CN103695442B - A kind of NFSAI gene coming from Lactobacterium acidophilum and its preparation method and application - Google Patents

Abstract

The invention discloses a NFSAI gene derived from Lactobacillus acidophilus and its preparation method and application. The nucleotide sequence of the NFSAI gene is as SEQ? ID? As shown in NO: 1, its coded amino acid sequence is shown as SEQ? ID? NO: shown in 2; the NFSAI gene is prepared by a gene synthesis method, and the NFSAI gene is transformed into a plant, which can improve the ability of the transgenic plant to degrade TNT. Therefore, the NFSAI gene of the present invention can promote the plant to participate in the degradation of TNT, Provide useful help for phytoremediation of TNT pollution.

Description

A kind of NFSAI gene derived from Lactobacillus acidophilus and its preparation method and application

technical field

The invention belongs to the field of crop genetics and breeding, and specifically relates to an NFSAI gene derived from Lactobacillus acidophilus, a preparation method and application thereof.

Background technique

2,4,6-Trinitrotoluene (TNT) is a widely used explosive with high toxicity and mutagenicity. It can enter the human body through the respiratory system, digestive system and skin, causing blood phase changes, cyanosis, Cataracts, methaemoglobinemia and toxic hepatitis. Because the soil has a strong adsorption effect on TNT, if the TNT in the wastewater is discharged directly without treatment, the TNT will quickly seep into the ground and accumulate in the soil, which will seriously endanger the environment. The pollution of water body and soil environment by TNT has long been paid attention to. When the content of TNT in the water reaches 1mg/L, the fish will die; TNT enters the soil and will interact with several main compounds in the soil to increase the concentration of organic matter in the soil. Therefore, it will cause far-reaching harm to the environment.

The physical and chemical treatment method proposed by Rodgers for TNT biological remediation is incineration, which is the most effective method at present, but it is expensive for soil pollution control, because incineration needs to excavate soil, transport and consume energy, which costs a lot. Several technologies have been proposed for the treatment of TNT in liquid pollutants, such as a class of related technologies developed by combining biochemical and physical chemical methods. First, good results have been achieved in the treatment of explosive wastewater. Biochemical treatment, anaerobic fluidized bed containing granular activated carbon, ozone oxidation plus biochemical method, warm decay, photocatalytic method, combination of photocatalytic and anaerobic, granular iron method, etc. Meanwhile, several bio-based technologies for treating TNT-contaminated soils have also been developed.

The basis of bioremediation is the biological metabolism of pollutants by microorganisms in nature, so early bioremediation mainly refers to microbial remediation, and a series of technologies have been developed for microbial remediation: such as on-site treatment, in-situ treatment and bioreactors. The research on biological degradation of NTT wastewater mainly focuses on three aspects of TNT degrading bacteria species, degradation pathway and degradation process. The bacteria that have been found to degrade TNT wastewater are mainly white rot fungi (phaneorhcaeetchyrsosporium). Zhang Jinglai, Chang Guanqin and others studied the influence of white rot fungi on the degradation of TNT explosive wastewater based on factors such as time, temperature, pH value, and lignin dosage. The results show that when the reaction time is 48h, the biochemical temperature is 15°C, the HP value is 5, and the lignin dosage is 3g, the CODcr removal rate of TNT wastewater is 99% [Zhang Jinglai et al., Journal of China University of Mining and Technology, 2001, 30 (2): 161-164]. He Dewen, Xiao Yutang et al. used white rot fungi to biochemically degrade two kinds of biodegradable organic wastewater. The experimental results showed that white rot fungi had good degradation performance on biodegradable TNT explosive wastewater and disperse dye wastewater, showing broad application prospects. [He Dewen et al., Industrial Water Treatment, 2000,20(3):16-18].

Regarding the degradation pathway of TNT, one of the sayings is that the nitro group is first reduced to an amino group, then converted into aminodinitrotoluene and diaminonitrotoluene, and condensed into tetranitroazotoluene and other products. Kaplan proposed a way to simulate the biochemical transfer of TNT in composting systems [Kaplanetal., EnvironMicrobio, 1982,44(3):757-760]. However, Spanggord proposed that the intermediate product of TNT biochemical degradation is 2-amino 4,6-dinitrobenzoic acid. The degradation pathway he proposed believed that the benzene ring was not opened, and detected dinitrophenol not mentioned by Kaplan in the experiment. And dinitroaniline and other compounds [Spanggord, AD-A138550, 1983]. At the same time, MCormick et al. also proposed a biochemical degradation pathway for 2,4-DNT: a nitro group of 2,4-dinitrotoluene is reduced to a nitroso group, which is then reduced to a fat, and then condensed into a dinitro group to be oxidized Compounds such as azotoluene may be reduced to aminonitrotoluene, and the latter is further degraded to 2,4-diaminotoluene. If degraded again, aminobenzene derivatives are obtained [McCormick, EnvironMicrobiol, 1978, 35 (5) :945-948].

