水稻OsAAA1基因超表达载体构建及遗传转化

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

中国农学通报 ›› 2020, Vol. 36 ›› Issue (12): 91-96.doi: 10.11924/j.issn.1000-6850.casb20191100881

所属专题：生物技术；水稻

水稻OsAAA₁基因超表达载体构建及遗传转化

宋斯敏, 郑启明, 李世娇, 邓仕琪, 李琨, 刘新琼()

中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北省重点实验室/生物技术国家民委重点实验室,武汉 430074

收稿日期:2019-11-27 修回日期:2019-12-25 出版日期:2020-04-25 发布日期:2020-04-21
通讯作者: 刘新琼
作者简介:宋斯敏,女,1995年出生,湖北鄂州人,硕士研究生,研究方向：遗传学。通信地址：430074 湖北省武汉市中南民族大学生命科学学院,E-mail： 2504807696@qq.com
基金资助:
国家自然科学基金面上项目“水杨酸应答新基因OsAAA-ATPase-1在水稻广谱抗病反应中的分子机理研究”(31370306)

OsAAA₁ Gene in Rice: Overexpression Vector Construction and Genetic Transformation

Song Simin, Zheng Qiming, Li Shijiao, Deng Shiqi, Li Kun, Liu Xinqiong()

Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China/Key Lab for Biotechnology of State Ethnic Affairs Commission/College of Life Science, South-Central University for Nationalities, Wuhan 430074

Received:2019-11-27 Revised:2019-12-25 Online:2020-04-25 Published:2020-04-21
Contact: Xinqiong Liu

摘要/Abstract

摘要：

为了构建带Flag标签的OsAAA₁超表达载体,获得阳性转基因植株并分析其OsAAA₁基因表达情况。以日本晴的cDNA为模板,将OsAAA₁克隆至pU1301-Flag载体;重组质粒通过PCR、酶切及测序鉴定正确后,以农杆菌为介导进行遗传转化至日本晴;转基因植株通过分子鉴定正确后,采用Real-time PCR检测OsAAA₁基因的mRNA表达水平变化。成功构建了OsAAA₁-pU1301-Flag重组载体;遗传转化后获得33株转基因植株;通过分子鉴定筛选出26株阳性转基因植株;荧光定量PCR结果分析发现23株阳性转基因植株OsAAA₁基因的表达出现不同程度上调。与日本晴相比,有8株表达量达到30倍以上,其中有5株表达量超过40倍。成功构建了带Flag标签的OsAAA₁超表达载体;遗传转化结果表明OsAAA₁-Flag能整合到日本晴的基因组DNA中,从而使OsAAA₁基因的表达水平增加。本研究为后续通过Flag标签,在水稻植物体内直接挖掘与OsAAA₁互作的功能蛋白奠定了基础。

关键词: 水稻, OsAAA₁基因, pU1301-Flag载体, 遗传转化, 分子鉴定

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

中图分类号:

S511

宋斯敏, 郑启明, 李世娇, 邓仕琪, 李琨, 刘新琼. 水稻OsAAA₁基因超表达载体构建及遗传转化[J]. 中国农学通报, 2020, 36(12): 91-96.

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.

图/表 8

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

表1. 本研究所使用的引物序列

图1. OsAAA1目的片段扩增 M：DL2000;1~18：单克隆。

图2. OsAAA1-pU1301-Flag质粒PCR鉴定 M：DL2000;1~3：Nip的cDNA

图3. OsAAA1-pU1301-Flag的BamHI与KpnI双酶切鉴定。。 M：DL15000;1~18：单克隆;19：pU1301-Flag空载体

图4. 农杆菌介导的水稻遗传转化的各时期 A：愈伤组织诱导,B：共培养,C：愈伤组织筛选,D：愈伤组织分化,E：生根培养,F：幼苗移栽

图5. 转基因植株的T0代的分子鉴定 M：DL2000;1~22,25~35：转基因植株;23,36：OsAAA1-pU1301-Flag阳性质粒;24,37：Nip

图6. 阳性转基因植株的cDNA的质量检测 M：DL2000;1~23：阳性转基因植株的cDNA;24：Nip的DNA

图7. 阳性转基因植株的OsAAA1的表达量检测

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水稻OsAAA₁基因超表达载体构建及遗传转化

OsAAA₁ Gene in Rice: Overexpression Vector Construction and Genetic Transformation

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