毛果杨PGR5基因转南林895杨的分子鉴定及表达量1）

毛果杨PGR5基因转南林895杨的分子鉴定及表达量1）

王丽娜;李开隆

【摘要】We introduced PGR5 gene from Populus trichocarpa to

Populus×euramericana cv. ‘Nanlin895 by agrobacterium medi⁃ated method, screened these transgenic plants by hygromycin resistance gene, and took the molecular detection and expres⁃sion analysis of these transgenic plants. With the data of PCR detection, 12 of 23 clones were positives, and the gene was integrated into the genome of poplars. By

RT⁃PCR detection, 12 clones had positive bands which were consistent

with the anticipated objective strap size, and the gene was expressed at

the transcripional level. By real time⁃PCR detection, the ex⁃pression of the 12 positive clones were 220 times as much as wild types.%采用农杆菌介导法将毛果杨（ Populus trichocarpa） PGR5基因转化南林895杨，经潮霉素抗性

基因筛选并对转基因植株进行了分子检测和表达量分析。普通PCR检测结果显示，有12株检测到阳性信号，表明该基因已整合到南林895杨基因组中；经RT－PCR检测，该12株转化子在目的基因处有阳性条带，表明该基因在转录水平表达；实时荧光定量PCR检测结果显示，12株阳性苗的表达量为野生型的2～20倍。【期刊名称】《东北林业大学学报》

【年(卷),期】2015(000)011

【总页数】3页(P6-8)

【关键词】PGR5;分子检测;实时荧光定量PCR;转基因杨树

【作者】王丽娜;李开隆

【作者单位】林木遗传育种国家级重点实验室东北林业大学，哈尔滨，150040;

林木遗传育种国家级重点实验室东北林业大学，哈尔滨，150040

【正文语种】中文

【中图分类】S792.11;Q78

We introduced PGR5 gene from Populus trichocarpa to

Populus×euramericana cv. ‘Nanlin895 by agrobacterium mediated method, screened these transgenic plants by hygromycin resistance gene, and took the molecular detection and expression analysis of these transgenic plants. With the data of PCR detection, 12 of 23 clones were positives, and the gene was integrated into the genome of poplars. By RT-PCR detection, 12 clones had positive bands which were consistent with

the anticipated objective strap size, and the gene was expressed at the transcripional level. By real time-PCR detection, the expression of the 12 positive clones were 220 times as much as wild types.

目前杨树转基因的研究主要集中在抗虫、抗除草剂、抗病、降低木质素、抗环境污染以及非生物胁迫等[1-3]，对光合作用环式电子传递链中的载体蛋白研究的较少，尤其是将光合与抗逆结合进行木本植物转基因的研究更少[4]。PGR5在C3植物杨树中的遗传转化是其功能性研究的重要方法，对PGR5转基因植株的分子鉴定和

表达量研究能有效地证明外源基因在转基因植株中的阳性转化效率及基因表达情况，是功能试验的关键环节[5]。笔者经过近一年的遗传转化获得了23个转基因抗性株系，对这些株系进行PCR、RT-PCR、qRT-PCR检测，获得12个在转录水平高表

达的转基因株系。

1.1 材料

毛果杨(Populus trichocarpa)组培苗由中国科学研究院植物研究所光合中心王佰臣教授提供。转基因所用材料为南林895杨

(Populus×euramericana‘Nanlin895’)组培苗，由上海植物生理生化研究所李莱庚教授提供。

大肠杆菌DH5a为东北林业大学林木遗传育种国家级重点实验室保存，pCAMBIA1300原始质粒、GV3101感受态菌株为中国科学研究院植物研究所王佰臣教授提供，Silica Bead DNA Gel Extraction Kit#K0513购于北京Thermo 公司，限制性内切酶购于北京NEB公司，高保真酶KOD-Plus-Neo购于TOYOBO公司，RNA提取试剂盒RNAprep Pure Plant Kit(DP441)购于TIANGEN公司，反转录试剂盒购于Invitrogen公司，克隆载体使用的北京全式金公司的pEASY-Blunt Simple Cloing Kit，TRANS2KTM DNA Marker购于全式金公司。

1.2 方法

1.2.1 普通PCR检测PGR5基因

对23个抗性株系提取总DNA，设计特异引物进行PCR扩增。PCR反应扩增条件为：94 ℃预变性5 min，94 ℃、1 min，60 ℃、1 min，72 ℃、1 min，35个循环，最后72 ℃延伸10 min，PGR5基因产物大小为1 000 bp，反应产物用1.2%的琼脂糖电泳进行检测[6-8]。

采用天根专门针对杨树多糖多酚特点的RNA提取试剂盒(RNAprep Pure Plant Kit)提取南林895杨总RNA，利用Invitrogen的反转录试剂盒(C28025-032)反转cDNA，采用特异性引物进行PCR扩增[9]。

1.2.2 实时荧光定量PCR检测