CN111733182B - Method for improving plant resistance to Fusarium graminearum by using AtALA1 gene - Google Patents

Abstract

The invention relates to a method for improving the resistance of plants to Fusarium graminearum, wherein the AtALA1 gene is integrated into the target plant, and the AtALA1 gene is expressed in the target plant to reduce the toxin content of Fusarium graminearum and improve plant For resistance to Fusarium graminearum, the nucleotide sequence of the AtALA1 gene is shown in SEQ ID NO.14. The invention utilizes the transporting and degrading mechanism of plants to pathogenic bacteria toxins, significantly reduces the toxin content in plants, and simultaneously improves the resistance to Fusarium graminearum. Therefore, it can be widely used to improve the resistance of different pathogenic bacteria and their toxins, and has wide applicability.

Description

Method for improving plant resistance to Fusarium graminearum by using AtALA1 gene

technical field

The invention belongs to the field of plant genetic engineering. Specifically, it relates to a method for improving plant resistance to Fusarium graminearum by using genetic engineering technology to reduce the content of Fusarium graminearum toxin.

Background technique

Rice and corn are the world's three major food crops and the main sources of food and feed in the world. The safety of their products is directly related to the survival of human beings. However, these cereal crops are often infected by a variety of pathogenic bacteria during their growth and development, which not only seriously reduces their yield and quality, but the grains contaminated by pathogenic bacteria often contain a variety of mycotoxins, which seriously endanger human and animal health (Zhen, 2010). According to the United Nations Food and Agriculture Organization (FAO), as many as 25% of the world's food crops are contaminated with mycotoxins, of which deoxynivalenol (DON, also known as vomitoxin) is the most common and important Fusarium Bacterial toxins. DON was first isolated by Japanese scientists Morooka and Yamamoto in 1972 from barley infected with head blight (Morooka & Yamamoto, 1972). According to the characteristics that it can cause sows to refuse to eat and vomit, it is named vomitoxin (Vomitoxin, VT), which is a type B trichothecene compound, mainly composed of Fusarium graminearum (F. graminearum) and yellow Fusarium (F. culmorum) production, including deoxynivalenol (DON) and its acetylated forms: 15-acetyl-deoxynivalenol (15-ADON) and 3-acetyl-deoxynivalene Alcohol (3-ADON). In 2018, the results of BIOMIN's assessment of the global mold contamination risk showed that DON was detected in 73% of the samples submitted for inspection, and the detection rate ranked first among all toxins. However, in the samples submitted for inspection in my country, the positive rate of DON in corn, flour, wheat or bran and rice bran exceeded 98%, and the positive rate of DON in pig feed and poultry feed was as high as 97%.

DON not only harms crops, but DON-contaminated food or feed enters human or animal bodies, and seriously endangers human and animal health (De Walle et al., 2010; Audenaert et al., 2014; Vidalet al., 2016). DON has a strong cytostatic effect and can inhibit the immune system of humans and animals, mainly inhibiting the synthesis of proteins, DNA and RNA, inhibiting the function of mitochondria, affecting cell division and cell membrane function (Goyarts et al., 2007; Seeling et al., 2006; Valenta et al., 2005). DON is toxic to humans and animals, and can damage the immune system of humans and animals, causing symptoms such as dizziness, vomiting, diarrhea, nausea, and abortion (De Walle et al., 2010; Audenaert et al., 2013; Pestka et al., 2004). DON is easily absorbed by the human body. After eating contaminated food by mistake, it will cause unsteady standing and unresponsiveness. Pigs will have symptoms such as food refusal, vomiting and decreased feed intake after eating DON-contaminated grains. In severe cases, abortion will occur. disease (Ebarbet al., 2018; Sayyari et al., 2018), so reducing DON in plants is of great significance.

At present, there are few reports on reducing the content of DON in plants. It is only found that UDP-glucosyltransferase can convert DON into non-toxic D3G, so that transgenic wheat and Arabidopsis show resistance to DON, thereby improving plant resistance to DON. Tolerance (Pasquet et al., 2016; Schweiger et al., 2013; Gardiner et al., 2010; Mandalà et al., 2019). More importantly, the regulatory genes in the disease resistance-related pathways are transformed into crops, which improves the tolerance to DON. The expression of the TaABCC gene can improve the tolerance of wheat to Fusarium graminearum, and also reduce the concentration of DON in wheat ears. Accumulation (Walter et al., 2015); TaFROG in wheat helps to increase host resistance to DON and Fusarium graminearum (Alexandre et al., 2015); Song et al found antibody CWP2 in the cell wall protein of Fusarium graminearum The recognized antigen GLX is located on the cell membrane of Gibberella and specifically binds to CWP2, thereby making plants resistant to disease (Song et al., 2016); expressing TaCYP72A helps to improve the host's tolerance to DON (Gunupuru et al., 2018 ); expression of TAD1 increased the resistance of wheat to Fusarium graminearum, suggesting that TAD1 may be a candidate gene for increasing the resistance of cereal crops to FHB (Sasakiet et al., 2016). The above methods basically improve the plant defense function, but cannot reduce the accumulation of DON in the grain.

Looking at the above methods, the physical and chemical methods have weak effects and a small range of action, cumbersome sample processing, and greater environmental hazards. Most of the biological methods start from the synthesis of toxins and other aspects to reduce the synthesis of toxins, and have not fundamentally improved the ability of plants to degrade toxins. With the continuous development of plant genetic engineering technology and the deepening understanding of plant-pathogen interaction, it has become an effective way to introduce exogenous resistance genes into plants to improve disease resistance. Practice has proved that the use of disease-resistant varieties is the only cost-effective way to reduce DON content and improve resistance to Fusarium graminearum.

Based on the in-depth study of the pathogenic mechanism of pathogenic bacteria, as well as the serious harm of toxins secreted by pathogenic bacteria and their important role in pathogenicity, it is important to use exogenous disease resistance genes to inhibit the infection of pathogenic bacteria on plants and improve the detoxification function of plants to pathogenic toxins. One of the effective ways to improve plant disease resistance. However, at present, most researchers mainly use the former method to achieve the purpose of improving plant resistance, and there is no report on the successful use of exogenous gene expression products to detoxify the toxins produced by pathogenic bacteria to improve resistance to pathogenic bacteria.

This study showed that the expression of AtALA1 alleviated the inhibitory effect of DON on maize root growth and improved the tolerance to DON. The detection of content found that the accumulation of DON in transgenic maize decreased by 105 ppb compared with the wild type, indicating that the expression of AtALA1 is a reduction. DON content in grains, a proven strategy to increase resistance to Fusarium graminearum, is of great importance.

Contents of the invention

An object of the present invention is to provide a method for improving the resistance of plants to Fusarium graminearum; Another object of the present invention is a method for preparing a transgenic plant resistant to Fusarium graminearum; The present invention also provides AtALA1 gene in Use for reducing Fusarium graminearum toxins in plants and increasing plant resistance to Fusarium graminearum.

AtALA1, a member of the P4-ATPases family, is a membrane protein containing 10 transmembrane domains. This protein family plays an important role in maintaining the asymmetric distribution of phospholipids in the cell membrane, initiating vesicle formation, and mediating intracellular protein transport. AtALA1 gene encodes AtALA1 protein, the nucleotide sequence of which is shown in SEQ ID NO.14. Using the AtALA1 gene in the model plant Arabidopsis thaliana as a model, and verifying it with the cereal plant maize which has the same detoxification mechanism of P4-ATPase protein, the research of the present invention has been completed.

According to one aspect of the present invention, a method for improving the resistance of plants to Fusarium graminearum, wherein the AtALA1 gene is integrated into the target plant, and the AtALA1 gene is expressed in the target plant to reduce Fusarium graminearum toxin content, and improve plant resistance to Fusarium graminearum, the nucleotide sequence of the AtALA1 gene is shown in SEQ ID NO.14.

The method of the present invention comprises the following steps:

1) constructing a recombinant plant expression vector containing the AtALA1 gene;

2) introducing the recombinant plant expression vector into the target plant, so that the AtALA1 gene is constitutively expressed in the target plant; and

3) Obtaining transgenic plants resistant to Fusarium graminearum.

In the method of the present invention, preferred target plants are Arabidopsis or cereals, such as maize.

In the method of the present invention, the structure of the recombinant plant expression vector used is shown in Figure 4A (Arabidopsis) or Figure 4B (maize).

According to another aspect of the present invention, a method for preparing a transgenic plant resistant to Fusarium graminearum comprises the following steps:

i) Obtaining the AtALA1 gene and operatively inserting it into a plant expression vector to construct a plant expression vector;

ii) transforming the host with the plant expression vector obtained in step i) to obtain a transformant;

iii) transforming plants with the transformants obtained in step ii) to obtain transgenic plants;

The nucleotide sequence of the AtALA1 gene is shown in SEQ ID NO.14.

