CN112941057B

CN112941057B - Application of ZmGGH gene, material and method for regulating and controlling content of corn folic acid

Info

Publication number: CN112941057B
Application number: CN202110141291.6A
Authority: CN
Inventors: 宋丽莉; 唐雪明; 施标; 郑洪建; 武国干; 王金斌; 黄艳娜; 吕贝贝; 刘华
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2021-11-02
Anticipated expiration: 2041-02-02
Also published as: CN112941057A

Abstract

The invention relates to application of ZmGGH gene, a material and a method for regulating and controlling the content of corn folic acid, belonging to the technical field of bioscience. The invention provides application of ZmGGH gene in regulating and controlling the content of maize folic acid, the over-expression ZmGGH gene reduces the content of maize folic acid by 48.1-70.3%, and ZmGGH gene mutation can improve the content of folic acid.

Description

Application of ZmGGH gene, material and method for regulating and controlling content of corn folic acid

Technical Field

The invention relates to the technical field of bioscience, in particular to application of a ZmGGH gene, a material and a method for regulating and controlling the content of corn folic acid.

Background

Folic acid is a water-soluble vitamin B (vitB9) and comprises Tetrahydrofolate (THF) and derivatives thereof, and is a micronutrient necessary for growth and development of animals and plants. Folate deficiency can lead to severe developmental disorders. Humans cannot synthesize folic acid, which is mainly supplemented from meals or folic acid tablets. The most important staple food crops (such as rice, wheat and corn) for human beings have low folic acid content. Currently, folic acid tablets and bio-fortified staple food crops are the primary means of addressing folate deficiency. For Chinese people, the mutation of the MTHFR gene (5, 10-methyltetrahydrofolate reductase) of methylenetetrahydrofolate reductase easily causes folic acid deficiency in vivo, and has poor absorption and metabolism effects on folic acid tablets, so that the biological strengthening of staple food crops is considered as a green and healthy method for resisting folic acid deficiency. At present, the population with global folic acid deficiency is still more common, and the solution of the global folic acid deficiency problem has important significance for human survival and health.

Corn is an important grain economic crop, researches influence on gene functions related to the stability of corn folic acid, defines the influence of the corn folic acid on the folic acid content, and has important significance on the research of the nutrition fortification of the corn folic acid.

Disclosure of Invention

The invention aims to provide application of ZmGGH gene, a material for regulating and controlling the content of corn folic acid and a method thereof. The over-expression ZmGGH gene can reduce the content of the maize folic acid, and the silencing ZmGGH gene can improve the content of the maize folic acid.

The invention provides an application of a ZmGGH gene in regulating and controlling the content of corn folic acid, wherein the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

The invention also provides application of the over-expressed ZmGGH gene in reducing the content of the maize folic acid, wherein the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

The invention also provides application of the silent ZmGGH gene in improving the content of the maize folic acid, wherein the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

The invention also provides a material for regulating and controlling the content of the corn folic acid, the material can over-express or silence the ZmGGH gene, and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

Preferably, the material comprises a carrier or bacteria.

The invention also provides an expression vector for regulating the content of the maize folic acid, the expression vector can over-express the ZmGGH gene or silence the ZmGGH gene, and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID No. 1.

Preferably, the backbone vector of the expression vector comprises pCAMBIA 3301.

The invention also provides a primer pair for detecting the ZmGGH gene, wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

The invention also provides a method for regulating and controlling the content of the corn folic acid, which comprises the following steps: constructing a maize strain over-expressing ZmGGH gene, and reducing the folic acid content in maize; or constructing a corn strain for silencing ZmGGH gene and increasing the folic acid content in the corn; the nucleotide sequence of the ZmGGH gene is shown in SEQ ID NO. 1.

Preferably, the construction method of the maize strain overexpressing the ZmGGH gene includes an Agrobacterium-mediated method; the construction method of the corn strain for silencing the ZmGGH gene comprises a gene editing method or an RNA interference method.