The use of plants to control soil pollution is a kind of energy-saving and relatively safe method for the environment. Compared with microbial remediation, phytoremediation is more suitable for in-situ remediation. It has the advantages of large remediation area, less investment, no damage to the site structure, and no secondary pollution of groundwater. It has been shown in the treatment of heavy metals and organic pollutants Obvious effectiveness, some have reached the level of field application.

Contents of the invention

The technical problem to be solved by the present invention is to provide a NFSAI gene derived from Lactobacillus acidophilus and its preparation method and application, transform the NFSAI gene into plants, and then improve the ability of the transgenic plants to degrade TNT.

Therefore, one of the technical problems to be solved by the present invention is to provide an NFSAI gene derived from Lactobacillus acidophilus.

The second technical problem to be solved by the present invention is to provide a method for preparing the NFSAI gene derived from Lactobacillus acidophilus.

The third technical problem to be solved by the present invention is to provide the application of the NFSAI gene derived from Lactobacillus acidophilus in improving plant degradation of TNT, and in cultivating and improving plant degradation organic pollution. That is, the NFSAI gene from Lactobacillus acidophilus is transformed into plants, and the function verification of the transgenic plants is carried out to prove that it has the ability to improve the plant's ability to repair TNT, and then can be applied to the technical field of cultivating and improving plant degradation of organic pollution.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

An NFSAI gene derived from Lactobacillus acidophilus, its nucleotide sequence is shown in SEQ ID No: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 2.

A preparation method of NFSAI gene derived from Lactobacillus acidophilus, which is prepared by gene synthesis method [Xiongetal., NuclAcidsRes, 2004, 32:e98], that is, the nicotinamide derived from Lactobacillus acidophilus is cloned according to the gene synthesis method Adenine dinucleotide phosphoflavin oxidoreductase (NADPH-flavinoxidoreductase, NFSA) gene is the NFSA gene. On the basis of keeping the amino acid sequence of the NFSA gene unchanged, design primers to synthesize the present invention derived from Lactobacillus acidophilus Nicotinamide adenine dinucleotide phosphoflavin oxidoreductase gene NFSAI is the NFSAI gene. The specifically designed primers are as follows:

