转HAL1基因大豆侧翼序列分析及特异性检测方法研究  

The T-DNA Flanking Sequence Analysis and Specific Detection Method of Transgenic Soybean with Gene HAL1

作　　者：蔡勤安[1] 尚丽霞[1] 姜志磊[1] 于志晶[1] 马瑞[1] 

机构地区：[1]吉林省农业科学院农业生物技术研究所,吉林长春130033

出　　处：《大豆科学》2018年第1期32-38,共7页Soybean Science

基　　金：农业部转基因生物新品种培育科技重大专项(2014zx08004-002-001)

摘　　要：为方便HAL1转基因大豆的研究和检测,以转HAL1基因大豆T3代基因组DNA为模板,使用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列,获得了插入片段DNA序列(T-DNA)的左翼序列和右翼序列。通过比对大豆基因组序列确定其整合位点,确定了HAL1基因的T-DNA在大豆基因组1号染色体非编码区49468395位点以单拷贝插入,转基因事件不影响大豆基因组功能基因的正常表达,而且在200 mmol·L^(-1)NaCl盐胁迫下转基因植株生长力明显强于对照材料。蛋白定量检测结果表明,在叶片中蛋白含量最高可达0.03 mg·g^(-1),在根茎花中也有表达,但是含量较低。根据整合位点处的左右侧翼序列设计两对特异性PCR检测引物,PCR检测结果显示只有转HAL1基因大豆阳性材料才能扩增出926和816 bp DNA片段。本研究建立的转HAL1基因大豆特异性定性检测方法,对转基因大豆的研究具有重要的意义,也为大豆转基因受体材料的管理与检测提供参考。For the convenience of research and testing of HAL1 trasformed soybeans, thermal asymmetric interlaced PCR （TAIL-PCR） method were used to separate the T-DNA flanking sequence, with the T3 genomie DNA of HAL1 transgentic soy- bean as the template, by comparing the soybean genomie data, the T-DNA was inserted in soybean genome chromosome 1 non-coding region 49468395 loci as single copy. In order to ensure that the transgenic events do not affect the normal expression of the functional genes of the soybean genome. Under the threaten of 200 mmol. L - ＇ NaC1, the growth force of transgenic plant was significantly stronger than that of the control materials, protein quantitative test results showed protein in leaf reached to 0. 03 mg＂ g- 1, protein content in root, stem and flower was lower. Two pairs of specific detection primers were designed based on the two flanking sequences and the insertion of the transgenic plant, and two DNA fragments of 926 and 816 bp were acquired by PCR. In this study, the established T-DNA flanking sequences analysis will provide effective method in transgenie soybean detection and management.

关 键 词：大豆 转基因 侧翼序列分析 

分 类 号：S565.1[农业科学—作物学]