According to another aspect of the present invention, there is provided the use of the AtALA1 gene in reducing Fusarium graminearum toxin in plants and improving the resistance of plants to Fusarium graminearum, wherein by integrating the AtALA1 gene into the target plant, a transgenic plant is obtained, and the The AtALA1 gene is expressed in plants to reduce the content of Fusarium graminearum toxin in the plant and improve the resistance of the target plant to Fusarium graminearum. The nucleotide sequence of the AtALA1 gene is shown in SEQ ID NO.14 Show.

SEQ ID NO.14

ATGGATCCCAGGAAATCAATTGATAAGCCGCCTCATCACGATCCAATTCTGGGTGTATCTTCAAGATGGAGCGTTTCTTCTAAAGACAACAAAGAAGTTACTTTCGGTGATTTGGGATCTAAGCGTATCCGTCATGGTTCAGCTGGAGCTGATTCTGAGATGCTAAGCATGTCTCAGAAAGAGATCAAAGACGAAGATGCTCGTTTGATTTATATTAACGATCCTGACAGAACTAACGAACGGTTTGAGTTCACTGGGAATTCTATCAAGACTGCTAAATACTCTGTCTTCACCTTCTTGCCTAGGAACTTGTTTGAACAGTTCCATAGAGTTGCTTACATTTACTTCCTTGTTATCGCTGTTCTCAATCAGCTTCCTCAGCTTGCAGTTTTTGGCAGAGGTGCATCCATCATGCCCCTTGCCTTTGTTCTCTTGGTCTCTGCTATCAAAGATGCTTACGAGGATTTCCGGAGACATAGGTCAGATAGAGTTGAGAACAATAGGTTGGCTTTAGTCTTTGAGGATCATCAGTTTCGAGAGAAGAAGTGGAAGCATATCCGGGTTGGGGAAGTCATTAAAGTCCAATCCAATCAGACTCTTCCCTGTGACATGGTGCTCTTGGCTACTAGTGATCCTACTGGGGTTGTCTACGTGCAGACGACTAATTTGGATGGTGAGTCGAATTTGAAGACCAGGTATGCCAAGCAGGAAACTCTTCTGAAAGCTGCTGATATGGAGTCGTTTAATGGATTTATCAAGTGTGAGAAACCTAACAGGAACATTTATGGGTTTCAAGCCAACATGGAGATTGATGGTAGAAGGCTCTCCCTTGGACCTTCTAATATTATTCTAAGAGGGTGTGAGCTTAAGAACACTGCTTGGGCTTTAGGGGTTGTTGTGTATGCTGGTGGTGAGACGAAAGCTATGCTCAACAACTCTGGAGCACCATCAAAGAGGAGTAGGCTAGAGACTCGAATGAATTTGGAGATCATTCTACTCT CTTTGTTTCTGATCGTCTTGTGTACAATCGCAGCCGCGACCGCTGCTGTGTGGTTGAGAACGCACAGGGATGACTTGGACACTATTCTCTTTTATAGAAGAAAGGACTACTCTGAGAGGCCAGGAGGGAAGAACTATAAATACTATGGTTGGGGGTGGGAGATATTCTTCACCTTCTTTATGGCAGTCATTGTGTACCAGATCATGATACCCATTTCTCTCTACATATCGATGGAGCTCGTCCGTATTGGTCAAGCATACTTCATGACCAATGATGATCAGATGTATGACGAGTCTTCAGATTCAAGTTTTCAATGCAGGGCTTTAAATATAAATGAAGATTTAGGGCAGATTAAGTATTTATTCTCTGATAAGACGGGTACACTCACGGACAACAAGATGGAGTTTCAATGTGCCTGCATCGAAGGCGTAGATTACTCTGACAGGGAACCTGCTGATAGCGAGCATCCTGGATACTCCATTGAAGTTGATGGAATTATTTTGAAGCCAAAGATGAGGGTGAGAGTTGATCCTGTGCTTCTTCAGTTAACGAAAACTGGCAAGGCAACAGAAGAAGCAAAACGTGCAAATGAGTTTTTCCTCTCACTGGCAGCTTGCAATACAATTGTGCCAATTGTTAGCAATACATCTGATCCCAATGTGAAACTGGTAGATTATCAAGGGGAGTCCCCTGATGAACAAGCATTGGTCTATGCAGCAGCTGCATATGGTTTCTTGCTCATAGAGAGAACCTCTGGTCATATAGTTATTAATGTGCGAGGAGAAACGCAAAGATTTAATGTTTTGGGATTGCATGAGTTCGATAGTGACCGAAAAAGAATGTCAGTGATACTGGGATGCCCCGACATGTCGGTGAAACTCTTTGTAAAAGGTGCAGACTCATCCATGTTTGGTGTCATGGATGAATCCTACGGTGGCGTCATACATGAGACCAAGATACAACTTCATGCTTACTCATCTGATGGTTTGAGAACACTTGT TGTTGGGATGAGAGAGCTGAACGATTCAGAGTTTGAGCAATGGCATTCTTCATTTGAGGCGGCAAGCACCGCCTTGATTGGTCGGGCTGGATTGCTAAGAAAAGTTGCTGGAAACATTGAGACTAACCTTAGGATAGTAGGAGCCACCGCAATTGAAGACAAATTGCAGCGTGGTGTCCCTGAAGCAATAGAATCTTTGAGGATTGCAGGGATAAAAGTCTGGGTCTTGACTGGTGACAAGCAAGAAACTGCCATATCCATTGGCTTCTCATCGAGGCTTCTGACAAGAAACATGAGGCAAATTGTAATAAATAGCAACTCGTTGGATTCATGTCGGAGGAGCTTAGAAGAAGCAAATGCCAGTATTGCAAGTAATGACGAAAGTGATAATGTGGCCTTGATTATTGACGGTACCAGCCTCATATATGTACTCGACAATGATCTTGAAGATGTGCTGTTCCAGGTGGCATGTAAGTGCTCTGCGATACTCTGCTGCCGGGTTGCTCCTTTCCAGAAAGCTGGAATCGTTGCACTTGTAAAGAACCGGACTTCTGACATGACTCTTGCCATTGGTGATGGTGCCAATGATGTCTCCATGATTCAAATGGCTGATGTTGGGGTAGGGATAAGCGGACAAGAAGGTCGCCAAGCTGTGATGGCATCTGATTTCGCAATGGGACAGTTCAGATTTTTAGTTCCGTTATTGCTCGTCCATGGACACTGGAATTACCAAAGGATGGGTTACATGATACTATATAATTTCTATAGAAATGCAGTTTTTGTTCTAATTTTATTTTGGTACGTTTTGTTTACTTGCTACACCTTGACAACTGCCATCACAGAATGGAGCAGTGTTTTGTACTCAGTCATATACACAGCAATCCCTACAATAATTATCGGTATTCTTGACAAAGACCTTGGAAGGCAGACTCTTCTTGATCATCCTCAGCTCTACGGTGTTGGCCAGAGGGCAGAGGGATATTCCACTACGCTCTTCTGG TATACAATGATTGACACAATCTGGCAAAGTGCAGCCATCTTCTTCATTCCTATGTTTGCTTATTGGGGCAGTACAATTGACACGTCGAGCCTAGGAGACCTATGGACGATAGCTGCAGTTGTGGTGGTTAATCTTCACTTGGCCATGGATGTGATCAGATGGAACTGGATCACACACGCCGCCATATGGGGATCCATTGTTGCAGCTTGTATATGTGTCATTGTGATTGATGTTATACCCACACTCCCTGGTTACTGGGCAATTTTCCAAGTGGGCAAGACATGGATGTTCTGGTTCTGCTTGCTAGCAATAGTTGTGACATCATTGCTTCCTAGATTCGCCATCAAGTTTCTAGTGGAGTATTACAGACCTTCCGATGTTCGGATAGCTAGGGAGGCTGAAAAGCTTGGAACTTTCAGAGAATCCCAACCCGTGGGAGTTGAAATGAACCTGATTCAGGATCCTCCACGGAGATGA

In a specific embodiment of the invention, the method of the present invention comprises the steps of:

1) Obtaining the Arabidopsis AtALA1 gene: introducing SmaI and SalI restriction sites to design primers, and then performing PCR amplification using the Arabidopsis cDNA as a template, and the amplified product is the AtALA1 gene sequence with restriction restriction sites added;

2) Construction of plant expression vectors constitutively expressing the AtALA1 gene: the amplified AtALA1 gene sequences were inserted into the dicotyledonous plant expression vector pLGN-35S-Nos and the monocotyledonous plant expression vector pCambia2300-Ubi1-Nos, respectively, to construct two new Plant expression vectors, named pLGN-35S-AtALA1-Nos and pCambia2300-Ubi1-AtALA1-Nos respectively;

3) Genetic transformation of plants: the pLGN-35S-AtALA1-Nos and pCambia2300-Ubi1-AtALA1-Nos plant expression vectors obtained in the above step 2) were integrated into the plant genome by using the Agrobacterium flower dipping method and the gene gun transformation method to realize Constitutive expression of AtALA1 gene in transgenic plants reduces DON content and improves resistance of transgenic plants to Fusarium graminearum;

4) Acquisition of AtALA1 transgenic plants with low DON content and resistance to Fusarium graminearum: the transgenic plants obtained in the above step 3) were further propagated, molecularly identified and identified for disease resistance to obtain AtALA1 transgenic plants with low DON content and resistance to Fusarium graminearum plants.