NFSAI-1

GGAGGATCCTCCATGATCCACAACAAGACTATTGATGCTCAACTCAATCACAGATCCATC

NFSAI-2

CAAGTTGTTCTTTGTTCAAGGTGATGTCCTTGAACTTTCTGATGGATCTGTGATTGAGTT

NFSAI-3

CTTGAACAAAGAACAACTTGAGACCTTGTACTCTGTCTTCGCTCAGACTCCAACCTCTAT

NFSAI-4

TGGATCGATGATGTGAACCAAGGAAGCGTTCTGCATGAACATAGAGGTTGGAGTCTGAGC

NFSAI-5

TGGTTCACATCATCGATCCAGAGCAGAAGAAGAAGATCAGAGAACTCTGCAACCAGAGAGT

NFSAI-6

TCAACGACGAAGATGAAGAGATCACCTTCAGCACCGACATACTTCTGGTTGCAGAGTTCT

NFSAI-7

CTCTTCATCTTCGTCGTTGACTTGTACAGAAACCAACAGATCAGAAAGCAACTTGGTAAG

NFSAI-8

CTTGGAAGAAGATGTCGATGGTGTGAACTCTGCCATCATCCTTACCAAGTTGCTTTCTGA

NFSAI-9

CATCGACATCTTCTTCCAAGCTATGGAAGACACCCTGCTTGCTTACCAGAACGTTGCCAA

NFSAI-10

ACCAAGTGGAACGTAACCCAGATCCATAGATTCGACAGCATTGGCAACGTTCTGGTAAGC

NFSAI-11

TGGGTTACGTTCCACTTGGTACTGTCAACGATCATCCACTTGAGATGCTCAAGACCCTTG

NFSAI-12

ACTTGCATACCAAGGACTGGAAAGGTCAGTTCTGGCAGGTCAAGGGTCTTGAGCATCTCA

NFSAI-13

CCAGTCCTTGGTATGCAAGTTGGTGTTCCAGACCAGAAACCACAACTGAAGCCAAGACTT

NFSAI-14

CCTTGTCATACTTACCTTCAAAGCAGATGAACTTGAGTGGAAGTCTTGGCTTCAGTTGTG

NFSAI-15

TGAAGGTAAGTATGACAAGGACTTCAACGTCAAGGACCTGAAGGACTACGATCAAGTCGT

NFSAI-16

GATTCTTCTGTTGGAGTCTCTCAGGTCGTAGTAGGTGGTGACGACTTGATCGTAGTCCTT

NFSAI-17

GAGACTCCAACAGAAGAATCGACTCCTTCACCAAGCAGATCACTGGTGCTAAGCTCGACA

NFSAI-18

TGAAGTTCCCTCTGGAAGTTCGTCTCTGTCAGTCTCATGGTTGTCGAGCTTAGCACCAGTG

NFSAI-19

GAACTTCCAGAGGAACTTCACAAGCAAGGCTTGTGCTTGGACTGGAAATAAGAGCTCGAGCTC

The NFSAI gene synthesized by the invention is transferred into plants, which can be applied to improve the ability of plants to degrade TNT, and further can be used in the technical field of cultivating and improving plants to degrade organic matter pollution. Specifically include the following steps:

1) Construction of recombinant plasmid AH608

After double digestion with BamHI and SacI, the NFSAI gene synthesized by the present invention was connected to the plant expression vector pCAMBIA-1301 containing double 35S promoters by T4 DNA ligase, and the recombinant protein containing the target gene NFSAI was obtained through enzyme digestion identification and sequence determination Plasmid AH608.

2) Transformation of Agrobacterium by electric shock method

The constructed recombinant plasmid AH608 was introduced into Agrobacterium tumefaciens by electric shock method, and the strain of Agrobacterium tumefaciens was EHA105, LBA4404, GV3101 or AGL-1.

3) Transformation of plants by Agrobacterium sticky flower method

Transformation of Agrobacterium tumefaciens with NFSAI gene into Arabidopsis, tobacco, tomato and rice [Cloughetal., Theplantjournal, 1998, 16(6):735-743] by using Agrobacterium sticky flower method, and then verified these Transgenic plants can significantly improve the plant's ability to degrade TNT.

Beneficial effect of the present invention

1. The present invention utilizes the gene synthesis method to artificially synthesize the NFSA gene from Lactobacillus acidophilus according to the plant preference code. On the basis of keeping the amino acid sequence of the NFSA gene unchanged, primers are designed to transform and synthesize the NFSAI gene of the present invention. The NFSAI gene can be continuously expressed in plants, and promotes plants to participate in the degradation of TNT, thereby improving the ability of plants to repair TNT pollution.

2. The plant containing the NFSAI gene of the present invention is safe and stable, has no adverse effect on plant growth, and has little environmental pollution.

Description of drawings

Fig. 1 is the construction schematic diagram of the recombinant plasmid AH608 containing NFSAI gene of the present invention;

Fig. 2 is the PCR electrophoresis figure of the seedling stage of transgenic Arabidopsis thaliana in Example 4 of the present invention, wherein, M is DNAmarker, WT is wild-type plant, and 1-7 are different strains of transgenic Arabidopsis respectively;

Fig. 3 is the PCR electrophoresis figure of the transgenic tobacco seedling stage of Example 5 of the present invention, wherein, M is DNAmarker, WT is wild-type plant, and 1-7 are respectively the different strains of transgenic tobacco;

Fig. 4 is the PCR electrophoresis figure of the transgenic tomato seedling stage of Example 5 of the present invention, wherein, M is a DNAmarker, WT is a wild-type plant, and 1-6 are respectively different strains of the transgenic tomato;

Fig. 5 is the PCR electrophoresis figure of the transgenic rice seedling stage of Example 6 of the present invention, wherein, M is DNAmarker, WT is wild-type plant, and 1-6 are respectively the different strains of transgenic rice;

