize endosperm AGPase under different substrates (ATP and 1-P-G) are shown in Table 1. Compared with wild-type maize endosperm AGPase, this The invented mutant AGPase-10-3-53 has an increased affinity to the substrate, and the cause of the increased affinity of the mutant to the substrate is analyzed, which may interact with the two subunits of Shrunken-2 and Brittle-2 after the mutation Enhancement, because the mutation site Ile from the 323rd position of the amino terminal of the small subunit is located in the "motif" structure formed by 55 amino acid residues, and the role of the "motif" structure is to regulate the size of the two subunits Interactions (Joanna M. Cross, Maureen Clancy, Janine R, Shaw et al. A polymorphic motif the small subunit of AGPase modulates interactions between the small and large subunits. The Plant Journal. 2005, 41, 501-511).

Table 1 Maize endosperm AGPase mutant AGPase-10-3-53 and wild type maize endosperm under different substrates

Enzyme Kinetic Constants of AGPase

substrate Km，mM wild-type AGPase Mutant AGPase-10-3-53 ATP1-P-G 0.048(0.006)0.0177(0.0024) 0.012(0.0056)0.0068(0.0003)

2) prepare the concentration of activator 3-phosphoglycerate (purchased from Sigma Company) and inhibitor inorganic phosphorus (purchased from Sigma Company) of different concentrations, namely 0mM, 0.05mM, 0.1mM, 0.25mM, 0.5mM, 1mM, 2.5mM, 5mM and 10mM, measure the enzyme activity of AGPase-10-3-53 and wild-type corn endosperm AGPase in the above-mentioned different concentrations of activators or inhibitors, respectively, to detect the effect of different concentrations of activators or inhibitors on enzyme activity Impact.

The test results show that the sensitivity of the corn endosperm AGPase mutant AGPase-10-3-53 of the present invention to activators and inhibitors is the same as that of the wild type corn endosperm AGPase, which proves that the corn endosperm AGPase mutant AGPase-10-3-53 is sensitive to the substrate The enhanced affinity is the reason for the improved enzyme activity and glycogen accumulation ability of the mutant.

sequence listing

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<210>1

<211>475

<212>PRT

<213> Zea mays L.