Further, the steps of constructing the constitutively expressed pLGN-35S-AtALA1-Nos plant expression vector include: after obtaining the cDNA of Arabidopsis thaliana AtALA1, design the upstream primer: 5'-TCC CCCGGG ATGGATCCCAGGAAATCAATTG(SmaI)-3', the downstream primer 5''-ACGC GTCGAC TCATCTCCGTGGAGGATCCTGA(SalI)-3' was amplified by PCR, and the amplified product and the pLGN-35S-Nos vector were subjected to SmaI and SalI double enzyme digestion, respectively, and the large fragments after enzyme digestion were recovered, and then recovered by ligase The fragments were connected to construct a new plant expression vector named pLGN-35S-AtALA1-Nos to realize the constitutive expression of AtALA1 gene in transgenic plants. The steps of constitutively expressed pCambia2300-Ubi1-AtALA1-Nos plant expression vector construction include: the pCambia2300-GUS vector is cut with Sal I/Kpn I, and the fragment of AtALA1 (Ubi1-AtALA1) is fused with the monocot constitutive promoter Ubi1 Recombinant plasmids were obtained by homologous recombination.

Without limiting any mechanism, the method provided by the present invention to reduce DON content and improve plant resistance to Fusarium graminearum is to transfer the AtALA1 gene from the plant into the target plant, and realize the constitutive expression of the gene in the transgenic plant , using P4 ATPases to transport and degrade DON and other Fusarium graminearum toxins, improve the detoxification ability of transgenic plants to Fusarium graminearum toxins, and then improve the resistance of transgenic plants to Fusarium graminearum.

According to the AtALA1 mutant that is sensitive to vomitoxin (DON) and loses the property of transporting it to the vacuole, combined with the characteristics of the pathogenic mechanism of Fusarium graminearum, the present invention creatively proposes to use the AtALA1 gene to constitutively express in transgenic plants to reduce the risk of transgenic Strategies to improve the DON content in plants, improve their detoxification ability to DON, and then improve the resistance of transgenic plants to Fusarium graminearum.

The present invention mainly uses the transfer and degradation mechanism of the transgenic product to Fusarium graminearum toxin, reduces the toxin content in the transgenic plant, improves its disease resistance to Fusarium graminearum, and further utilizes the transgenic product in the same way to improve the plant's resistance to The ability to detoxify other mycotoxins is also within the scope of the present invention. At the same time, the plant expression vectors and transgenic plants involved in the present invention are also within the protection scope of the present invention.

The AtALA1 transgenic Arabidopsis thaliana obtained in the present invention, T3 homozygous transgenic line OE-25 was treated with 50 μg/mL DON for 17 days, and the DON content was 5.49 ng/g, which was 3.42 ng/g lower than that of the wild type (WT) control. Treated with DON (5 μg/mL) and FM4-64 (8 μM) for 10 h, 5-FAM-DON in the overexpression lines OE-25 and OE-15 all entered the vacuoles labeled with FM4-64, and the fluorescence intensities were 6.56, 6.52 , which were 3.53 and 3.49 higher than WT respectively. The results showed that excess AtALA1 promoted the transport and degradation of DON to the vacuole, and improved its tolerance. Using the transgenic corn T2 generation plants obtained by the present invention, the corn kernels (radicle 0.2 cm) were treated with 30 μM DON for 2 days, and the DON was washed away to continue culturing for 3 days. The radicle lengths of 13-3 and 15-2 transformants reached 1.7 cm and 1.4 cm respectively. cm, 1.4 and 1.1 cm higher than WT, respectively. The results of DON content detection showed that the 13-3 and 15-2 transformants were 56% and 52% lower than WT, respectively. In addition, 7 days after being inoculated with Fusarium graminearum, the lesion areas of 13-3 and 15-2 transformants were 6 mm ² and 11 mm ² , which were 60% and 27% less than those of WT; the number of spores was 1.3×10 ⁵ /mL , 2.4×10 ⁵ cells/mL, which were 87% and 76% less than WT, respectively, indicating that the expression of AtALA1 improved the resistance of maize to Fusarium graminearum. The research results show that the transgenic Arabidopsis and corn obtained by the method of the invention can significantly reduce the DON content and improve the resistance to Fusarium graminearum. It shows that the method provided by the invention for reducing the DON content in plants and improving the resistance of plants to Fusarium graminearum has remarkable effects. This method is not only applicable to Arabidopsis and corn, all plants that can be infected by Fusarium graminearum can use this method to reduce their DON content and improve their resistance to Fusarium graminearum.

The method provided by the invention mainly utilizes the transporting and degrading mechanism of the pathogen toxin by plants, reduces the content of the toxin of the pathogen to achieve the purpose of detoxification, and further improves the resistance of the plant, so it does not have the characteristics of race resistance of the pathogen. Therefore, this method does not have the limitation of race-specific resistance, and can be widely used to improve the resistance of different physiological races of pathogenic bacteria.

Description of drawings

Figure 1 shows the acquisition of Arabidopsis ala1-12 mutant

A is the mutation site of the ala1-12 mutant, which is located in the exon region of AtALA1 (the triangle position in the figure); exon is an exon, and intron is an intron; LP, RP, LBb1.3: Identification Primers for homozygous mutants; B is PCR identification of ala1 homozygous mutants, a: LP+RP amplification product, about 1100bp; b: LBb1.3+RP amplification product, about 700bp; DNA molecular weight standard is Marker 2000; 4 and 12: obtained homozygous mutants; Col-0: wild-type Arabidopsis; C is real-time fluorescent quantitative PCR detection of AtALA1 expression in wild-type (Col-0) and mutants.

Figure 2 is the screening of Arabidopsis mutants sensitive to DON

A is the sensitivity detection results of wild type (Wild Type: Col-0) and different Arabidopsis mutants to 100 μg/mL DON; ala1, ala10, ala6, ala3 and ala7: different P4-ATPases mutants of Arabidopsis; B is the ratio of Arabidopsis albino leaves under DON treatment.

Figure 3 shows the translocation of DON by different Arabidopsis P4-ATPases mutants

Wild type (Wild Type: Col-0); ala1, ala10, ala6, ala3 and ala7: different P4-ATPases mutants in Arabidopsis; FM4-64: a membrane dye, used to stain cell membrane and tonoplast membrane; 5- FAM: DON is labeled with the fluorescent group 5-FAM (5-carboxyfluorescein), which emits green fluorescence under 488nm excitation light.

Figure 4 is a structural diagram of a plant expression vector;

A: pLGN-35S-AtALA1-Nos plant expression vector diagram, which contains a 2×35S-promoted GUS::NPTⅡ fusion gene expression cassette and a 35S-promoted AtALA1 gene expression cassette;

B: pCambia2300-Ubi1-AtALA1-Nos plant expression vector diagram, which contains an expression cassette of NPTII gene initiated by 2×35S, and an expression cassette of AtALA1 gene initiated by Ubi1 constitutively expressed in monocotyledons.

Figure 5 shows the results of pLGN-35S-AtALA1-Nos vector plasmid digestion verification

M: DNA molecular weight standard Marker15; ALA1: ALA1 target fragment obtained after digestion of pLGN-35S-AtALA1-Nos vector plasmid.

Figure 6 shows the tolerance of Arabidopsis thaliana complementary to AtALA1 in ala1 to DON

Col-0: wild-type Arabidopsis; ala1: AtALA1 homozygous mutant; ala1/ALA1: complementary AtALA1 Arabidopsis in ala1; photographed after 50 μg/mL DON treatment for 7 days, Bar=1cm.

Figure 7 is the expression level of AtALA1 gene in transgenic Arabidopsis

Col-0: wild type Arabidopsis; 4, 7, 9, 10, 11, 13, 15, 24, 25, 27, 28 and 29: independent transformants of AtALA1 transgenic Arabidopsis.

Figure 8 shows the tolerance test of ala1 and excessive AtALA1 Arabidopsis thaliana to DON. A, B: DON (50 μg/mL) treatment results for 5 days, Bar=5 mm; C: DON (50 μg/mL) treatment results for 14 days, Bar=1cm; ala1-12: Arabidopsis AtALA1 homozygous mutant; OE 15-4, OE 25-1: T ₃ generation homozygous plants with excessive expression of AtALA1 Arabidopsis.