Fig. 6 is a graph showing TNT tolerance of NFSAI-transferred Arabidopsis of the present invention, wherein CK is a wild-type plant, and others are different strains of NFSAI-transferred Arabidopsis.

detailed description

Embodiment 1: Synthesis of NFSAI enzyme gene derived from Lactobacillus acidophilus by gene synthesis

Using the gene synthesis method [Xiongetal., NuclAcidsRes, 2004, 32:e98] to clone the NFSA gene derived from Lactobacillus acidophilus, carry out chemical synthesis, and design according to the plant preference code on the basis of keeping the amino acid sequence of the NFSA gene unchanged Primers were de novo synthesized from the gene encoding the Lactobacillus acidophilus NFSAI enzyme. The designed primers are as follows:

NFSAI-1

GGAGGATCCTCCATGATCCACAACAAGACTATTGATGCTCAACTCAATCACAGATCCATC

NFSAI-2

CAAGTTGTTCTTTGTTCAAGGTGATGTCCTTGAACTTTCTGATGGATCTGTGATTGAGTT

NFSAI-3

CTTGAACAAAGAACAACTTGAGACCTTGTACTCTGTCTTCGCTCAGACTCCAACCTCTAT

NFSAI-4

TGGATCGATGATGTGAACCAAGGAAGCGTTCTGCATGAACATAGAGGTTGGAGTCTGAGC

NFSAI-5

TGGTTCACATCATCGATCCAGAGCAGAAGAAGAAGATCAGAGAACTCTGCAACCAGAGAGT

NFSAI-6

TCAACGACGAAGATGAAGAGATCACCTTCAGCACCGACATACTTCTGGTTGCAGAGTTCT

NFSAI-7

CTCTTCATCTTCGTCGTTGACTTGTACAGAAACCAACAGATCAGAAAGCAACTTGGTAAG

NFSAI-8

CTTGGAAGAAGATGTCGATGGTGTGAACTCTGCCATCATCCTTACCAAGTTGCTTTCTGA

NFSAI-9

CATCGACATCTTCTTCCAAGCTATGGAAGACACCCTGCTTGCTTACCAGAACGTTGCCAA

NFSAI-10

ACCAAGTGGAACGTAACCCAGATCCATAGATTCGACAGCATTGGCAACGTTCTGGTAAGC

NFSAI-11

TGGGTTACGTTCCACTTGGTACTGTCAACGATCATCCACTTGAGATGCTCAAGACCCTTG

NFSAI-12

ACTTGCATACCAAGGACTGGAAAGGTCAGTTCTGGCAGGTCAAGGGTCTTGAGCATCTCA

NFSAI-13

CCAGTCCTTGGTATGCAAGTTGGTGTTCCAGACCAGAAACCACAACTGAAGCCAAGACTT

NFSAI-14

CCTTGTCATACTTACCTTCAAAGCAGATGAACTTGAGTGGAAGTCTTGGCTTCAGTTGTG

NFSAI-15

TGAAGGTAAGTATGACAAGGACTTCAACGTCAAGGACCTGAAGGACTACGATCAAGTCGT

NFSAI-16

GATTCTTCTGTTGGAGTCTCTCAGGTCGTAGTAGGTGGTGACGACTTGATCGTAGTCCTT

NFSAI-17

GAGACTCCAACAGAAGAATCGACTCCTTCACCAAGCAGATCACTGGTGCTAAGCTCGACA

NFSAI-18

TGAAGTTCCCTCTGGAAGTTCGTCTCTGTCAGTCTCATGGTTGTCGAGCTTAGCACCAGTG

NFSAI-19

GAACTTCCAGAGGAACTTCACAAGCAAGGCTTGTGCTTGGACTGGAAATAAGAGCTCGAGCTC

Using NFSAI-1 and NFSAI-19 as outer primers, and NFSAI-2～NFSAI-18 as inner primers, PCR was used to amplify NFSA gene fragments. In a 50 μl reaction system, the inner primer concentrations were 1.5 pmol, and the outer two The concentration of each primer was 30 pmol. The amplification conditions are: preheating at 94°C for 1min; 94°C for 30s, 50°C for 30s, 72°C for 1min. A total of 25 cycles. After PCR, 1% agarose gel was used to recover, and 10 μl of the recovered product was directly connected to the T/A cloning vector, and ligated overnight at 4°C to obtain DNA ligation products, which were efficiently transformed into DH5α competent cells.