<400>1

Met Asp Met Ala Leu Ala Ser Lys Ala Ser Pro Pro Pro Trp Asn Ala

1 5 10 15

Thr Ala Ala Glu Gln Leu Ile Pro Lys Arg Asp Lys Ala Ala Ala Asn

20 25 30

Asp Ser Thr Tyr Leu Asn Pro Gln Ala His Asp Ser Val Leu Gly Ile

35 40 45

Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Tyr Pro Leu Thr Lys Lys

50 55 60

Arg Ala Lys Pro Ala Val Pro Leu Gly Ala Asn Tyr Arg Leu Ile Asp

65 70 75 80

Ile Pro Val Ser Asn Cys Leu Asn Ser Asn Ile Ser Lys Ile Tyr Val

85 90 95

Leu Thr Gln Phe Asn Ser Ala Ser Leu Asn Arg His Leu Ser Arg Ala

100 105 110

Tyr Gly Ser Asn Ile Gly Gly Tyr Lys Asn Glu Gly Phe Val Glu Val

115 120 125

Leu Ala Ala Gln Gln Ser Pro Asp Asn Pro Asn Trp Phe Gln Gly Thr

130 135 140

Ala Asp Ala Val Arg Gln Tyr Leu Trp Leu Phe Glu Glu His Asn Val

145 150 155 160

Met Glu Phe Leu Ile Leu Ala Gly Asp His Leu Tyr Arg Met Asp Tyr

165 170 175

Glu Lys Phe Ile Gln Ala His Arg Glu Thr Asn Ala Asp Ile Thr Val

180 185 190

Ala Ala Leu Pro Met Asp Glu Lys Arg Ala Thr Ala Phe Gly Leu Met

195 200 205

Lys Ile Asp Glu Glu Gly Arg Ile Ile Glu Phe Ala Glu Lys Pro Lys

210 215 220

Gly Glu Gln Leu Lys Ala Met Met Val Asp Thr Thr Ile Leu Gly Leu

225 230 235 240

Asp Asp Val Arg Ala Lys Glu Met Pro Tyr Ile Ala Ser Met Gly Ile

245 250 255

Tyr Val Phe Ser Lys Asp Val Met Leu Gln Leu Leu Arg Glu Gln Phe

260 265 270

Pro Glu Ala Asn Asp Phe Gly Ser Glu Val Ile Pro Gly Ala Thr Ser

275 280 285

Ile Gly Lys Arg Val Gln Ala Tyr Leu Tyr Asp Gly Tyr Trp Glu Asp

290 295 300

Ile Gly Thr Ile Ala Ala Phe Tyr Asn Ala Asn Leu Gly Ile Thr Lys

305 310 315 320

Lys Pro Met Pro Asp Phe Ser Phe Tyr Asp Arg Phe Ala Pro Ile Tyr

325 330 335

Thr Gln Pro Arg His Leu Pro Pro Ser Lys Val Leu Asp Ala Asp Val

340 345 350

Thr Asp Ser Val Ile Gly Glu Gly Cys Val Ile Lys Asn Cys Lys Ile

355 360 365

Asn His Ser Val Val Gly Leu Arg Ser Cys Ile Ser Glu Gly Ala Ile

370 375 380

Ile Glu Asp Ser Leu Leu Met Gly Ala Asp Tyr Tyr Glu Thr Glu Ala

385 390 395 400

Asp Lys Lys Leu Leu Ala Glu Lys Gly Gly Ile Pro Ile Gly Ile Gly

405 410 415

Lys Asn Ser Cys Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala Arg Ile

420 425 430

Gly Asp Asn Val Lys Ile Leu Asn Ala Asp Asn Val Gln Glu Ala Ala

435 440 445

Arg Glu Thr Asp Gly Tyr Phe Ile Lys Gly Gly Ile Val Thr Val Ile

450 455 460

Lys Asp Ala Leu Leu Pro Ser Gly Thr Val Ile

465 470 475

<210>2

<211>1428

<212>DNA

<213> Zea mays L.

<400>2

atggacatgg ctttggcgtc taaagcctcc cctccgccct ggaatgccac cgccgccgag 60

cagctaattc caaagcgtga caaagccgct gcaaatgatt caacatacct caatcctcaa 120

gctcatgata gtgttcttgg aatcattctg ggaggtggtg ctgggactag attgtacccc 180

ttgacaaaga agcgtgccaa gcctgcagtg ccattgggtg ccaactatag actgattgat 240

attcctgtca gcaattgtct caacagcaac atatccaaga tctatgtgct aacgcaattt 300

aactctgctt ccctcaaccg tcacctctca agagcctacg ggagcaacat tggagggtac 360

aagaatgaag ggtttgttga agtcttagct gcacagcaga gccgataa tccaaactgg 420

tttcagggta ctgcagatgc tgtaaggcag tacttgtggt tgtttgagga gcataatgtg 480

atggaatttc taattcttgc tggcgatcac ctgtaccgga tggactatga aaagttcatt 540

caggcacaca gagaaacaaa tgctgatatt accgttgctg ccctaccgat ggatgagaaa 600

cgtgcaactg catttggcct catgaaaatt gatgaagaag ggaggatcat tgagtttgct 660

gagaaaccga aaggagagca gttgaaagca atgatggttg acaccaccat acttggcctt 720

gatgacgtga gggcaaagga aatgccttat attgctagca tgggtatcta tgttttcagc 780

aaagatgtaa tgcttcagct cctccgtgaa caatttcctg aagccaatga ctttggaagt 840

gaggttatc caggtgcaac cagcattgga aagagggttc aggcttatct gtatgatggt 900

tactgggaag atatcggtac cattgcggca ttttataatg caaacttggg aataaccaag 960

aagccaatgc cagatttcag cttctatgac cgttttgctc caatttatac acaacctcga 1020

cacctgccac cttcaaaggt tcttgatgct gatgtgacag acagtgttat tggtgaagga 1080

tgtgtttatta aaaactgcaa gataaaccat tctgtagttg gactccgatc ttgcatatct 1140

gaaggtgcta tcatagagga cagtttacta atgggtgcgg actactatga gacagaagct 1200

gataaaaaac tccttgccga aaaaggtggc attcctattg gtattgggaa aaattcatgc 1260

atcaggagag caatcattga caagaatgct cgaattggag acaatgttaa gatactcaat 1320

gctgacaatg ttcaagaagc tgcaagggag acagacgggt acttcatcaa aggtggaatt 1380

gtcacagtga tcaaggatgc tttactccct agtggaacag ttatatga 1428

Claims (10)

1, a kind of method of screening corn embryosperm ADP-glucose pyrophosphorylase mutant may further comprise the steps:

