Molecular Characterization and Inheritance Stability Analysis of Herbicide-resistant Cotton GV-2

doi:10.13304/j.nykjdb.2021.0581

Abstract

Abstract:

Molecular characterization and inheritance stability are important data for safety evaluation of genetic modified crop in China. The whole genome sequencing was used to analyze the T-DNA insertion site， copy number and flanking sequence of the herbicide-resistant transgenic cotton GV-2. The stability of target gene in three successive generations （T₃~T₅） GV-2 were verified by qRT-PCR （quantitative real time polymerase chain reaction）， Southern hybridization， colloidal gold dipstick and ELISA （enzyme linked immunosorbent assay）. The results showed that the T-DNA in GV-2 was inserted in the form of single copy， and the insertion site was located in 2 092 124~2 092 194 bp on chromosome A06 of upland cotton， resulting in the deletion of 70 bp DNA in the cotton genome. The insertion site and flanking sequence were further confirmed by the specific PCR of the transformant and Sanger sequencing. In addition， the target gene and its expressed protein， as well as the target character could be inherited stably in different generations of transformants. The results provided effective support for safety evaluation of cotton GV-2 transformant.

Key words: transgenic cotton, herbicide resistance, G10evo, molecular characterization, inheritance stability

摘要：

分子特征和遗传稳定性是我国转基因植物安全评价流程所需考察的重要数据。利用基因组测序技术对抗除草剂棉花GV-2的T-DNA插入位点、拷贝数及侧翼序列进行解析，并采用实时荧光定量PCR（quantitative real time polymerase chain reaction，qRT-PCR）、Southern杂交、胶体金试纸及ELISA（enzyme linked immunosorbent assay）方法对连续3代（T₃~T₅）GV-2转化体中目的基因的表达的稳定性进行验证。结果表明，转化体GV-2中T-DNA以单拷贝形式插入到陆地棉A06染色体2 092 124~2 092 194 bp之间，造成棉花基因组中70 bp DNA的缺失。转化体特异性PCR及Sanger测序结果进一步验证了插入位点的正确性。此外，目的基因及其表达蛋白在不同世代转化体中均可稳定遗传，为转基因棉花GV-2转化体的安全评价提供了有效的支撑。

关键词: 转基因棉花, 抗除草剂, G10evo, 分子特征, 遗传稳定性

CLC Number:

S562

Guoqing LU, Caixia MA, Guoqing SUN, Huiming GUO, Hongmei CHENG. Molecular Characterization and Inheritance Stability Analysis of Herbicide-resistant Cotton GV-2[J]. Journal of Agricultural Science and Technology, 2023, 25(1): 42-49.

陆国清, 马彩霞, 孙国清, 郭惠明, 程红梅. 抗除草剂棉花GV-2的分子特征和遗传稳定性分析[J]. 中国农业科技导报, 2023, 25(1): 42-49.

Figures/Tables 6

Table 1 Prime sequences in this study

引物名称 Prime name	引物序列 Prime sequences（5’-3’）	扩增产物大小 Size of amplification product/bp
LF	TTAAGTCGCGGATCGTATTGT	742
LR	TACCCTCAGTTCTTCGCTCA	742
RF	GCTTCCTCGTGCTTTACGGT	673
RR	TTGGCATGGGATGTTTCCGT	673

Fig. 1 Insertion site analysis of GV-2A： Genome sequencing and insertion site of GV-2； B： Specific PCR validation of the insertion site； C： Verification by Sanger sequencing of the specific PCR amplification products of transformant

Fig. 2 Relative expression level of target gene in different generations

Fig. 3 Integrative stability analysis of target geneA： Composition of each element and the relative position of probe in T-DNA； B： Southern hybridization analysis of G10evo； C： Southern hybridization analysis of nptⅡ； the blue bar represents the relative position of nptⅡ probe； the orange bar represents the relative position of G10evo probe； M—Nucleic acid maker； 1 and 5—R15 is digested by Hind Ⅲ and SpeⅠrespectively； 2~4—T3~T5 generation of GV-2 is digested by Hind Ⅲ； 6~8—T3~T5 generation of GV-2 is digested by SpeⅠ； 9—Mixtures of R15 and plasmid digested with Hind Ⅲ.

Fig. 4 Qualitative and quantitative analysis of target proteinA： Qualitative analysis of target protein by the colloidal gold strip； B： Standard curve for quantitative ELISA detection； C： Quantitative analysis of target protein by ELISA

Fig. 5 Test of glyphosate resistance

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