A 771 fragment was obtained by PCR reaction, and the product was recovered, cloned, sequenced and analyzed, and the obtained sequence was the NFSAI gene of the present invention, its nucleotide sequence was shown as SEQ ID No: 1, and its encoded amino acid sequence was shown as SEQ ID NO: 2.

Embodiment 2: Construction of NFSAI gene plant expression vector

Double enzyme digestion was performed with BamHI and SacI respectively, and the DNA fragments were recovered, and the NFSAI enzyme gene was connected to the pCAMBIA-1301 vector containing double 35S promoters by T4 DNA ligase, and the recombinant plasmid containing the NFSAI enzyme gene was obtained through enzyme digestion identification and sequence determination AH608, as shown in Figure 1. The expression vector also contains a GUS reporter gene and a kanamycin resistance marker gene with an intron.

Example 3: Agrobacterium cultivation and plant transformation

The Agrobacterium strain was Agrobacterium tumefaciens LBA4404 strain. The recombinant plasmid AH608 obtained above was introduced into Agrobacterium LBA4404 by electroporation. Pick a single bacterium into 25ml YEB medium (50mg/l rifampicin) for overnight culture, transfer 5ml of the bacterial liquid to 100ml of YEB medium (50mg/l rifampicin), cultivate to OD600=0.7-0.8, put the bacterial liquid on ice Leave it for 10 minutes, centrifuge at 5000rpm for 10min at 4°C, collect the bacteria, add 100ml sterile double distilled water to wash twice. Add 4ml of 10% glycerol to suspend the bacteria and transfer to a 50ml centrifuge tube. Centrifuge at 5500rpm for 10min at 4°C. Collect the bacteria, add 500 μl of 10% glycerol to suspend the bacteria, transfer to a 1.5ml centrifuge tube, and obtain Agrobacterium competent cells. Take 70 μl of competent cells, add 1 μl of recombinant plasmid AH608, mix well with a yellow pipette tip with the head removed, and transfer to a 0.1 cm electric shock cup. Electric shock parameters: 200Ω, 1.7KV, 2.5F, add 800μl SOC culture solution immediately after electric shock. After culturing for 1 hour, take 100 μl of the resistant plate and incubate overnight at 28°C to screen for strains successfully introduced with the recombinant plasmid AH608.

Embodiment 4: Arabidopsis thaliana transformation by sticking flower method

Inoculate a single colony of the screened Agrobacterium strain containing the target plasmid in 5 ml of LB medium containing the corresponding antibiotic and culture it at 28°C for 2 days. Transfer 5 ml of bacterial liquid to 500 ml of liquid LB medium and incubate at 28°C for 16-24 hours (OD=1.5-2.0), and the liquid can be stored at 4°C for 30 days. The bacterial cells were collected by centrifugation at room temperature, and centrifuged at 4000g for 10 minutes. Suspend with an equal volume of 5% fresh sucrose solution. Add 0.02% Silwet-77 and mix well, then transfer to a beaker to obtain the transformed bacteria liquid. Transform each strain with 300 ml and turn 2-3 bowls. Transform once every 7 days. Put Arabidopsis thaliana (ecotype columbia ) upside down and immerse in the transformation solution for 10 seconds to infect both rosettes and inflorescences. Air-dry the transformed plant bacterial liquid for 3-5 seconds after infection. Circle the transformed plants with plastic wrap and lay them flat for 16-24 hours. Do not place under high temperature and strong light after transformation. Remove the plastic wrap, keep a certain humidity, and harvest the seeds after another month of growth. Use 50 μg/mL hygromycin to screen the transformed plants, transplant seedlings that grow normally on the hygromycin plate, harvest the seeds, and identify positive seedlings.

Example 5: Tobacco and Tomato Transformation

Select plump tobacco seeds K326 and tomato seeds Hupan 1429 respectively, wash with 75% alcohol for 1 minute, add 1 drop of sodium hypochlorite to sterilize with soil temperature for 10 minutes, spread the seeds on MS0 medium, and cultivate them at 28°C until they germinate. Young tobacco leaves, tomato cotyledons or hypocotyls were cut into 1 cm ² , placed in MS0+NAA1 (1ug/ml)+BA2 (4ug/ml) medium, and cultured at 22°C for 1 day. Agrobacterium containing the target plasmid was cultured at OD0.8-1.0, centrifuged at 5000g for 8 minutes, washed once with sterile water, suspended and infected with an equal volume of MS culture medium for 8 minutes, blotted and placed in MS0+NAA1(1ug/ml)+ In BA2 (4ug/ml) medium, co-cultivate at 22°C for 3 days. Then transfer to screening medium MSO+IAA1(0.1ug/ml)+ZT(2ug/ml)+Cb(500ug/ml)+Km(50ug/ml) for 2-3 weeks, then transfer to differentiation medium MS0+ IAA1(0.1ug/ml)+ZT(2ug/ml)+Cb(500ug/ml)+Km(100ug/ml) was cultured for 2-3 weeks, and finally transferred to rooting medium 1/2MS+IAA1(0.1ug/ml )to cultivate.

