Construction of a chalcone reductase expression vector and transformation of soybean plants.
Construction of a chalcone reductase expression vector and transformation of soybean plants.
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查尔酮还原酶表达载体的构建和大豆植物的转化。
DOI：
10.3892/mmr.2017.7382
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发表时间：
2017
影响因子：
3.4
通讯作者：
J. Qu
中科院分区：
文献类型：
--
作者：
Nan Wu;Pi;Nan Lin;Shi Lu;Yong;Jie Rong;Zhuo Zhang;J. Qu
本研究旨在克隆大豆查尔酮还原酶3（CHR 3），构建含有Bar抗性基因作为选择标记的重组表达载体pCAMBIA 3300-CHR 3，然后利用农杆菌感染获得转基因大豆植株。将植物表达载体pCAMBIA 3300-CHR 3转入大豆受体植物吉农17和吉林30中。用PCR和Southern杂交对转基因植株进行了鉴定。此外，逆转录-定量PCR（RT-qPCR）用于检测CHR 3的表达，并使用高效液相色谱（HPLC）测定转基因后代中异甘草素的含量。将大豆CHR 3（932 bp片段）克隆到植物表达载体pCAMBIA 3300-CHR 3中，并将其转化到大豆受体植株中。T1代经PCR鉴定为阳性植株，其中8株为吉农17，5株为吉林30，Southern杂交证明pCAMBIA 3300-CHR 3载体的功能成分已整合到大豆基因组中; RT-qPCR结果表明，与非转基因大豆相比，转基因大豆中CHR 3 mRNA的表达量增加了2 - 20倍植物高效液相色谱检测结果表明，转基因吉农17的异甘草素含量比对照提高了8.56%。CHR 3基因可以产生大豆苷元的前体异甘草素，从而提高大豆抗疫霉根腐病的能力。
The present study aimed to clone the soybean chalcone reductase 3 (CHR3) and create a recombinant expression vector pCAMBIA3300‑CHR3 containing Bar resistance gene as a selection marker, and then obtain transgenic soybean plants using Agrobacterium infection. The plant expression vector pCAMBIA3300‑CHR3 was transferred into soybean receptor plants, Jinong 17 and Jilin 30. Polymerase chain reaction (PCR) and Southern blotting were used to confirm the positive transgenic plants. Additionally, reverse transcription‑quantitative PCR (RT‑qPCR) was used to detect CHR3 expression and isoliquiritigenin content was measured using high‑performance liquid chromatography (HPLC) in the transgenic offspring. Soybean CHR3 (932 bp fragment) was successfully cloned into the plant expression vector pCAMBIA3300‑CHR3, which was subsequently transferred into soybean receptor plants. In the T1 generation positive plants were validated by PCR analysis, including eight Jinong 17 and five Jilin 30 transgenic plants; Southern blotting demonstrated that the functional components of the pCAMBIA3300‑CHR3 vector had been integrated into the soybean genome; RT‑qPCR results demonstrated that the expression of CHR3 mRNA was increased by 2 to 20‑fold in the transgenic plants compared with the non‑transgenic soybean plants. Furthermore, the isoliquiritigenin content was increased by 8.56% in the transgenic Jinong 17, compared with control plants, as detected by HPLC. The CHR3 gene can produce isoliquiritigenin, a precursor of daidzein, which in turn can improve the ability of soybean to resist phytophthora root rot.
影响因子：
7.4
作者：
Shimada, N;Aoki, T;Ayabe, S
通讯作者：
Ayabe, S