doi:

DOI: 10.3724/SP.J.1006.2019.84118

Acta Agronomica Sinica (作物学报) 2019/45:5 PP.683-692

Creation and analysis of marker free transgenic soybean germplasm with low phosphate tolerance transcription factor GmPTF1 based on Cre/loxP system


Abstract:
The screening marker genes in transgenic crops have potential safety risks, which are necessarily eliminated in the improvement of transgenic plants. The transcription factor PTF1 has the effect improving phosphorus uptake by plants under low phosphorus stress. So Cre/loxP-GmPTF1 was transferred into soybean cultivar Yudou 22[Glycine max (L.) Merr.] via Agrobacterium-mediated cotyledonary node transformation method. And then, the marker-free transgenic soybean with GmPTF1 was obtained by means of induced Cre/loxP site specific recombination with β-estradiol. After the marker gene was deleted, the recombinant sequences were amplified by PCR and then sequenced. The sequencing results indicate that the marker genes were completely deleted out of the transgenic soybean genome. The recombinant maintained with correct ORF correct target gene sequence, and a new splicing type appeared in loxP recombination. Two loxP sequences were missing in the recombination. The length of the new recombinant in splicing site was 38 bp which was no homology with other sequences in NCBI database. And the recombination was involved in the flank sequence outside the two loxP sites, which lead partially deleted in the outer flank sequences of Cre/loxP cassette. The results of RT-PCR and Western blot showed that GmPTF1 could be transcribed and translated normally in marker-free transgenic soybean plants. The expression level of GmPTF1 in roots, stem and leaves was higher than that in wild type, which was not significantly different in seeds. in the sand culture experiment under low phosphorus condition, the root indexes, dry biomass, chlorophyll content and phosphorus content in marker-free transgenic soybean were significantly higher than there in the wild type, while the MDA content was lower than that in the control. We conclude that screening marker genes in transgenic soybean could be effectively deleted by Cre/loxP recombinant system.

Key words:Cre/loxP,GmPTF1,marker-free,transgenic soybean

ReleaseDate:2019-11-05 15:30:00



[1] Hinchee M A W, Connorward D V, Newell C A, McDonnell R E, Sato S J, Gasser C S, Fischhoff D A, Re D B, Fraley R T, Horsch R B. Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Nat Biotechnol, 1988, 6:915-922.

[2] 杨乐坤. 中国转基因农产品的标识规制策略. 南京大学硕士学位论文, 江苏南京, 2016. Yang L K. China's Strategic Labeling of GM Food Based on Two-stage Dynamic Game Model. MS Thesis of Nanjing University, Nanjing, Jiangsu, China, 2016(in Chinese with English abstract).

[3] 崔宁波, 宋秀娟. 国外转基因大豆种植与育种研究进展国外转基因大豆种植与育种研究进展. 东北农业大学学报, 2015, 46(8):103-108. Cui N B, Song X J. Research progress on development and planting of foreign genetically. J Northeast Agric Univ, 2015, 46(8):103-108(in Chinese with English abstract).

[4] 陈晓亚, 杨长青, 贾鹤鹏, 凌飞. 中国转基因作物面临的问题. 华中农业大学学报, 2014, 33(6):115-117. Chen X Y, Yang C Q, Jia H P, Ling F. Issues confronting GMO crops in China. J Huazhong Agric Univ, 2014, 33(6):115-117(in Chinese with English abstract).

[5] 石红璆, 查代明. 转基因食品的道德风险及其解决对策. 安徽农业科学, 2018, 46(8):7-9. Shi H Q, Zha D M. Moral risks of genetically modified foods and their countermeasures. J Anhui Agric Sci, 2018, 46(8):7-9(in Chinese with English abstract).

[6] 黄大昉. 我国转基因作物育种发展回顾与思考. 生物工程学报, 2015, 31:892-900. Huang D F. Review of transgenic crop breeding in China. Chin J Biotechnol, 2015, 31:892-900(in Chinese with English abstract).

