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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (5): 683-692.doi: 10.3724/SP.J.1006.2019.84118

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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

Xiao-Fang ZHANG1,Qiu-Ping DONG1,Xiao QIAO2,Ya-Ke QIAO1,*(),Bing-Bing WANG1,Kai ZHANG1,Gui-Lan LI1,*()   

  1. 1 College of Agronomy and Biotechnology, Hebei Normal University of Science and Technology, Changli 066600, Hebei, China
    2 Department of Physics, Hebei Normal University of Science and Technology, Qinhuangdao 066004, Hebei, China
  • Received:2018-08-27 Accepted:2018-12-24 Online:2019-05-12 Published:2019-01-16
  • Contact: Ya-Ke QIAO,Gui-Lan LI E-mail:qiaoyake@126.com;lgl63@126.com
  • Supported by:
    This study was supported by the National Transgenic Major Project of China(2014ZX0800404B);Modern Agricultural Industrial Technology System Hebei Province Innovation Team Construction Project(HBCT2018090203)

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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

Fig. 1

Insertion sequence of transgenic plant before (A) and after (B) deletion of marker genes and PCR identification diagram"

Fig. 2

PCR detection of transgenic plants prior to deletion of markers 1: 100 bp DNA marker ladder; 2: blank control; 3: wild type control; 4, 5: PCR products of P1/P2; 6, 7: nptII PCR products of P3/P4; 8, 9: GmPTF1 gene PCR products of P5/P6."

Fig. 3

PCR detection of transgenic plants after deletion of markers 1, 12: 100 bp DNA marker ladder; 2: blank control; 3: wild type control; 4, 5: PCR products of P1/P2; 6, 7: nptII PCR products of P3/P4; 8, 9: GmPTF1 gene PCR products of P5/P6; 10, 11: PCR product of P1/P6, recombinant DNA fragment after deletion of markers."

Fig. 4

Analysis of recombinant sequence PCR amplification product of P1/P6 sequencing results after deletion of marker genes The shaded parts were sequences of P1 and P6 primer, and the recombination splice sites were in the frame."

Fig. 5

Southern blot figure of marker free transgenic soybean of T5 generation 1: Southern blot of transgenic soybean digested by Xba I; 2: Southern blot of transgenic soybean digested by Kpn I; WT: Southern blot of wild type controls digested by Xba I; M: λDNA/Hind III marker."

Fig. 6

GmPTF1 RT-PCR of marker free transgenic soybean H: blank control; WT: wild type control; 1-7: transgenic soybean; P: plasmid positive control; M: 100 bp DNA marker ladder."

Fig. 7

Analysis of GmPTF1 transcription level in marker-free transgenic soybeans 1: wild type controls; 2: transgenic soybean plants."

Fig. 8

Western blot results of GmPTF1 in different organs of marker free transgenic soybean 1: wild type controls; 2: transgenic plants; A: GmPTF1 protein; B: Actin protein."

Fig. 9

Relative expression of GmPTF1 protein in different organs of marker free transgenic soybean 1: wild type controls; 2: transgenic soybean plants; A: root; B: leaf, C: stem; D: seed."

Table 1

Root character of different soybean seedlings under different phosphorus concentrations"

材料
Material		 根总长
Root length (cm)		 根表面积
Root surface area (cm2)		 根平均直径
Root average diameter (mm)		 根体积
Root volume (cm3)
正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P
Transgenic line 2515±99 aA 2175±49 aA 146±4.8 aA 113±4.1 aA 0.58±0.04 aA 0.52±0.01 aA 6.67±0.71 aA 4.48±0.29 aA
WT 2269±14 bA 1632±29 bB 147±6.5 aA 85±2.2 bB 0.56±0.06 aA 0.48±0.01 bB 6.25±0.95 aA 3.51±0.15 bB

Table 2

Effects of low phosphorus stress on over ground parts of different soybean seedlings"

材料
Material		 株高 Plant height (cm) 第一展开叶片面积 The first developed blade area (cm2)
正常磷
Normal-P		 低磷
Low-P		 降幅
Drop (%)		 正常磷
Normal-P		 低磷
Normal-P		 降幅
Drop (%)
Transgenic line 30.3±0.6 aA 24.0±1.2 Aa 20.0 20.28±0.67 aA 19.69±1.51 aA 2.96
WT 37.7±1.5 aA 23.8±2.3 aA 36.9 21.61±1.86 aA 16.06±1.25 bA 25.7

Table 3

Effects of low P concentrations on dry matter accumulation in shoots and roots of transgenic soybean plants"

材料
Material		 根干重 Dry weights of roots 茎叶干重 Dry weights of stems and leaves
正常磷
Normal-P		 低磷
Low-P		 降幅
Drop (%)		 正常磷
Normal-P		 低磷
Normal-P		 降幅
Drop (%)
Transgenic line 0.88±0.13 aA 0.61±0.04 aA 30.7 2.81±0.1 aA 1.38±0.06 aA 51
WT 0.85±0.04 aA 0.38±0.06 bB 55.3 2.6±0.25 aA 0.85±0.05 bB 68

Fig. 10

Contents of MDA and photosynthetic pigment in soybean leaves"

Table 4

Amplitude of MDA and photosynthetic pigments in soybean leaves"

材料
Material		 光合色素降幅
Drop of photosynthetic pigments (%)		 丙二醛增幅
Increasing range of MDA (%)
Transgenic line 20.81 ± 0.03 bB 29.43 ± 3.13 bB
WT 49.76 ± 0.02 aA 64.57 ± 2.09 aA

Fig. 11

P content of soybean leaves under different phosphorus concentration treatments 1: wild-type control; 2: transgenic line."

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