1) with the oxammonium hydrochloride mutagens the big small subunit cDNA of corn embryosperm AGPase is carried out random mutation under 70-80 ℃ of warm bath condition; Described oxammonium hydrochloride mutagens prescription is: the 0.15-0.25g oxammonium hydrochloride, and the trisodium phosphate 1.0-1.6mL of 125mM pH7.0, the NaCl 0.2-0.4mL of 1M, the EDTA 10-14 μ l of 500mM pH8.0 is settled to 3mL with deionized water;

2) with the big small subunit cDNA difference of big small subunit cDNA of corn embryosperm AGPase through suddenling change and wild-type corn embryosperm AGPase in contrast in step 1) transformed into escherichia coli glgC mutant strain, screening transforms respectively to be had through the big small subunit cDNA of corn embryosperm AGPase of sudden change and the positive monoclonal of the big small subunit cDNA of wild-type corn embryosperm AGPase;

3) with step 2) positive colony of screening is inoculated in Conberg and adds on the rich solid culture flat board, cultivated 12-24 hour down at 35-39 ℃; Described Conberg adds rich solid culture based formulas: potassium primary phosphate 8-9g, and dipotassium hydrogen phosphate 10-12g, yeast extract 5-7g, glucose 8-12g, agar 15-25g, water is settled to 1L, final pH 7.0;

4) with Wagner's reagent Conberg in step 3) being added the single bacterium colony that grows on the rich solid culture flat board dyes, compare with the color of contrast bacterium colony, it is mutants which had that comparison is shone saturate, through sequence verification, obtain corn embryosperm ADP-glucose pyrophosphorylase mutant again; Described Wagner's reagent is the aqueous solution that contains 0.08-0.12mol/L iodine and 0.25-0.35mol/L potassiumiodide.

2, method according to claim 1 is characterized in that: the warm bath temperature in the described step 1) is 75 ℃, and the oxammonium hydrochloride mutagens is for containing mass percentage concentration 6.9% oxammonium hydrochloride, 50mM trisodium phosphate, 0.3M NaCl, the aqueous solution of 2mM EDTA; Step 2) big small subunit cDNA of corn embryosperm AGPase through suddenling change in and the big small subunit cDNA of wild-type corn embryosperm AGPase in contrast are by containing through the big small subunit cDNA of corn embryosperm AGPase of sudden change or the prokaryotic expression carrier transformed into escherichia coli glgC mutant strain of the big small subunit cDNA of wild-type corn embryosperm AGPase; Vaccination ways in the step 3) is line, and culture temperature is 37 ℃, and the prescription that Conberg adds rich solid medium is: potassium primary phosphate 8.5g, dipotassium hydrogen phosphate 11g, yeast extract 6g, glucose 10g, agar 20g, water are settled to 1L, final pH 7.0; Wagner's reagent in the step 4) is the aqueous solution that contains 0.1mol/L iodine and 0.3mol/L potassiumiodide.

3, method according to claim 1 is characterized in that: with described step 1)-4) repeat 1-3 time.

4, the corn embryosperm ADP-glucose pyrophosphorylase mutant that obtains with each described method of claim 1-3.

5, corn embryosperm ADP-glucose pyrophosphorylase mutant according to claim 4 is characterized in that:

Described corn embryosperm ADP-glucose pyrophosphorylase mutant is one of following amino acid residue sequences:

1) the SEQ ID NO:1 in the sequence table;

2) amino acid residue sequence of SEQ ID NO:1 in the sequence table had the active protein of AGPase through replacement, disappearance or the interpolation of one to ten amino-acid residue.

6, the gene of coding claim 4 or 5 described corn embryosperm ADP-glucose pyrophosphorylase mutants.

7, gene according to claim 6 is characterized in that: described gene is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:2 in the sequence table;

2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;

3) with sequence table in the dna sequence dna that limits of SEQ ID NO:2 have 90% above homology and have the active nucleotide sequence of AGPase;

4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID NO:2 in the sequence table.

8, contain claim 6 or 7 described expression carrier, transgenic cell line and host bacterium.

9, a kind of method that improves the corn seed starch content is that claim 6 or 7 described ADP-glucose pyrophosphorylase mutant genes are imported corn tissue or cell, and the starch content of corn seed is improved.

10, method according to claim 9 is characterized in that: described ADP-glucose pyrophosphorylase mutant gene imports corn tissue or cell by the plant expression vector that contains described ADP-glucose pyrophosphorylase mutant gene.