[7] 逄金辉, 马彩云, 封勇丽, 胡瑞法. 转基因作物生物安全:科学证据. 中国生物工程杂志, 2016, 36(1):122-138. Peng J H, Ma C Y, Feng Y L, Hu R F. Biosafety of genetically modified crops:scientific evidence. Chin Biotechnol, 2016, 36(1):122-138(in Chinese with English abstract).

[8] 祁永斌, 刘庆龙, 陆艳婷, 金庆生. 转基因植物中删除选择标记基因的研究进展. 浙江农业学报, 2014, 26:1387-1393. Qi Y B, Liu Q L, Lu Y T, Jin Q S. Research progress of the selectable marker genes eliminated in the transgenic plants. Acta Agric Zhejiangensis, 2014, 26:1387-1393(in Chinese with English abstract).

[9] Guo F, Gopaul D N, Van Duyne G D. Structure of Cer recombinase complexes with DNA in a site-specific recombination synapse. Nature, 1997, 389:40-46.

[10] Dale E C, Ow D W. Intra-and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene, 1990, 91:79-85.

[11] Ma B G, Duan X Y, Niu J X, Ma C, Hao Q N, Zhang L X, Zhang H P. Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker. Biotechnol Lett, 2009, 31:163-169.

[12] Zuo J, Niu Q, Moller S, Chua N H. Chemical-regulated, site-specific DNA excision in transgenic plants. Nat Biotechnol, 2001, 19:157-161

[13] 李喜焕, 常文锁, 张彩英. 提高植物磷营养效率(候选)基因研究进展. 植物遗传资源学报, 2012, 13:83-97. Li X H, Chang W S, Zhang C Y. Research progress of candidate genes for improving plant phosphorus-effiency. J Plant Genet Resour, 2012, 13:83-97(in Chinese with English abstract).

[14] 张全琪, 朱家红, 倪燕妹, 张治礼. 植物bHLH转录因子的结构特点及其生物学功能. 热带亚热带植物学报, 2011, 19:84-90. Zhang Q Q, Zhu J H, Ni Y M, Zhang Z L. The structure and function of plant bHLH transcription factors. J Trop Subtrop Bot, 2011, 19:84-90(in Chinese with English abstract).

[15] 杨致荣, 毛雪, 李润植. 植物次生代谢基因工程研究进展. 植物生理与分子生物学学报, 2005, 31:11-18. Yang Z R, Mao X, Li R Z. Progress in studies on genetic engineering of secondary metabolites in plant. J Plant Physiol Mol Biol, 2005, 31:11-18(in Chinese with English abstract).

[16] 吴冰. 大豆耐低磷转录因子GmPTF1与GmPHR1功能分析. 河北农业大学硕士学位论文, 河北保定, 2013. Wu B. Functional Analysis of Transcription Factor GmPTF1 and GmPHR1 Responsed to Low Phosphorus in Soybean. MS Thesis of Agricultural University of Hebei, Baoding, Hebei, China, 2013(in Chinese with English abstract).

[17] 李桂兰, 刘晨光, 乔潇, 杨晓倩, 王迪, 乔亚科. 共培养条件对农杆菌转化大豆子叶节的影响. 核农学报, 2014, 28:1567-1575. Li G L, Liu C G, Qiao X, Yang X Q, Wang D, Qiao Y K. Conditions of co-culture affecting on the efficiency of Agrobacterium-mediated transformation of cotyledonary node of soybean. J Nucl Agric Sci, 2014, 28:1567-1575(in Chinese with English abstract).

[18] 李桂兰, 乔亚科, 杨少辉, 靳朝霞, 李明刚. 农杆菌介导大豆子叶节遗传转化的研究. 作物学报, 2005, 31:170-176. Li G L, Qiao Y K, Yang S H, Jin Z X, Li M G. Study of the Agrobacterium-mediated transformation systems of soybean cotyledonary node. Acta Agron Sin, 2005, 31:170-176(in Chinese with English abstract).

[19] 王关林, 方宏筠. 植物基因工程原理与技术. 北京:科学出版社, 1998. pp 634-636. Wang G L, Fang H J. Plant Gene Engineering Principle and Technique. Beijing:Science Press, 1998. pp 634-636(in Chinese).