The invention provides an application of ZmGGH gene in regulating and controlling the content of maize folic acid. The over-expression ZmGGH gene can reduce the content of the maize folic acid, the silencing ZmGGH gene can improve the content of the maize folic acid, and the silencing (mutation) of the ZmGGH gene has important value for improving the content of the folic acid. Test results show that the maize ZmGGH gene is synthesized and connected to pCAMBIA3301, and an overexpression vector pCAMBIA3301-ZmGGH is constructed through enzyme digestion, connection and transformation. The gene is transferred to corn B104 by an agrobacterium-mediated method to obtain resistant materials. The ZmGGH gene is identified by PCR detection, and the Bar gene is detected by a test strip, so that 5 positive strains are obtained. Detecting the content of the over-expressed corn kernels by liquid phase mass spectrometry. Compared with the wild type, the folic acid content of 3 over-expression strains is reduced by 48.1-70.3%.

Drawings

FIG. 1 is a diagram of pCAMBIA3301-ZmGGH vector provided by the present invention;

FIG. 2 is a PCR identification chart of pCAMBIA3301-ZmGGH transferred to Agrobacterium provided by the present invention;

FIG. 3 is a diagram showing the result of PCR identification of leaves of an overexpression strain provided by the present invention;

FIG. 4 is a diagram showing the identification results of the over-expressed strain and the wild-type test strip provided by the present invention;

FIG. 5 is a diagram showing the screening results of the overexpression lines and the wild-type glufosinate provided by the present invention;

FIG. 6 is a graph comparing the folate content of the overexpression lines provided by the invention with that of wild-type.

Detailed Description

The invention provides an application of a ZmGGH gene (corn coding glutamyl hydrolase gene ZmGGH (gamma-glutamyl hydrolases)) in regulating and controlling the content of corn folic acid, wherein the nucleotide sequence (CDS sequence) of the ZmGGH gene is shown as SEQ ID NO. 1: ATGGACTCGCGCTGCCCCCATCTGCTACTGCTCCTCCCTCTCCTCCTCTTGGCCGCCCTCCTGCCCCCGCCGTCGTCGGGGGTGCCGGAGGTCATCTGGCTGCCGACGAGCGGGGGAGCGGGGCCCTTGTCCTGCGCGCCGCCGGACCCCGCCGTGTACGACCGCCCCGTCATCGGCATCGTCTCGCACCCAGGGGACGGTGCGGGCGGCAGGATCAGCAACACCACCGCGACCTCCTACATCGGCGCCTCCTACGTCAAGTTCGTCGAGGCCGCCGGCTCGCGCGTCATCCCACTCGTCTACAACGAGCCCGAAGACCGCCTCCTCGAGAAATTGAGTTTGTTGAATGGAGTGCTGTTCACTGGTGGGTCAGAGAAGGAAGGTGTATATTTTGACACGATAAAAAGAGTATTTCAGTATGTTTTGGACAAGAATGATGCAGGTGAACCATTTCCTTTGTTTGCCCAATGTCTTGGCTTTGAGCTTGTAAGCATGATTGTCAGCAAGGACAATAATATCTTGGAGGCATTTGACGCCCAGGATCAAGCATCAACTCTTCAGTTTCCCAGCTATGCTTTGGAGGGTTCAGTTTTTCAAAGGTTTGACACTGACCTCATCAAGAAAGTCAGCACCAACTGTCTTGTCATGCAAAATCACAGATATGGTATATCACCAAGGCGATATCGAGAGAACGATGCACTATCAAGTTTCTTCAAAATCTTGACTACATCACCTGATGAAAACGGCAAGGTCTATGTCTCAACTGTACAAGCCAATAATTATCCAATCACTTGCACGCAATGGCATCCTGAGGCTTTAACCTCTATTATTATACAATTCAAGTTGGCAGTACATCATGTTGGTTTAAGCATATGCAAGAAAGGACCTTGCTTTGATGAAACTTGCAAGAGTGTTAGACTAATGCGTGTATTTTTGGTAGGAAATCGTTTGAGGAGGTCTACATCTTCTCCTGAGACTGGATTGAACGCCTGA, respectively; the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4: MDSRCPHLLLLLPLLLLAALLPPPSSGVPEVIWLPTSGGAGPLSCAPPDPAVYDRPVIGIVSHPGDGAGGRISNTTATSYIGASYVKFVEAAGSRVIPLVYNEPEDRLLEKLSLLNGVLFTGGSEKEGVYFDTIKRVFQYVLDKNDAGEPFPLFAQCLGFELVSMIVSKDNNILEAFDAQDQASTLQFPSYALEGSVFQRFDTDLIKKVSTNCLVMQNHRYGISPRRYRENDALSSFFKILTTSPDENGKVYVSTVQANNYPITCTQWHPEALTSIIIQFKLAVHHVGLSICKKGPCFDETCKSVRLMRVFLVGNRLRRSTSSPETGLNA are provided. The over-expression ZmGGH single gene can realize that the folic acid content of corn grains is reduced by 70.3 percent at most. The invention has no special limitation on the obtaining method of ZmGGH gene, and the artificial synthesis method is adopted.