Figure 9 shows the detection results of DON content in wild type, mutant ala1 and overexpressed AtALA1 Arabidopsis

The following Arabidopsis seedlings were treated with DON (50 μg/mL) for 7 days, and the DON content was detected by ELISA; WT: wild-type Arabidopsis; ala1: AtALA1 homozygous mutant; 11, 15, 4 and 24 were excess AtALA1 Different transformants of A. thaliana.

Figure 10 is a flow chart for the construction of pCambia2300-Ubi1-AtALA1-Nos plant expression vector

The map of pCambia2300-Ubi1-AtALA1-Nos plant expression vector, which contains a 2×35S-promoted NPTII gene expression cassette and a monocotyledon constitutively expressing Ubi1-promoted AtALA1 gene expression cassette.

Figure 11 is the electrophoresis diagram of pCambia2300-Ubi1-AtALA1-Nos vector restriction enzyme digestion

M: DNA molecular weight standard DM3100; Ubi1-AtALA1: the target fragment obtained after the pCambia2300-Ubi1-AtALA1-Nos vector plasmid was digested with SalI/KpnI.

Figure 12 is the PCR identification of Ubi::AtALA1 transgenic maize

M: DNA marker 2000; 1: wild-type maize DNA as template, as negative control; 2: successfully constructed Ubi::AtALA1 plasmid as template, as positive control; 3-18: different transgenic maize DNA as template Amplify results.

Figure 13 shows the expression level of AtALA1 in each transformant of Ubi::AtALA1 transgenic maize

The internal reference gene for expression analysis is EF1a, and the data are the average value of three repetitions; 2～N-1: Ubi::AtALA1 transgenic maize different transformants.

Figure 14 is the phenotype of AtALA1 transgenic maize under DON treatment

A is the phenotype of maize kernels treated with DON; B is the statistical result of maize root length under DON treatment; C is the content of DON in maize kernels; DON treatment concentration is 50 μM; WT: wild-type maize kernels; Transgenic plant control; 13-3, 15-2: corn kernels expressing high AtALA1 expression; scale bar = 1 cm.

Figure 15 shows the tolerance results of AtALA1 transgenic maize to T-2 toxin

A is the effect of T-2 toxin (30μM) on maize root length; B is the statistical result of maize root length under T-2 toxin; WT: wild-type maize kernels; 13-3: No. 13-3 _T1 generation transgenic maize kernels ; 15-2: No. _15-2 T1 generation transgenic corn kernels; scale = 0.5 cm.

Figure 16 shows the tolerance analysis of AtALA1 transgenic maize to Fusarium graminearum

A: Phenotype of Fusarium graminearum inoculation; B: Lesion area statistics; C: Spore number statistics; WT: wild-type corn kernels; 13-3: No. 13-3 _T1 transgenic corn kernels; 15-2: No. 15-2 _T1 generation transgenic corn kernels; scale bar = 0.5 cm.

Figure 17 shows the resistance of AtALA1 transgenic maize to Fusarium graminearum

A is the inoculation of Fusarium graminearum on young maize grains by soaking method; B is the statistical result of spore count 3 days after inoculation. WT: wild-type corn kernels; 13-3: No. 13-3 T1 AtALA1 transgenic corn kernels; 15-2: No. 15-2 T1 AtALA1 transgenic corn kernels; scale = 0.3 cm.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings, but the following description does not limit the present invention.

The pharmaceutical reagents in the implementation examples of the present invention are domestic conventional chemical reagents that are not specified, and all materials and methods that are not specified are referred to "Molecular Cloning Experiment Guide" (Sambrook and Russell, 2001).

1. Proposal of the core strategy of the invention technology

1.1 Obtaining homozygous mutants of Arabidopsis thaliana with deletion of AtALA1 gene

1.1.1 Arabidopsis mutant acquisition and planting

Arabidopsis mutants were purchased from Arabidopsis Biological Resource Center (Arabidopsis Biological ResourceCentre, ABRC) T-DNA insertion mutants, numbers are: ala1 (SALK_056947), ala3 (SALK_082157), ala6 (SALK_150173), ala7 (SALK_125598 ), ala10 (SALK_024877).

Arabidopsis seed culture method: divide the seeds into 1.5mL centrifuge tubes, oscillate repeatedly with 75% alcohol and 0.1% Tween 80 for 25 minutes for surface disinfection, rinse the seeds with sterilized distilled water for 3-5 times, and then wash the seeds with 0.2% agarose Resuspend the seeds in sterile medium, and spread the seeds on 1/2 MS solid medium (2.2g/L MS salt, 15g/L sucrose, 2g/L Gelrite, pH 6.0) with a pipette. After cold treatment at 4°C for 2 days, culture according to the following conditions:

16h of light/8h of darkness, light intensity of 150 μmolm ^-2 S ^-1 , relative humidity of 70%, and temperature of 22°C. When the seedlings grew for 10 days, they were moved to the soil (the ratio of nutrient soil to vermiculite was about 1:1), and the plants were cultivated by adding 1.5L of water and 1L of B5 nutrient solution. Greenhouse lighting conditions: 16h light/8h dark, temperature 22°C.

1.1.2 Obtaining homozygous mutants of Arabidopsis thaliana

About 0.1 g of Arabidopsis leaves were taken, and DNA was extracted using a new plant genomic DNA rapid extraction kit (Aidlab product, catalog number: DN15), and the operation was carried out in strict accordance with the instructions. Mutant screening PCR primers were designed according to Signal website (http://signal.salk.edu/tdnaprimers.2.html).

The total PCR reaction system was 10 μL: 1 μL template, 0.5 μL each primer (20 mmol·L ⁻¹ ), 5 μL 2×Taq PCRMaster Mix, 3 μL ddH ₂ O. The reaction program was: denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min, 30 cycles; and extension at 72°C for 10 min.

The amplification primers are:

LP-ala1:5'-GCCATTGGTGATGGTAATGAC-3' (SEQ ID NO.1)

RP-ala1:5'-ACCAGAACATCCATGTCTTGC-3' (SEQ ID NO.2)

LBb1.3:5'-ATTTTGCCGATTTCGGAAC-3' (SEQ ID NO.3)

1.1.3 Obtaining DON-sensitive Arabidopsis mutants

Plant wild-type (Col-0) and homozygous mutant Arabidopsis thaliana in 1/2 MS solid medium, and transfer to 1/2 MS solid medium containing 75 μg/mL DON after the roots grow to about 2 cm, after 7 days Observing, photographing and counting the proportion of albino leaves showed (Fig. 2), the albino leaves of ala1 were more serious, and the proportion of white flower leaves was nearly 90%, while the other mutants had no significant difference compared with the wild type. The wild type and mutant Arabidopsis were treated with 5-FAM-labeled DON and the membrane dye FM4-64 at the same time. The results showed (Figure 3) that there was no 5-FAM-labeled DON in the vacuole with only ala1 after 20 h of treatment, indicating that AtALA1 may Play an important role in improving the tolerance of Arabidopsis to DON.

1.2 The proposal of the core strategy of the invention technology

The research results of Example 1.1.3 show that the AtALA1 gene T-DNA mutant ala1 is more sensitive to DON and loses the function of transporting DON into the vacuole. AtALA1, a member of the P4-ATPases family, is a membrane protein containing 10 transmembrane domains. This protein family plays an important role in maintaining the asymmetric distribution of phospholipids in the cell membrane, initiating vesicle formation, and mediating intracellular vesicle transport. Therefore, we speculate that DON may be transported to the vacuole for storage or degradation through AtALA1-mediated vesicular transport, thus playing an important role in improving the tolerance of Arabidopsis to DON. Fusarium graminearum secretes many kinds of toxins, and many toxins are similar in structure to DON. Combined with the serious harm of Fusarium graminearum toxins to humans and animals, the present invention creatively proposes the core strategy of the invention, "using plant-derived P4-ATPases to reduce graminearum toxins such as DON, and at the same time increase the resistance of transgenic plants to Fusarium graminearum".

2. Acquisition of AtALA1 gene

2.1 Arabidopsis RNA extraction and cDNA synthesis

RT reagent Kit with gDNA Eraser试剂盒合成cDNA，操作严格按说明书进行。Take an appropriate amount of leaves of wild-type Arabidopsis thaliana (Col-0), use the liquid nitrogen grinding method, and follow the instructions of the EASYspinRNA rapid extraction kit from Aidlab to extract the total RNA of Arabidopsis thaliana, and the quality of the obtained RNA is detected by 1% agarose electrophoresis. . Reuse of TaKaRA's PrimeSc-

RT reagent Kit with gDNA Eraser kit was used to synthesize cDNA, and the operation was carried out strictly according to the instructions.