[20] 龙定沛, 谭兵, 赵爱春, 许龙霞, 向仲怀. Cre/lox位点特异性重组系统在高等真核生物中的研究进展. 遗传, 2012, 34:177-189. Long D P, Tan B, Zhao A C, Xu L X, Xiang Z H. Progress in Cre/lox site-specific recombination system in higher eu-karyotes. Hereditas, 2012, 34:177-189(in Chinese with English abstract).

[21] Cheng Y A, Jee J, Hsu G, Huang Y Y, Chen C, Lin C P. A markerless protocol for genetic analysis of Aggregatibacter actinomycetemcomitans. J Formosan Med Assoc, 2014, 113:114-123.

[22] Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z. Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep, 2005, 24:86-94.

[23] Togawa Y, Nunoshiba T, Hiratsu K. Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus. Mol Genet Genom, 2018, 293:277-291.

[24] Zhang Y, Li H, Ouyang B, Ye Z. Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects. Biotechnol Lett, 2006, 28:1247-1253.

[25] Li Z, Xing A, Moon B P, Burgoyne S A, Guida A D, Liang H, Lee C, Caster C S, Barton J E, Klein T M, Falco S C. A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants. Plant Mol Biol, 2007, 65:329-341.

[26] 张彦丽, 贾健辉, 赵月琪, 谷思玉, 许景钢. 大豆苗期耐低磷筛选指标的研究. 安徽农业科学, 2010, 38:5506-5507. Zhang Y L, Jia J H, Zhao Y Q, Gu S Y, Xu J G. Screening index of soybean for low phosphorus tolerance at seedling stage. J Anhui Agric Sci, 2010, 38:5506-5507(in Chinese with English abstract).

[27] 梁泉, 尹元萍, 严小龙, 廖红. 不同磷水平下大豆根系性状的遗传特性分析. 分子植物育种, 2009, 7:321-329. Liang Q, Yin Y P, Yan X L, Liao H. Genetic analysis of root characters in soybean using a recombinant inbred line population at two phosphorus levels. Mol Plant Breed, 2009, 7:321-329(in Chinese with English abstract).

[28] 敖雪, 谢甫绨, 刘婧琦, 张惠君. 不同磷效率大豆品种光合特性的比较. 作物学报, 2009, 35:522-529. Ao X, Xie F T, Liu J Q, Zhang H J. Comparison of photosynthetic characteristics in soybean cultivars with different phosphorus efficiencies. Acta Agron Sin, 2009, 35:522-529(in Chinese with English abstract).

[29] 李俊, 张春雷, 秦岭, 马霓, 李峰. 不同磷效率基因型油菜对低磷胁迫的生理响应. 中国油料作物学报, 2010, 32:222-228. Li J, Zhang C L, Qin L, Ma N, Li F. Physiological response to low phosphorus stress for different P-efficiency genotype of rapeseed. Chin J Oil Crop Sci, 2010, 32:222-228(in Chinese with English abstract).

[30] 王晶, 韩晓日, 站秀梅, 侯玉慧, 赵伟力. 低磷胁迫对番茄叶片脂膜过氧化及保护酶活性的影响. 植物营养与肥料学报, 2005, 11:851-854. Wang J, Han X R, Zhan X M, Hou Y H, Zhao W L. Influence of low-phosphorus stress on membrane lipid peroxidation and protective enzyme activities in tomato leaves. Plant Nutr Fert Sci, 2005, 11:851-854(in Chinese with English abstract).

[31] 钟磊, 乔亚科, 乔潇, 李桂兰, 王林红, 刘晨光. 转GmPTF1基因大豆在低磷胁迫下的表现. 核农学报, 2013, 27:1041-1047. Zhong L, Qiao Y K, Qiao X, Li G L, Wang L H, Liu C G. Performance of transgenic soybean with GmPTF1 gene under low phosphorus stress. J Nucl Agric Sci, 2013, 27:1041-1047(in Chinese with English abstract).