The invention also provides application of the over-expressed ZmGGH gene in reducing the content of the maize folic acid, wherein the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1. The method of the present invention for the overexpression is not particularly limited, and a conventional gene overexpression method well known to those skilled in the art may be used.

The invention also provides application of the silent ZmGGH gene in improving the content of the maize folic acid, wherein the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1. The method of silencing in the present invention is not particularly limited, and a conventional method of silencing gene expression, which is well known to those skilled in the art, may be employed. The silencing method of the present invention preferably comprises gene editing or RNA interference (RNAi) or mutation of the ZmGGH gene to silence it.

The invention also provides a material for regulating and controlling the content of the corn folic acid, the material can over-express or silence the ZmGGH gene, and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1. In the present invention, the material preferably includes a carrier or a bacterium.

The invention also provides an expression vector for regulating the content of the maize folic acid, the expression vector can over-express the ZmGGH gene or silence the ZmGGH gene, and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID No. 1. In the present invention, the backbone vector of the expression vector includes pCAMBIA3301, and the expression vector can be used to overexpress the ZmGGH gene. When silencing the ZmGGH gene, the material used preferably comprises maize B104, B73.

The invention also provides a primer pair for detecting the ZmGGH gene, wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.2(CTTGACCATGGACTCGCGCTGCCCCCAT) and SEQ ID NO.3 (TGTAATTCACACGTGTCAGGCGTTCAATCCAGTCT), and the nucleotide sequence of the ZmGGH gene is shown as SEQ ID NO. 1.

In the present invention, the construction method of a maize strain overexpressing the ZmGGH gene preferably includes an Agrobacterium-mediated method. The method for mediating the agrobacterium is not particularly limited, and a conventional agrobacterium-mediated method is adopted. The invention preferably adopts an agrobacterium-mediated method to carry out genetic transformation on the maize immature embryos. In the present invention, the construction method of the maize line silencing the ZmGGH gene preferably includes gene editing or RNA interference (RNAi) or mutation of the ZmGGH gene to silence it. The silencing method of the invention preferably comprises mutating the maize ZmGGH gene to obtain maize material with increased folate content.

The application of the ZmGGH gene and the material and method for regulating the content of the maize folic acid are further described in detail with reference to the following specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

Example 1

Construction of overexpression vector pCAMBIA3301-ZmGGH

(1) Construction of expression vector pCAMBIA 3301-ZmGGH:

the corn ZmGGH gene is synthesized and coded by a chemical synthesis method, and is constructed on a T carrier, and the correct plasmid is obtained by sequencing verification and is named as pMD 18T-ZmGGH. Constructing an overexpression vector through enzyme digestion, connection and transformation. Firstly, the pMD18T-ZmGGH plasmid is cut by NcoI and PmlI to obtain a target gene ZmGGH (1808001), then pCAMBIA3301 cut by NcoI and PmlI is used for ligation and transformation, a single clone is picked to extract a plasmid, and a recombinant expression vector pCAMBIA3301-ZmGGH is obtained after sequencing verification. The recombinant expression vector pCAMBIA3301-ZmGGH comprises a BIpR (bar) screening marker gene and a KanR (kan kanamycin) screening marker gene, and a specific vector diagram is shown in figure 1.

The digestion reaction system is shown in Table 1.

TABLE 1 digestion reaction System

Supplementing water to 30 mu L, carrying out enzyme digestion at 37 ℃ for 1h, and recovering and purifying reaction products after agarose gel electrophoresis.

And (3) connection reaction:

and (3) transformation: adding 100 μ L DH5a competent cells into the ligation product, ice-cooling for 30min, heat-shocking at 42 deg.C for 1min, standing on ice for 2min, adding 200 μ L non-resistant LB culture solution, recovering by shaking table at 37 deg.C for 45min, and plating (LB solid culture medium containing kanamycin). Incubated at 37 ℃ overnight.

Sequencing: and (4) extracting plasmids after selecting the monoclone, and obtaining correct plasmids through sequencing verification.