2.2 Cloning of AtALA1 gene

Using the Arabidopsis cDNA obtained in Example 2.1 as a template, the upstream primer of the AtALA1 gene upstream primer: 5'-TCC CCCGGG ATGGATCCCAGGAAATCAATTG(SmaI)-3'(SEQ ID NO.4), the downstream primer 5'-ACGC GTCGAC TCATCTCCGTGGAGGATCCTGA( SalI)-3' (SEQ ID NO.5) was used as a primer, and KOD-Plus-Neo (TOYOBO) enzyme was used for PCR amplification, and the amplified product was recovered to obtain an AtALA1 gene fragment with a SmaI/SalI restriction site.

PCR amplification system: 5 μL of 10×PCR Buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , 2 μL of 10 μM upstream and downstream primers, 4 μL of template (50 μg/μL), 1 μL of KOD-Plus-Neo (1U/μL), add water to 50 μL;

The amplification program was 94°C for 2min; 98°C for 10s, 68°C for 4min, and cycled 35 times.

3. Construction of dicot plant expression vector pLGN-35S-AtALA1-Nos and Agrobacterium transformation

3.1 Acquisition of pLGN-35S-Nos plant expression vector

pLGN-35S-Nos is a binary plant expression vector transformed from pCambia2300 in our laboratory. Its T-DNA segment (the region between RB and LB) was replaced with a fusion gene expression cassette of the reporter gene GUS and the marker gene NPTII controlled by the constitutive promoter CaMV35S-P, and a LoxpFRT was added at both ends of the expression cassette A recombinase recognition site, and another expression cassette controlled by CaMV35S-P.

3.2 Construction of pLGN-35S-AtALA1-Nos plant expression vector

Carry out double enzyme digestion with SmaI/SalI on the fragment obtained in Example 2.2 above and the vector plasmid in Example 3.1 of pLGN-35S-Nos, respectively, and recover the digested fragment, and then use T4-DNA ligase to ligate the recovered AtALA1 gene fragment and pLGN-35S-Nos vector fragment. The ligated product was heat-shock transformed Escherichia coli DH5α, and the positive clones were screened and verified by enzyme digestion (as shown in Figure 5). The results showed that the AtALA1 gene fragment had been successfully ligated into the pLGN-35S-Nos vector. The sequencing result is consistent with the published AtALA1 gene sequence, and the specific sequence is shown in SEQ ID NO.14. The correctly sequenced plant expression vector pLGN-35S-AtALA1-Nos monoclonal was stored at -80°C for future use, and the vector had the structure shown in Figure 4 .

3.3 Acquisition of plant expression vector pLGN-35S-AtALA1-Nos recombinant Agrobacterium

Using the electroporation method, transfer the pLGN-35S-AtALA1-Nos plant expression vector obtained in step 3.2 into Agrobacterium GV3101 competent cells, and use kanamycin and rifampicin as selection marker genes for resistance screening to obtain positive clones , then extract the Agrobacterium plasmid and carry out double enzyme digestion with SmaI/SalI, and carry out electrophoresis through 1% agarose gel, and obtain the target fragment of 3477bp (the enzyme digestion result is the same as that in Figure 5), indicating that the enzyme containing pLGN has been obtained. -35S-AtALA1-Nos plant expression vector for recombinant Agrobacterium.

4. Genetic transformation of Arabidopsis

4.1 Planting

Arabidopsis was planted in 1/2MS medium, moved to culture soil when it grew 2 true leaves, and grown in a light incubator. The culture conditions were: temperature 22°C/20°C, 16h light/8h dark, 70% relative humidity, light intensity 150 μmol/m ² /s. When the primary inflorescence is about 5-15 cm long and the secondary inflorescence has just formed a flower bud shape, remove the primary inflorescence, which is beneficial to the growth and development of the secondary inflorescence and facilitates infiltration and transformation. The osmotic transformation should be completed within 3-5 days after removing the primary inflorescences, and the plants should be fully watered 1 day before the osmotic transformation, so that the stomata of the plants can fully open during the transformation.

Infect Col-0 to obtain AtALA1 transgenic Arabidopsis; infect ala1 homozygous mutant to obtain complementary AtALA1 Arabidopsis (ala1/AtALA1).

4.2 Culture of bacteria liquid

Take the Agrobacterium strain containing the pLGN-35S-AtALA1-Nos carrier stored at -80°C, and put it on the YEB solid medium containing 50mg/L Rif and 50mg/L Km (5g/L sucrose, 1g/L yeast extract, 10g /L tryptone, 0.5g/LMgSO ₄ 7H ₂ O, 15g/L agar powder, pH 7.0) streak culture to obtain a single colony, pick a single colony, inoculate into additional 50mg/LKm and 125mg/L Sm (sulfuric acid Streptomycin) YEB medium (5g/L sucrose, 1g/L bacterial yeast extract, 10g/L tryptone, 0.5g/L MgSO ₄ 7H ₂ O, pH 7.0), 28°C, Shake culture at 200rmp overnight, until the OD600 value of the bacterial solution reaches 0.8, carry out a large amount of overnight culture (Rif: 25mg/L; Kana: 50mg/L) at the ratio of 1:100 the night before transformation. When it turns from dark red to orange, add acetosyringone (50 mg/L) and continue culturing for 2 hours until the OD ₆₀₀ is about 2.0. Centrifuge at 5000r/min at room temperature for 15min, discard the supernatant, and suspend the Agrobacterium pellet in an equal volume of osmotic medium (sucrose 50g/L; MS salt 2.37g/L; Silwet L-77 500μL/L), so that the OD600 is 0.8 or so, for Arabidopsis infection.

4.3 Transformation of Arabidopsis by soaking flowers

Infect the above-ground part of Arabidopsis with the Agrobacterium suspension carrying the target gene, cover it with a black film cover after infecting for 1 minute, and cultivate it in the dark for about 12 hours, uncover the black film and cultivate it normally, and water it after about 1 week of infection According to its growth condition, it can infect again 1-2 times, continue to cultivate until the plant matures, and harvest T ₀ generation seeds for screening positive transformants.

4.4 Molecular identification of transgenic Arabidopsis

4.4.1 Identification of AtALA1 transgenic Arabidopsis by GUS histochemical staining

Referring to the method of Jefferson et al. (1987), take the young leaves of transgenic plants with tweezers and put them in GUS staining solution (500mg/L X-Gluc, 0.1mol/LK ₃ Fe(CN) ₆ , 0.1mol/LK ₄ Fe(CN) ₆ , 1% Triton X-100 (v/v), 0.01mol/L Na ₂ EDTA, 0.1mol/L phosphate buffer (pH7.0), react at 37°C for 3h. After staining, decolorize with 75% ethanol, and replace the decolorizing solution every 4 hours until the color of the uncolored part fades completely. Regenerated materials with no blue leaves are non-transgenic plants, and those with blue color are transgenic plants.

4.4.2 PCR identification of AtALA1 transgenic Arabidopsis

For all the regenerated transgenic plants with positive GUS histochemical staining, about 0.1 g of the seedling leaves were taken, and the DNA was extracted using the new plant genomic DNA rapid extraction kit of Aidlab Company according to the operating procedures in the manual. After the DNA was obtained, 1% of the The quality of the DNA was detected by agarose gel electrophoresis, and then the extracted DNA was used as a template to amplify the vector-specific fragment to detect whether the regenerated plants integrated the AtALA1 gene.

PCR amplification primers are:

Upstream primer: 5'-AACAGGCCGATAATGCGCTA-3'; (SEQ ID NO.6)

Downstream primer: 5'-GTCTCGGGAAGTCCTTGCTT-3' (SEQ ID NO.7).

25μL amplification system includes: 10×LAPCR Buffer, 2.5μL; 25mmol/L MgCL ₂ , 2.5μL; 2.5mmol/L dNTP Mixture, 2μL; template DNA, 1μL (about 10ng); 5μmol/L upstream primer, 1μL; 5μmol /L downstream primer, 1 μL; LA Taq enzyme, 0.2 μL; ddH ₂ O, 14.8 μL.

PCR temperature cycle parameters: 94°C, 5min; 94°C, 30s, 57°C, 30s, 72°C, 30s, 30 cycles of amplification; 72°C, 10min. Finally, the amplified products were detected by 1% agarose gel electrophoresis.

5 Detection of AtALA1 transcript level in transgenic plants

5.1 RNA extraction and cDNA synthesis

RT reagent Kitwith gDNA Eraser试剂盒(TaKaRa产品)合成cDNA，再以cDNA为模板扩增目标基因，RNA中植物基因组DNA的去除以及cDNA的合成均严格按说明书进行。Plant RNA was extracted using a plant RNA rapid extraction kit (Aidlab product), all operations were strictly performed according to the instructions, and the quality of the obtained RNA was detected by 1% agarose electrophoresis. Reuse Prime

RT reagent Kit with gDNA Eraser kit (TaKaRa product) was used to synthesize cDNA, and then the cDNA was used as a template to amplify the target gene. The removal of plant genomic DNA in RNA and the synthesis of cDNA were carried out strictly according to the instructions.

