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

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

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