(2) Agrobacterium transformation is carried out by a freeze-thaw method, 10 muL of pCAMBIA3301-ZmGGH with correct sequencing is taken, 10 muL of EHA105 is added for competence, ice bath is carried out for 10min, the pCAMBIA is taken out and put into liquid nitrogen for 5min, then the pCAMBIA is taken out and put into 37 ℃ for heat shock for 5min, then the pCAMBIA is put into ice for standing for 5min, 200 muL of non-resistant YEB is added, resuscitation is carried out for 4h at 28 ℃, a plate (YEB solid culture medium added with kanamycin) is coated, and culture is carried out for 48h at 28 ℃. 18 colonies were picked from EHA105 plates for PCR validation. The PCR identification primer is:

1808001-F:CTTGACCATGGACTCGCGCTGCCCCCAT(SEQ ID NO.2)

1808001-R:TGTAATTCACACGTGTCAGGCGTTCAATCCAGTCT(SEQ ID NO.3)

reaction system:

and (3) PCR reaction conditions:

1-18 using colony as template, CK as blank control (template is water), PCR product is 993 bp. Lanes 1-14, 16-18, which are positive clones, and CK lane has no bands, and PCR identification is detailed in FIG. 2, wherein the positive clone strain can be used for subsequent corn genetic transformation.

Example 2

Construction of maize overexpression lines

(1) Agrobacterium mediated transformation of maize immature embryo

Activating agrobacterium: adding 100 mu L of pCAMBIA3301-ZmGGH positive agrobacterium into YEB culture medium containing Rif, Str and Kan, and culturing overnight at 28 ℃; 5mL of the culture was added to 50mL of Agrobacterium liquid medium and cultured at 28 ℃ to OD₆₀₀0.4-0.6; centrifuging at 5000rpm for 10min, and collecting thallus.

Infecting maize immature embryos by an agrobacterium-mediated method:

selecting an explant: taking waxy corn young ears pollinated for 9-14 days, removing wrapped leaves, sterilizing, and selecting young embryos as explants;

induction of callus: inoculating the explant into a callus induction culture medium for induction culture, expanding a scutellum after culturing for 7-10 days, and timely removing buds and root bodies; after 15 days of induction culture, selecting the callus with light yellow color, compact structure and vigorous growth for subculture, and subculturing for 1 time every 15-20 days and subculturing for 2-3 times to obtain the embryogenic callus.

Differentiation culture: and transferring the embryonic callus to a differentiation culture medium for culturing, and subculturing for 1 time every 15-20 days until a differentiation regeneration seedling is obtained.

Rooting culture and seedling hardening: transferring the differentiated and regenerated seedlings growing to 3-5 cm into a rooting culture medium for culturing to obtain rooted seedlings; and (3) when the root length of the rooted seedling is 2-3 cm, removing the sealing film of the culture bottle, hardening the seedling, washing the culture medium on the rooted seedling, and transplanting.

(2) PCR detection of maize material ZmGGH gene

The CDS sequence of the maize ZmGGH gene is taken as a template, primers for identifying the maize ZmGGH gene are designed, and the primer sequences are respectively as follows:

F:ACAACGAGCCCGAAGACC(SEQ ID NO.5)

R:CCATTGCGTGCAAGTGAT(SEQ ID NO.6)

DNA of T2 generation over-expression corn material leaf is extracted by CTAB method, and PCR amplification is carried out by taking the leaf DNA as a template, and the PCR identification result is shown in figure 3. Lane 0 is water, blank control; lane WT is wild type, negative control; lane M is the DNA molecular weight standard of DL2000 DNAmarker, wherein the

numbers

2, 3, 5, 8 and 11 are different strains of ZmGGH gene-transferred corn, the size of the PCR product of the plant successfully transferred with the ZmGGH gene is 500bp strip, and the PCR positive rate of the over-expressed corn material of T2 generation determined by PCR identification is 100%.

(3) Bar gene for detecting corn material test strip

The T2 generation over-expression corn material leaf is taken, the surface is cleaned, and the leaf is sucked dry by filter paper. Cutting leaves, placing in a mortar, adding 1ml of sterile water, grinding, fully grinding, placing the test strip in grinding liquid, standing for 2min, and observing the result. In FIG. 4, WT (wild type),

overexpression lines

2, 3, 5, 8, 11, respectively, are shown from left to right. As a result, it was found that the wild type (control material WT) had only one band and no Bar gene; the test strip of the overexpression strain (2, 3, 5, 8, 11) is provided with two strips which contain the Bar gene, and the Bar gene is proved to be integrated into the genome of the overexpression strain.