5.2 AtALA1 gene transcription level detection

Real-time PCR method was used to detect the expression level of the transgene in the plants. To normalize the cDNA concentration, the Arabidopsis gene Atactin2 was used as an internal standard.

The 10 μL reaction system includes: 1 μL of cDNA, 0.5 μL of upstream and downstream primers, 5 μL of 2×iQ SYBR Green Supermix, and make up 10 μL with RNase-free double distilled water.

The amplification program was: 95°C pre-amplification for 3 minutes; 94°C for 10s, 56°C for 30s, 72°C for 30s, a total of 40 cycles of amplification. After the amplification was completed, the relative expression of the gene was analyzed using Gene Study software.

6. DON treatment and content detection of AtALA1 transgenic Arabidopsis

6.1 DON processing

Plant wild-type (Col-0), homozygous mutant ala1 and complementary AtALA1 Arabidopsis (ala1/AtALA1) on 1/2 MS solid medium. After the root grows to about 2 cm, transfer to 1/2 medium containing 50 μg/mL DON In 2MS solid medium, after 7 days, it was found that albino leaves were the most serious, and its cotyledons and some true leaves were yellowed and wilted; some cotyledons of wild-type Arabidopsis were yellowed and wilted, but no yellowed leaves were observed in the recovered plants (Figure 6) , indicating that complementary AtALA1 restored tolerance to DON.

Transformants 25 and 15 with high expression levels in AtALA1 transgenic Arabidopsis were selected to observe their tolerance to DON. It was found that the leaves of mutant ala1-12 were chlorotic, yellow, or even albino and dead after 50 μg/mL DON treatment for 5 days, while the overexpressed Most of the leaves of lines 25 and 15 were still green (Fig. 8A, B); after treatment 9d, the leaves of the mutant ala1-12 were shrunken, chlorotic and yellow, and the lower leaves were albino and dead, while the overexpression line 25 had a few The leaves were yellowed, but most of the leaves were still green and stretched without shrinking, and some plants even bolted and bloomed (Fig. 8C), indicating that overexpression of AtALA1 improved tolerance to DON.

6.2 Detection of DON content

6.2.1 Sample preparation

The wild-type (Col-0), mutant ala1 and AtALA1 transgenic Arabidopsis with a root length of about 2 cm were transferred to 1/2 MS solid medium containing 100 μg/mL DON for 7 days, samples were taken and immediately frozen with liquid nitrogen, Store in -80°C refrigerator for DON content detection.

6.2.2 DON extraction method

Strictly follow the method in the ELISA kit (Beijing Huaan Maike Technology Co., Ltd.) to detect the DON content. The specific method is as follows:

(1) Take the required reagents out of the refrigerated environment and place them at room temperature (20-25°C) to equilibrate for more than 1 hour. Note that each liquid reagent must be shaken well before use;

(2) Take out the required number of microwell plates, put the unused microwells into the original tin foil bag and reseal it together with the provided desiccant, store at 2-8°C, do not freeze;

(3) The washing working solution also needs to be warmed up before use;

(4) Please note that the reaction temperature should not be too high, and the temperature should be controlled at 20-25°C;

(5) Numbering: Number the microwells corresponding to the samples and standards in sequence, make 2 parallel holes for each sample and standard, and record the positions of the standard holes and sample holes;

(6) Add standard/sample: Add 50 μL of standard/sample to the corresponding microwell, add 50 μL/well of enzyme marker, then add 50 μL/well of anti-reagent and shake gently to mix, cover the plate with cover film Put it in the environment of 25℃ and avoid light for 15min;

(7) Plate washing: Carefully uncover the cover plate membrane, dry the liquid in the well, wash 4-5 times with washing solution 250 μL/well, with an interval of 10 s between each time, and pat dry on absorbent paper (not dry after pat dry). Removed air bubbles can be punctured with an unused pipette tip);

(8) Color development: Add 100 μL/well of the substrate solution, cover the plate with a cover film, and place it in a dark environment at 25°C for 5 minutes;

(9) Determination: Add 50 μL/well of stop solution, shake and mix gently, set the microplate reader at 450nm (dual wavelength 450/630nm is recommended for detection, please read the data within 5min), measure the OD value of each well .

6.2.3 Quantitative analysis

(1) Calculation of the percent absorbance, the percent absorbance of the standard or sample is equal to the average (double hole) of the percent absorbance of the standard or sample divided by the absorbance of the first standard (0 standard), then multiply by 100% that is

B—the average absorbance value of the standard or sample solution;

The average absorbance value of B0—0 (μg/kg) standard.

(2) Drawing and calculation of standard curve

Draw the standard curve with the percent absorbance of the standard substance as the vertical axis and the logarithm of the concentration of the standard substance of vomitoxin (μg/kg) as the horizontal axis. Substitute the percent absorbance of the sample into the standard curve, read the corresponding concentration of the sample from the standard curve, and multiply it by the corresponding dilution factor to get the actual amount of vomitoxin in the sample.

According to the above method to detect DON content in different plants of Arabidopsis thaliana, it was found that the content of DON in the deletion mutant ala1 was higher than that of the control; while the content of DON in the overexpression AtALA1 plants was lower than that of the control (Figure 9), and in the detected overexpression Among AtALA1 plants, plant No. 15 with the highest expression level had the lowest DON content (Fig. 7, Fig. 9), indicating that the reduction of toxin content was positively correlated with the expression level of this gene.

7. Construction of monocot expression vector pCambia-Ubi1-AtALA1-Nos and Agrobacterium transformation

7.1 Obtaining the Fusion Fragment of Monocot Constitutive Promoter Ubi1 and AtALA1 Gene

Use the following primers to amplify Ubi1 and AtALA1 respectively, and obtain the Ubi1-AtALA1 fusion fragment by fusion PCR.

Amplify Ubi1 upstream primer: 5'-AAGCTTGCATGCCTGCAG GTCGAC TCGCAGTGCAGCGTGACCCG-3'(SalI) (SEQ ID NO.8)

Downstream primer: 5'-ATTGATTTCCTGGGATCCATAAGGCCTTTGCAGAAGTAACACCAA-3' (SEQ ID NO.9)

AtALA1 upstream primer: 5'-TTGGTGTTACTTCTGCA AAGGCC TTATGGATCCCAGGAAATCAATTG-3' (StuI) (SEQ ID NO.10)

AtALA1 downstream primer: 5'-TCGGGGAAATTCGAGCTC GGTACC TCATCTCCGTGGAGGATCCTG-3'(KpnI) (SEQ ID NO.11)

PCR amplification system: 5 μL of 10×PCR Buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , 2 μL of 10 μM upstream and downstream primers, 4 μL of template (50 μg/μL), 1 μL of KOD-Plus-Neo (1U/μL), add water to 50 μL. The amplification program was 2 min at 94°C; 10 s at 98°C, 4 min at 68°C, 40 cycles. The amplified product was detected by 1% agarose gel electrophoresis, and the correct fragment was recovered for fusion PCR.

Fusion PCR amplification system: 2 μL of Ubi1 recovered fragment, 2 μL of AtALA1 recovered fragment, 5 μL of 10×PCR Buffer, 5 μL of 2mM dNTPs, 3 μL of 25mM MgSO ₄ , 1 μL of KOD-Plus-Neo (1U/μL), add water to 46 μL, follow the procedure below 15 cycles of amplification: 94°C for 2min; 98°C for 10s, 68°C for 4min. Add 2 μL of Ubi1 upstream primer and 2 μL of AtALA1 downstream primer to the above amplification system, and follow the following PCR amplification program: 94°C for 2 min; 98°C for 10 s, 68°C for 6 min, and cycle 30 times. The amplified product was detected by 1% agarose gel electrophoresis, and the correct Ubi1-AtALA1 fusion fragment was recovered.

7.2 Construction of pCambia-Ubi1-AtALA1-Nos monocot plant expression vector

pCambia2300-GUS is preserved in our laboratory. Its T-DNA segment (the region between RB and LB) contains a reporter gene GUS and a NPTII marker gene controlled by 2×CaMV35S.

The pCambia2300-GUS vector plasmid was digested by SalI/KpnI double enzymes and the large fragment was recovered, connected with the fragment obtained in the above embodiment 7.1 by homologous recombination, transformed into Escherichia coli DH5α by heat shock, screened positive clones and carried out SalI/KpnI KpnI double enzyme digestion verification (as shown in Figure 11), the results show that the Ubi1-AtALA1 fragment has been successfully ligated into the pCambia2300-GUS vector. The sequencing results are consistent with the published sequences of Ubi1 and AtALA1. The specific sequence of AtALA1 is shown in SEQ ID NO.14, and the specific sequence of Ubi1 is shown in SEQ ID NO.15. The monocot expression vector pCambia-Ubi1-AtALA1-Nos with correct sequencing was stored at -80°C for future use. The vector has the structure shown in FIG. 10 .