Embodiment 6: rice transformation

N6 medium is the basic medium, and the hulled rice varieties are the seeds of Zhonghua. The immature embryos 12-15 days after pollination are surface-sterilized and inoculated into N6D2 medium to induce callus (N6 medium, hydrolyzed milk Protein 500mg/L, sucrose 30g/L, 2,4-D2mg/L, plant gel 2.5g/L, pH5.8); after 4-7 days of culture, callus was taken for transformation. Agrobacterium containing the target plasmid was cultured at OD0.8-1.0, centrifuged at 5000g for 8 minutes, washed once with DDH ₂ O, suspended and infected with an equal volume of MS culture medium for 8 minutes, blotted and placed in MS0+NAA1(1ug/ml)+ In BA2 (4ug/ml) medium, co-cultivate at 22°C for 3 days. Then change to selection medium MSO+NAA1 (1ug/ml)+BA2 (4ug/ml)+Cb (500ug/ml)+HAT (50ug/ml) (add cephalosporin Cb (500ug/ml) and hygromycin HAT ( 50ug/ml)), the transformed callus was cultured on the resistant medium MS0+Cb (500ug/ml)+HAT (50ug/ml) for 3 to 4 generations, and transferred to the differentiation medium MS0+NAA1 (1ug/ml )+BA2(4ug/ml)+KT(2ug/ml)+Cb(500ug/ml)+HAT(50ug/ml) (2mg/LKT); the young shoots grow to 2mm and transferred to the rooting medium (1/2MS +0.5mg/LIBA). Add 500mg/L enzyme-hydrolyzed milk protein (CH), 0-700mg/L glutamine or arginine, 30-80g/L sucrose, 6g agar, pH 5.8 to the above medium respectively. The subculture period is 25d. The light yellow embryogenic callus was transferred to the differentiation medium, and it differentiated and sprouted in about 30 days. The light intensity is 1500~2000lx, 12~14h/d.

Part of the leaves were taken from the transformed plants of Arabidopsis thaliana, tobacco, tomato and rice obtained in Examples 4-6, and invaded into the staining solution containing X-GLUC, and the transgenic plants whose leaves turned blue were screened for molecular detection. Extract the total DNA of the leaves, refer to the method of "Molecular Cloning", and use NFSAI-1 and NFSAI-19 as primers to perform PCR detection on the transformed plants. The amplification conditions are: 94°C preheating for 1min; 94°C for 30s, 60°C for 30s , 72°C, 4min. A total of 25 cycles, the results are shown in Figures 2, 3, 4, and 5. It can be seen from the figure that the target bands were not detected in the wild-type plants, but there were very bright target bands in the transgenic plants, which proved that these lines were all positive seedlings, and proved that the target gene had been detected at the molecular level. Imported successfully.

Example 7: Detection of the degradation ability of TNT after the synthetic NFSAI gene transforms the plant

The above-mentioned transgenic plants that have been successfully transferred into the target gene are selfed and homozygous for three generations to obtain homozygous transformants, and the seeds are harvested. Seeds were cultured in MS medium containing 0.15mMTNT, and non-transgenic plants were cultivated at the same time as a control experiment. Observe the germination, growth and fresh weight of the plants. The specific experimental results are as follows:

The germination rate of transgenic plants in the triangular flask containing TNT is 2 times higher than that of non-transgenic plants. In the Erlenmeyer flask medium containing 0.15mM TNT, the fresh weight of the transgenic plants was 65% higher than that of the non-transgenic plants, as shown in FIG. 6 .

On the TNT-containing plate, the germination rate of transgenic tobacco seeds is 3.5 times higher than that of non-transgenic plants, the root length is 60% higher than that of non-transgenic plants, and the fresh weight is 45% higher than that of non-transgenic plants.