(4) Corn material glufosinate screening

The German Bayer test shows that the glufosinate ammonium (concentration is 18%) is diluted 1000 times by water, the diluted solution is sprayed on leaves of wild type and over-expression strains, and after 48 hours of spraying, the leaves of the wild type are observed to turn yellow, but the leaves of the over-expression strains are still green, which shows that the over-expression strains have resistance to the glufosinate ammonium, and a glufosinate ammonium screening phenotype chart is shown in figure 5.

The invention can quickly and efficiently identify and determine the over-expression strain from three aspects. In a first aspect, the invention uses glufosinate spraying to identify phenotypes, i.e. to identify foliar changes after spraying; after spraying, the wild type leaves turn yellow, dry and die; the color of the leaves of the over-expression strain is unchanged, and the over-expression strain can grow normally and become seedlings. The glufosinate-ammonium spraying can be used for rapidly and preliminarily identifying whether the gene is transformed into a plant body through the leaf state. In the second and third aspects, the target gene and the selection marker gene are identified separately on a molecular level. Identifying a target gene by PCR, wherein if the gene is successfully introduced into a plant, a target strip can be seen by running electrophoresis of a PCR product, and a wild type cannot be amplified; the test paper strip identifies and screens the marker gene, and is rapid and efficient. Only the leaves need to be ground, if the plant has the screening marker gene (Bar gene), two bands (positive) can appear on the test strip, and if the wild type does not have the screening marker gene, one band (negative) can appear on the test strip. By utilizing the PCR method and the test paper strip, whether the target gene and the screening marker gene are integrated on the genome of the transformed plant can be rapidly identified.

Example 3

Grain folic acid content reduction of maize overexpression strain

Preparation of samples: weighing the seeds of the wild corn strain and the overexpression strain respectively, and quickly freezing the seeds in liquid nitrogen.

And (3) extracting a sample: grinding a sample by using a ball mill, and accurately weighing 200mg of the sample; adding 500 μ LPBS solution (containing 0.2% beta-mercaptoethanol and 1% ascorbic acid) extractive solution, and mixing; adding 35 μ L of rat serum, mixing, and standing at 37 deg.C for 4 hr; boiling the sample in boiling water for 10 min; cooling on ice for 10 min; centrifuging at 4 deg.C and 13300rpm for 10min, and collecting supernatant;

liquid phase mass spectrometry determination: the sample extract was used for LCMS to determine the folate and derivative content.

Quantitative results: calculating the content of each component according to the standard curve (see the following formula)

The content of each component in the sample (ng/mg) ═ C V/M

Wherein C is the instrument reading concentration; v is the volume of the sample extracting solution; m is the total amount of the sample to be weighed.

As shown in Table 2, the folic acid content of the wild type strain is 1.29 mug/100 g, the folic acid content of the over-expression strain is 0.38-0.67 mug/100 g, and the folic acid content of the over-expression strain is reduced by 48.1-70.3% compared with that of the wild type strain (see figure 6). The result shows that the gene influences the content of the maize folic acid, has important research significance, and the research on the ZmGGH gene mutation has important value in improving the content of the folic acid.

TABLE 2 maize kernel folate content

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. ZmGGHApplication of gene in regulating and controlling content of corn folic acid, and application of gene in regulating and controlling content of corn folic acidZmGGHThe nucleotide sequence of the gene is shown as SEQ ID NO. 1; over-expressionZmGGHThe gene reduces the content of the corn folic acid; silencingZmGGHThe gene improves the content of the corn folic acid.

2. A method for regulating and controlling the content of corn folic acid comprises the following steps: construction of overexpressionZmGGHThe gene corn strain reduces the folic acid content in the corn; or to construct silenceZmGGHThe folic acid content in the corn is improved by the genetic corn strain; the above-mentionedZmGGHThe nucleotide sequence of the gene is shown in SEQ ID NO. 1.

3. According to claim2, characterized in that it is overexpressedZmGGHThe construction method of the corn strain of the gene comprises an agrobacterium-mediated method; silencingZmGGHThe construction method of the corn strain of the gene comprises a gene editing method or an RNA interference method.