7.3 Acquisition of monocot expression vector pCambia-Ubi1-AtALA1-Nos recombinant Agrobacterium

Using the electroporation method, transfer the pCambia-Ubi1-AtALA1-Nos plant expression vector obtained in step 7.2 into Agrobacterium EHA105 competent cells, and use kanamycin and rifampicin as selection marker genes for resistance screening to obtain positive clones , and then extract the Agrobacterium plasmid and verify it with SalI/KpnI double enzyme digestion, and perform electrophoresis on 1% agarose gel. The enzyme digestion result is the same as that in Figure 11, indicating that the plant expression vector of recombinant Agrobacterium.

8. Genetic transformation of maize

8.1 Medium for Maize Transformation

N6E (callus induction medium): N6 salt + N6 organic + 2,4-D (2mg/L) + sucrose 30g/L + 100mg/L inositol + proline 2.76g/L + hydrolyzed casein 100mg/L +Gelrite 2.5g/L, pH 5.8, add AgNO ₃ 1ml/L (mother solution 25mM) after high temperature and high pressure sterilization.

N6OSM (osmotic medium): N6 salt + N6 organic + 2,4-D (2mg/L) + sucrose 30g/L + proline 0.69g/L + hydrolyzed casein 100mg/L + sorbitol 36.4g/L + mannitol 36.4 g/L+Gelrite 2.5g/L, pH 5.8, add AgNO ₃ 1ml/L (mother solution 25mM) after high temperature and high pressure sterilization.

N6S (screening medium): N6 salt + N6 organic + 2,4-D (2mg/L) + 100mg/L inositol + sucrose 30g/L + Gelrite 2.5g/L, pH 5.8, added after high temperature and high pressure sterilization AgNO ₃ 0.2 ml/L (mother solution 25mM) and reagents required for screening.

25D1B (transition medium): MS salt + MS organic + inositol 100mg/L + 2,4-D (0.25mg/L) + sucrose 30g/L + Gelrite 2.5g/L, pH 5.8, after high temperature and high pressure sterilization Add reagents required for screening.

Reg1 (regeneration medium 1): MS salt + MS organic + inositol 100mg/L + sucrose 60g/L + Gelrite 3g/L, pH5.8, add the reagents required for screening after high temperature and high pressure sterilization.

Reg2 (regeneration medium 2): MS salt + MS organic + inositol 100mg/L + sucrose 30g/L + Gelrite 3g/L, pH5.8, add the reagents required for screening after high temperature and high pressure sterilization.

8.2 Sterilization of corn transformation materials and acquisition of immature embryos

Corn is planted in the test field, and the young corn embryos 7-10 days after pollination are selected for callus induction. However, under natural conditions, corn can be automatically pollinated 1-4 days after earing, and it is difficult to accurately calculate the specific time of corn pollination. Artificial pollination is carried out during the day when the light is good. The number of days of pollination is calculated from the artificial pollination. The corn immature embryos 7-15 days after pollination can induce the required callus, but the longer the days, the less thorough the sterilization of corn seeds.

Separation steps of corn immature embryos:

(1) the whole corn cob head and tail are respectively 2 cm long parts transversely cut off, and only the corn kernels in the middle part of the corn cob are used to peel off the immature embryo;

(2) The middle part of the fruit ear is divided into several sections, put into a jar commonly used for laboratory tissue culture, add 75% alcohol, and pour out the alcohol after sterilizing for 2 minutes;

(3) Clean the ear segment at least twice with sterile water;

(4) add 0.1% mercuric chloride solution to sterilize for 6-8 minutes, and then wash with water at least six times;

(5) Use tweezers and a scalpel to peel off the complete corn kernels into a sterilized petri dish, clamp the endosperm-rich tail of the corn kernels with tweezers, then gently pick out the young corn embryos from the corn head with a knife, and place It was placed in N6E medium and cultured in the dark at 28°C for 3 days.

Note: The healthy callus induced by N6E medium is generally in the form of light yellow massive hard granules, and the granules are generally clustered into lumps, and rarely grow into individual granules; when growing in 25D1B medium, divide the callus into about 3- For small groups with a diameter of 5 cm, about 5 small groups can be placed in a jar; similarly, this method is also used when growing in Reg1 medium.

8.3 Preparation of reagents for gene gun transformation

8.3.1 Preparation of gold powder

(1) Weigh 60mg of gold powder (Bio-Rad M-10) and add it to a 1.5mL EP tube;

(2) Add 70% ethanol (freshly prepared);

(3) Vigorously oscillate (vortex) for 3-5 minutes and let stand for 15 minutes;

(4) Centrifuge to precipitate particles (12000g, 1min), discard the supernatant;

(5) Add 1mL ddH ₂ O, shake and vortex for 1min, and let stand for 1min;

(6) Centrifuge to precipitate particles (12000g, 1min), discard the supernatant;

(7) Repeat steps 6-9 twice for a total of 3 washes;

(8) Add 1 mL of 50% glycerol, vortex fully, aliquot into 50 μL, and store at -20°C for later use.

8.3.2 Encapsulation of microparticles and DNA

(1) Vortex the prepared gold powder for 5 minutes to disperse it as much as possible;

(2) Take 50 μL of gold powder, shake vigorously for 2 minutes, and add in order:

Plasmid DNA 10 μL; Calcium chloride (2.5M) 35 μL; Spermidine (0.1M) 15 μL;

(3) Vortex for 2 minutes, let stand for 5 minutes, and centrifuge at 1000rpm for 3-5s;

(4) Aspirate the supernatant, leaving 10 μL, pipette or vortex to mix;

(5) Take 2 μL of uniformly mixed sample on the carrier membrane (macrocarrier), put two drops on each carrier membrane (one for the control and one for the sample to be tested), and align it with the gun holes of the double gun;

(6) The distance from the stopping screen to the target shelf is 6cm, the distance from the rupture disk to the carrier film is 1cm, the pressure of the rupture disk is 650psi, and the vacuum is 28psi. is the default setting;

(7) After each bombardment, wipe the tip of the double gun with 70% alcohol and let it dry naturally;

8.4 Biolistic transformation of corn immature embryo callus

(1) Put the immature corn embryos to be transformed into N6OSM medium with filter paper, and cultivate them in dark at 28°C for 4 hours for transformation;

(2) When transforming, put about 10 immature embryos in the middle of the petri dish, and put the callus initiation germination upwards, 6 cm away from the carrier membrane;

(3) The pressure of the ruptured membrane is 650psi;

(4) The method of using the gene gun is strictly in accordance with the instruction of the reference gene gun (Bio-Rad He/1000 particledelivery system);

(5) The transformed material was taken out in time, and cultured in dark at 28°C for 16-20h in N6OSM medium lined with filter paper;

(6) Finally, the material was transferred to N6E medium for dark culture.

Note: The transformation efficiency of the gene gun is relatively high, but the pollution is serious, so the internal sterilization of the gene gun must be careful before use; when transforming, it is best to place a wet filter paper in the center of the petri dish, and put the material on it to prevent The material is left for too long to lose water; when placing the material on the medium, be sure to place the callus initiation site upwards.

8.5 Tissue culture after transformation of maize material

(1) The corn immature embryos that have been induced and cultured in N6E medium for 10-14 days have part of the callus growing out, and at this time, the material can be moved to N6S medium to continue dark culture;

(2) The tissue material cultured in dark in N6S medium is generally subcultured once every 15-20 days, and a callus with selection resistance will grow after about 6 to 8 weeks;

(3) If the callus produced is less, before transferring to Reg1 medium, it can be put into 25D1B medium for dark culture for about 3 weeks, which can help produce more healthy callus for plant regeneration culture;

(4) When corn is cultured in dark on Reg1 medium for 2 to 3 weeks, it can be found that more white somatic embryos grow;

(5) Put the white somatic embryos into the Reg2 medium for light culture for about 3 weeks, and then the regenerated seedlings can be obtained.

8.6 Molecular verification of AtALA1 transgenic maize

8.6.1 PCR amplification verification

Maize genetic transformation was carried out by gene gun, and T ₀ generation transgenic maize plants were obtained, with a total of 23 independent transformants. The DNA was extracted by using the new plant genome DNA rapid extraction kit of Plant Aidlab Company, the AtALA1 transgenic maize DNA was extracted, and the quality of the DNA was detected by 1% agarose gel electrophoresis. The extracted maize DNA was used as a template to detect whether the AtALA1 gene was integrated in the transgenic maize.

PCR amplification primers are:

Upstream primer: 5'-ATGGATCCCAGGAAATCAATTG-3' (SEQ ID NO.12);

Downstream primer: 5'-AGCCAACCTATTGTTCTCAACTCTA-3' (SEQ ID NO.13).

The 10 μL amplification system includes: 2×TaqMaster mix 5 μL; template DNA, 1 μL; 5 μmol/L upstream primer 0.5 μL; 5 μmol/L downstream primer 0.5 μL; ddH ₂ O 3 μL.

