Callery pear ascorbate peroxidase gene and use thereof in resisting heavy metal stress

[0027] Example 1: Isolation, sequence analysis and expression characteristics of ascorbate peroxidase gene from pear pear.

[0028] After 100μmol·L -1 CdCl 2 2.5H 2 Total RNA was extracted from leaves of 9-month-old pear seedlings treated with O for 24 hours using RNA plant plus Reagent from Tiangen Biochemical (Beijing) Technology Co., Ltd., and the first strand of cDNA was synthesized according to the instructions of PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Company). Using the Chinese white pear (Pyrus×bretschneideri) cytoplasmic ascorbate peroxidase gene (XM_018647890) as an electronic probe, searched the soybean pear transcriptome database constructed earlier in our laboratory, obtained the transcript Pbr03394 as a candidate gene, and designed the primer Pc .APX-S: 5'-ATGGGGAAGTGCTACCCTA-CCGTG-3' and Pc.APX-F: 5'-TTAGGCCTCAGCAAACCCAAGCTC-3', amplified the cDNA sequence of the Pc.APX gene. The DNA extraction of soybean pear leaves used the plant genome DNA extra...

[0033] Example 2: Prokaryotic expression test.

[0034] Extracted by 100μmol·L -1 CdCl 2 2.5H 2 O treatment of 24h soybean pear 9-month-old seedling leaf total RNA, reverse transcription synthetic cDNA first strand (concrete operation process is the same as embodiment 1), adopts primer Pc.APX-YHBD-S1 (5'-CCG GAATTC ATGGGGAAGTGCTACCCTACC-3', the underlined part is the EcoRI restriction site, the italic part is the protected base) and Pc.APX-YHBD-F1 (5'-ATAAGAAT GCGGCCGC GGCCTCAGC AAACCCAAGCTC-3', the underlined part is the NotⅠ restriction site, and the italicized part is the protected base) to amplify the cDNA of the leaf of Pear pear, and obtain the coding region of the cDNA sequence of the Pc.APX gene (excluding the stop codon), After the PCR product was recovered, ligated and sequenced (the specific operation procedure was the same as in Example 1), the recombinant plasmid containing the Pc.APX gene was named pClone007-Pc.APX. The pClone007-Pc.APX recombinant plasmid ...

[0038] Example 3: Transgenic experiment.

[0039] Construction of plant expression vectors:

[0040] Design Pc.APX gene specific primer Pc.APX-ZWBD-S: (The underlined part is the Nde I restriction site, and the italic part is the protected base) and Pc.A PX-ZWBD-F: Extract total RNA from soybean pear leaves, synthesize the first-strand cDNA by reverse transcription, amplify the Pc.APX gene with a high-fidelity enzyme, and connect it to the pCl one007BluntVector vector, transform it into Escherichia coli DH5α and sequence it (the specific operation process is the same as in Example 1 ).

[0041] Extract and purify the recombinant plasmid and digest it with Nde I, recover and purify the Pc.APX gene, and simultaneously digest the plant expression vector pRI201-AN-GUS (TaKaRa company) with Nde I, recover the linear plant expression vector pRI201-AN-GUS, and The Pc.APX gene was connected with the linear plant expression vector pRI201-AN-GUS, and transformed into Escherichia ...