OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation

doi:10.11924/j.issn.1000-6850.casb20191100881

Abstract

Abstract:

The objectives are to construct the overexpression vector with Flag tagged OsAAA₁, obtain positive transgenic plants and detect expression of OsAAA₁, in positive transgenic rice. The sequence of OsAAA₁, was amplified by polymerase chain reaction (PCR) using the cDNA template of Nip. The PCR product was cloned into Flag-tagged pU1301; after confirmation of PCR, enzyme digestion and sequencing, recombinant plasmid was transformed to Nip by agrobacterium; when positive transgenic plants were verified by molecular identification, the expression of OsAAA₁, was detected by Real-time PCR. The recombinant plasmid of OsAAA₁,-pU1301-Flag was successfully constructed; after genetic transformation, thirty-three plants were differentiated; twenty-six transgenic plants were verified to be positive by molecular identification; the Real-time PCR results indicated that the expression of OsAAA₁, was up-regulated to different degrees in twenty-three plants. Compared with the Nip, the expression of OsAAA₁, was over 30 times in eight positive transgenic plants, in five of which the expression was even over 40 times. The overexpression vector with Flag tagged OsAAA₁, was successfully constructed. Genetic transformation results showed that OsAAA₁, with Flag-tag could be integrated into the genomic DNA of Nip, which could increase the expression of OsAAA₁,. This study provides a basis for discovering functional proteins interacting with OsAAA₁, in rice by using the Flag tag.

Key words: rice, OsAAA₁ gene, pU1301-Flag vector, genetic transformation, molecular identification

CLC Number:

S511

Song Simin, Zheng Qiming, Li Shijiao, Deng Shiqi, Li Kun, Liu Xinqiong. OsAAA₁ Gene in Rice: Overexpression Vector Construction and Genetic Transformation[J]. Chinese Agricultural Science Bulletin, 2020, 36(12): 91-96.

Figures/Tables 8

References 21

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引物名称	序列(5’-3’)
OsAAA₁KpnIF	CGGGGTACCATGGAGGCGACGTCGTCGTCGTCGT
OsAAA₁BamHIR	CTTGGATCCCTTATCCTTCCCGACCACTTCTACATC
UbiF	TTGTCGATGCTCACCCTGTT
actinF	CTCAACCCCAAGGCTAACAG
actinR	ACCTCAGGGCATCGGAAC
eEF1aQRTF	TTTCACTCTTGGTGTGAAGCAGAT
eEF1aQRTR	GACTTCCTTCACGATTTCATCGTAA
OsAAA₁QRTF	GGCCAAGACATACCTCGACGT
OsAAA₁QRTR	GCTCTTGGGCGTCAGGTTCT

引物名称	序列(5’-3’)
OsAAA₁KpnIF	CGGGGTACCATGGAGGCGACGTCGTCGTCGTCGT
OsAAA₁BamHIR	CTTGGATCCCTTATCCTTCCCGACCACTTCTACATC
UbiF	TTGTCGATGCTCACCCTGTT
actinF	CTCAACCCCAAGGCTAACAG
actinR	ACCTCAGGGCATCGGAAC
eEF1aQRTF	TTTCACTCTTGGTGTGAAGCAGAT
eEF1aQRTR	GACTTCCTTCACGATTTCATCGTAA
OsAAA₁QRTF	GGCCAAGACATACCTCGACGT
OsAAA₁QRTR	GCTCTTGGGCGTCAGGTTCT

OsAAA₁ Gene in Rice: Overexpression Vector Construction and Genetic Transformation

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