PCR temperature cycle parameters: 94°C, 5min; 94°C for 30s, 56°C for 30s, 72°C for 30s, 30 cycles; 72°C, 10min. Finally, the amplified products were detected by 1% agarose gel electrophoresis.

PCR results showed that the positive control and the selected 16 transformants could amplify the target band ( FIG. 12 ), but the wild type could not amplify the target band, indicating that the AtALA1 gene was successfully expressed in maize.

8.6.2 Transcript level detection of AtALA1 transgenic maize

Real-time PCR method was used to detect the expression level of the transgene in the plants. To normalize the cDNA concentration, the maize EF1a gene was used as an internal standard.

The 10 μL reaction system includes: 1 μL of cDNA, 0.5 μL of upstream and downstream primers, 5 μL of 2×iQ SYBR Green Supermix, and make up 10 μL with RNase-free double distilled water.

The amplification program was: 95°C pre-amplification for 3 minutes; 94°C for 10s, 56°C for 30s, 72°C for 30s, a total of 40 cycles of amplification. After the amplification was completed, the relative expression of the gene was analyzed using Gene Study software. The results showed that the AtALA1 gene was expressed in different degrees, and the transformants No. 4 and No. 13-3 had higher expression levels ( FIG. 13 ).

9. Resistance of AtALA1 transgenic maize to Fusarium graminearum and its toxin

9.1 Resistance of AtALA1 transgenic maize to Fusarium graminearum toxins DON and T-2

9.1.1 Resistance of AtALA1 transgenic maize to Fusarium graminearum toxin DON

AtALA1 transgenic and wild-type corn kernels that germinated to about 0.2 cm were selected, treated with 30 μM DON for 2 days, washed with distilled water and then normal culture was resumed. After 3 days, it was found that the transgenic corn could still grow normally, with longer roots and stems; The growth of roots in the type and the negative control was significantly inhibited, and some grains were rotten to varying degrees (Fig. 14A). The statistical results of root length showed (Fig. 14B) that the root length of wild-type maize was 0.3 cm, which was not significantly increased compared with that before treatment; the root length of 13-3 transformant and 15-2 transformant were significantly longer than that of wild type, of which The length of 13-3 with higher expression reached 1.7 cm, which was 1.3 cm longer than that of the wild type; the root length of the 15-2 transformant with the second highest expression reached 1.4 cm, which was 1 cm longer than that of the wild type, indicating that the expression of AtALA1 alleviated the effect of DON on DON. Inhibition of maize root length. The results of DON content detection showed that the DON content of No. 13-3 transformant with higher expression level was 105, 110ppb lower than that of wild type and control group respectively; The group was lower by 95, 100 ppb ( FIG. 14C ), indicating that expression of AtALA1 reduced DON accumulation in maize kernels and increased resistance to it.

9.1.2 Resistance of AtALA1 transgenic maize to Fusarium graminearum T-2 toxin

To observe whether AtALA1 transgenic maize is also tolerant to the A toxoid T-2 toxin of Fusarium graminearum. The germinated grains were also treated with 30 μM T-2 toxin for 2 days, and normal culture was resumed. As a result, it was found (Fig. 15A) that the root length of wild-type grains had no significant change compared with that before treatment, while the root length of No. 13-3 transformants with higher expression levels reached 0.58cm, which was about 93% longer than that of wild-type. The root length of the next transformant No. 15-2 was about 0.6 cm, which was 100% longer than that of the wild type and the control ( FIG. 15B ), indicating that the expression of AtALA1 improved the resistance of maize to the T-2 toxin pair.

9.2 Resistance of AtALA1 transgenic maize to cereal sickle

Wild-type and AtALA1 transgenic corn kernels were selected 18-20 days after pollination, and the corn kernels were inoculated by injection and soaking methods, respectively, for disease resistance analysis. After inoculating Fusarium graminearum for 7 days by injection, it was found that the inoculation site of wild-type corn kernels was obviously blackened, covered with a large number of white hyphae, and the lesions were larger; while the inoculation sites of 13-3 and 15-2 transgenic The amount of fungal hyphae was significantly less than that of the wild type (Figure 16), and the lesion area was counted (Figure 16B). The results showed that the diseased area of the wild type was 15mm ² after inoculation, and the lesion area of the No. 13-3 transformant was 6mm ² , Reduced by 60% compared with the wild type; No. 15-2 transformant lesion area was 11mm ² , which was reduced by 27% compared with the wild type; counting the number of spores on the grain (Fig. 16C), the results showed that the number of spores on the grain of the wild type was 1.0×10 ⁶ cells/mL, No. 13-3 transformant was 1.3×10 ⁵ cells/mL, 87% less than wild type; No. 15-2 transformant was 2.4×105 cells/mL, 76% less than wild type %. The above results indicated that the expression of AtALA1 enhanced the resistance of maize to Fusarium graminearum.

The young corn kernels were inoculated with Fusarium graminearum by soaking method, and it was found that the yellow epidermis of 13-3 and 15-2 transgenic corn kernels was mostly visible, with only a small amount of white hyphae, while the surface of wild-type corn kernels was covered with white bacteria silk, and the yellow skin of corn can hardly be seen (Fig. 17A); the statistics of spore counts (Fig. 17B) showed that the spore counts of ^No. type (1.5×10 ⁶ cells/mL), indicating that the expression of AtALA1 inhibited the ability of Fusarium graminearum to infect maize.

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SEQUENCE LISTING

<110> Southwest University

<120> Method for Improving Plant Resistance to Fusarium graminearum Using AtALA1 Gene

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Claims (8)

1. A method for improving the resistance of plants to fusarium graminearum is implemented, wherein the resistance of the plants to the fusarium graminearum is improved by integrating an AtALA1 gene into a target plant and expressing the AtALA1 gene in the target plant so as to reduce the content of fusarium graminearum toxin, and the nucleotide sequence of the AtALA1 gene is shown as SEQ ID No.14.

2. The method of claim 1, comprising the steps of:

1) Constructing a recombinant plant expression vector containing the AtALA1 gene;

2) Introducing the recombinant plant expression vector into a target plant to make AtALA1 gene expressed in the target plant in a constitutive mode; and

3) Transgenic plants resistant to fusarium graminearum are obtained.

3. The method according to any one of claims 1 to 2, wherein the target plant is Arabidopsis thaliana.

4. The method according to any one of claims 1 to 2, wherein the target plant is a cereal plant.

5. The method of claim 4, wherein the target plant is maize.

6. A preparation method of a transgenic plant resisting fusarium graminearum comprises the following steps:

i) Obtaining an AtALA1 gene, operably inserting the AtALA1 gene into a plant expression vector, and constructing the plant expression vector;

ii) transforming a host with the plant expression vector obtained in step i) to obtain a transformant;

iii) Transforming a plant with the transformant obtained in step ii) to obtain a transgenic plant;

the nucleotide sequence of the AtALA1 gene is shown as SEQ ID NO.14.

7. The method of claim 6, comprising the steps of:

a) Obtaining a plant AtALA1 gene: introducing SmaI and SalI enzyme cutting site design primers, and then carrying out PCR amplification by taking the cDNA of the plant as a template to obtain an amplification product, namely an AtALA1 gene sequence added with the enzyme cutting site;

b) Constructing a plant expression vector for constitutive expression of the AtALA1 gene: respectively inserting the amplified AtALA1 gene sequence into a dicotyledonous plant expression vector pLGN-35S-Nos and a monocotyledonous plant expression vector pCambia2300-Ubi1-Nos, and constructing two new plant expression vectors which are respectively named as pLGN-35S-AtALA1-Nos and pCambia2300-Ubi1-AtALA1-Nos;

c) Genetic transformation of plants: integrating the pLGN-35S-AtALA1-Nos or pCambia2300-Ubi1-AtALA1-Nos plant expression vector obtained in the step b) into a plant genome by utilizing an agrobacterium tumefaciens flower soaking method and a gene gun transformation method, realizing the constitutive expression of the AtALA1 gene in a transgenic plant, reducing the content of fusarium graminearum toxin and improving the resistance of the transgenic plant to fusarium graminearum;

d) Obtaining of low-toxin-content anti-fusarium graminearum AtALA1 transgenic plants: and (c) further carrying out propagation, molecular identification and disease resistance identification on the transgenic plant obtained in the step c) to obtain the low-toxin-content fusarium graminearum resistant AtALA1 transgenic plant.

The application of the AtALA1 gene in reducing fusarium graminearum toxins and improving the fusarium graminearum resistance of plants is realized, wherein the AtALA1 gene is integrated into a target plant to obtain a transgenic plant, and is expressed in the plant body, so that the content of the fusarium graminearum toxins in the plants is reduced, and the fusarium graminearum resistance of the target plant is improved, and the nucleotide sequence of the AtALA1 gene is shown as SEQ ID NO.14.

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