On the plate containing TNT, the germination rate of transgenic tomato seeds is 2.5 times higher than that of non-transgenic plants, the root length is 50% higher than that of non-transgenic plants, and the fresh weight is 50% higher than that of non-transgenic plants

On the plate containing TNT, the germination rate of transgenic rice seeds is 3 times higher than that of non-transgenic plants, the root length is 55% higher than that of non-transgenic plants, and the fresh weight is 40% higher than that of non-transgenic plants.

In summary, the present invention remodeled the NFSA gene derived from Lactobacillus acidophilus by designing 19 primers and using gene synthesis method, and obtained the NFSAI gene whose nucleotide sequence is shown in SEQIDNo1. At the same time, the NFSAI gene was successfully introduced into Arabidopsis thaliana, tobacco, tomato and rice, and the obtained transgenic plants were found to have strong TNT resistance ability, indicating that the NFSAI gene transformed and synthesized by the present invention has the ability to improve plant resistance to TNT , can be used to cultivate and improve the technical field of plant degrading organic matter pollution.

Claims (1)

1. A primer for synthesizing the NFSAI gene derived from Lactobacillus acidophilus, the nucleotide sequence of the NFSAI gene is as shown in SEQ ID NO: 1, and its encoded amino acid sequence is as shown in SEQ ID NO: 2, characterized in that , the primers include NFSAI-1～NFSAI-19, and the specific primer sequences are as follows: NFSAI-1 GGAGGATCCTCCATGATCCACAACAAGACTATTGATGCTCAACTCAATCACAGATCCATC NFSAI-2 CAAGTTGTTCTTTGTTCAAGGTGATGTCCTTGAACTTTCTGATGGATCTGTGATTGAGTT NFSAI-3 CTTGAACAAAGAACAACTTGAGACCTTGTACTCTGTCTTCGCTCAGACTCCAACCTCTAT NFSAI-4 TGGATCGATGATGTGAACCAAGGAAGCGTTCTGCATGAACATAGAGGTTGGAGTCTGAGC NFSAI-5 TGGTTCACATCATCGATCCAGAGCAGAAGAAGAAGATCAGAGAACTCTGCAACCAGAGAGT NFSAI-6 TCAACGACGAAGATGAAGAGATCACCTTCAGCACCGACATACTTCTGGTTGCAGAGTTCT NFSAI-7 CTCTTCATCTTCGTCGTTGACTTGTACAGAAACCAACAGATCAGAAAGCAACTTGGTAAG NFSAI-8 CTTGGAAGAAGATGTCGATGGTGTGAACTCTGCCATCATCCTTACCAAGTTGCTTTCTGA NFSAI-9 CATCGACATCTTCTTCCAAGCTATGGAAGACACCCTGCTTGCTTACCAGAACGTTGCCAA NFSAI-10 ACCAAGTGGAACGTAACCCAGATCCATAGATTCGACAGCATTGGCAACGTTCTGGTAAGC NFSAI-11 TGGGTTACGTTCCACTTGGTACTGTCAACGATCATCCACTTGAGATGCTCAAGACCCTTG NFSAI-12 ACTTGCATACCAAGGACTGGAAAGGTCAGTTCTGGCAGGTCAAGGGTCTTGAGCATCTCA NFSAI-13 CCAGTCCTTGGTATGCAAGTTGGTGTTCCAGACCAGAAACCACAACTGAAGCCAAGACTT NFSAI-14 CCTTGTCATACTTACCTTCAAAGCAGATGAACTTGAGTGGAAGTCTTGGCTTCAGTTGTG NFSAI-15 TGAAGGTAAGTATGACAAGGACTTCAACGTCAAGGACCTGAAGGACTACGATCAAGTCGT NFSAI-16 GATTCTTCTGTTGGAGTCTCTCAGGTCGTAGTAGGTGGTGACGACTTGATCGTAGTCCTT NFSAI-17 GAGACTCCAACAGAAGAATCGACTCCTTCACCAAGCAGATCACTGGTGCTAAGCTCGACA NFSAI-18 TGAAGTTCCCTCTGGAAGTTCGTCTCTGTCAGTCTCATGGTTGTCGAGCTTAGCACCAGTG NFSAI-19 GAACTTCCAGAGGAACTTCACAAGCAAGGCTTGTGCTTGGACTGGAAATAAGAGCTCGAGCTC.