Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants

[0018] DETAILED DESCRIPTION: The present invention will be further described in detail below in conjunction with the accompanying drawings:

[0019] The methods used in the following examples are conventional methods unless otherwise specified.

[0020] experiment material

[0021] 1. Strains and vectors: Escherichia coli strain DHα and Agrobacterium strain EHA105 were purchased from Huamei Biological Company; expression vector pBI121 was purchased from Clontech Company.

[0022] 2. Main reagent: SMART TM PCR cDNA Synthesis Kit and Clontech PCR-Select cDNA Subtraction Kit were purchased from Clontech; Taq DNA polymerase was purchased from Beijing Quanshijin Company; DNA standard molecular weight was purchased from Takara; restriction endonuclease, T4DNA ligase, M-MLV reverse The transcriptase was purchased from Promega; the RNeasy Plant Mini Kit was purchased from Qiagen; other chemical reagents were all domestically pure.

[0023] 3. The primer sequence designed by the present invention:

[0024] The primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd., and the sequence is as follows:

[0025] IbMT-BamHI: 5′-AA GGATCC ATGTCTTCCGGTTGCAAG-3′ (Introduction of BamH I restriction site)

[0026] IbMT-Sac I: 5′-AT GAGCTC CTAACAGTTGCAAGGGTC-3′ (Introduction of Sac I restriction site)

[0027] 4. The culture medium used in the present invention:

[0028] LB medium: each liter contains 10g tryptone, 5g yeast extract, 10g NaCl, adjust the pH to 7.0 with NaOH, and sterilize for use. Add 1.5% agar to make a solid medium.

[0029] YEB medium: each liter contains 5g tryptone, 1g yeast extract, 5g sucrose, MgSO 4 0.5g, adjust pH to 7.5 with NaOH, sterilize for use. Add 1.5% agar to make a solid medium.

[0030] MS medium: purchased from Sigma Company, added 8% agar to make a solid medium.

[0031] MS co-culture medium: 6-benzylaminopurine (6-BA, 2mg/L) and naphthaleneacetic acid (NAA, 0.2mg/L) were added to MS medium.

[0032] MS selection medium: Carbenicillin (250mg/L) and kanamycin (100mg/L) were added to MS co-culture medium.

[0033] MS rooting medium: Add NAA (0.3mg/L), carbenicillin (250mg/L) and kanamycin (50mg/L) to 1/2MS medium. 1/2MS medium means MS half.

[0034] 5. Plant material: sweet potato (Ipomoea batatas)

[0035] 6. Main equipment: room temperature centrifuge and refrigerated centrifuge are from Eppendorf; PCR machine and gel imager are purchased from Biorad.

[0036] Example 1: Obtaining the sweet potato metallothionein gene IbMT1

[0037] 1. Treatment of sweet potato seedlings: select the shoot tips or undifferentiated axillary buds of sweet potato plants, cut into sections and inoculate them on MS solid medium for culture, culture temperature is 27 ± 2 ℃, light is about 2000 Lux. When the sterile seedlings grow to a height of about 3 cm, the healthy sterile seedlings that grow vigorously are selected and transferred to MS solid medium containing 200 mM NaCl for inducing culture for 24 hours.

[0038] 2. Extraction of total RNA: Use RNeasy Plant Mini Kit (Qiagen Company) to extract the total RNA of NaCl-treated and untreated sweet potato seedlings, and refer to the kit instructions for specific operations.

[0039] 3. Construction of salt stress suppression subtractive library: 2μg of total RNA of NaCl-treated and untreated sweet potato seedlings was reverse-transcribed into cDNA to construct a salt stress suppression subtractive library of sweet potato. The specific operations were performed with reference to the instructions of the SMARTTM PCR cDNA Synthesis Kit (Clontech Company) and PCR-Select cDNA Subtraction Kit (Clontech Company) kits.

[0040] 4. Acquisition of IbMT1 gene of sweet potato metallothionein

[0041] The clones in the library were randomly selected and cultured overnight in LB liquid medium (containing 100 mg/L of ampicillin), and 1 mL of the overnight culture broth was taken to Beijing Huada Gene Research Center for sequencing. Use the VecScreen program in the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) to remove the vector sequence from the sequencing results, and then use the Blast program to remove the bases of the cloned fragments. The sequence was searched for sequence similarity in GenBank. Through the above screening and analysis, a 561bp cDNA sequence was obtained with a poly-A tail at the 3'end, containing a 201bp open reading frame, encoding 66 amino acid sequences, which are similar to those of metallothionein from other species published in the Gene Bank. The amino acids have high homology, indicating that the sweet potato metallothionein gene obtained after the above-mentioned screening step is named IbMT1. The nucleotide sequence of the IbMT1 gene and the encoded amino acid sequence are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0042] Example 2: Construction of PBI121-IbMT1 plant expression vector

[0043] 1. Amplification of the coding region of IbMT1cDNA: primers IbMT-BamH I and IbMT-Sac I were designed according to the sequences at both ends of the coding region of IbMT1 cDNA, and the bacterial liquid containing IbMT1 was used as a template for PCR amplification. Add sequentially to the 25μL reaction system: 10×buffer(Mg 2+ ) 2.5μL, dNTP (10mM) 1μL, each primer 1μL (10μM), template 1μL, Taq enzyme 0.2μL, the rest use ddH 2 O make up.

[0044] The PCR amplification conditions were: 94°C for 5 minutes after pre-denaturation, 94°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, a total of 35 cycles. The results of agarose gel electrophoresis showed that a specific band with a size of about 200 bp was amplified, which was sent to the Beijing Huada Gene Research Center to confirm that it was the full sequence of the IbMT1 cDNA coding region.

[0045] 2. Enzyme digestion and ligation: Combine the amplified IbMT1 fragment and PBI121 vector (see the vector map figure 1 ) Double digestion with BamH I (Takara company) and Sac I (Takara company). The double digestion reaction was performed according to Takara's instructions. The IbMT1 fragment and the PBI121 vector fragment were recovered and purified, and the ratio was 2-10:1 under the action of T4DNA ligase at 16°C overnight.

[0046] 3. Transformation: The preparation method of Escherichia coli DHα competent cells was carried out according to F. Osber et al. ("Compiled Molecular Biology Experiment Guide", 1998, Science Press). The ligation product was introduced into E. coli DHα competent cells by heat shock at 42°C, and resistant colonies were screened on LB plates containing kanamycin (50mg/L), plasmids were extracted, and the enzymes were digested for identification. The correct one is recombination Plasmid PBI121-IbMT1.