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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 789-798.doi: 10.3724/SP.J.1006.2021.04169

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

Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance

LI Jie-Hua1,3(), DUAN Qun1,3(), SHI Ming-Tao1,3(), WU Lu-Mei1,3, LIU Han2, LIN Yong-Jun1, WU Gao-Bing3, FAN Chu-Chuan1,3,*(), ZHOU Yong-Ming1,3,*()   

  1. 1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    2Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China
    3College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-07-25 Accepted:2020-11-01 Online:2021-05-12 Published:2020-12-14
  • Contact: FAN Chu-Chuan,ZHOU Yong-Ming E-mail:1023629768@qq.com;554246233@qq.com;843121993@qq.com;fanchuchuan@mail.hzau.edu.cn;ymzhou@mail.hzau.edu.cn
  • Supported by:
    National Major Project for Developing New GM Crops(2018ZX08020001)

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Abstract:

Glyphosate is the most widely used broad-spectrum herbicide in the world. However, at present there is no glyphosate-tolerant rapeseed variety with independent intellectual property rights in China. In the study, a novel glyphosate-resistant genes I. variabilis EPSPS was transferred to the Brassica napus pure line J9707 via the Agrobacterium tumefaciens-mediated hypocotyl method, and 126 T0-positive transgenic plants with 97.0% positive rate were generated. The T-DNA insertion with a single copy (44.8%) is dominant. The insertion locations of T-DNA in the lines of EPS-2, EPS-6, and EPS-7 were identified by inverse PCR method. The stability of the T-DNA insertion in these lines were further confirmed by insertion-specific PCR in their T0 to T3 plants. The gene expression analysis revealed that the I. variabilis EPSPS gene and its protein was stably expressed in different generations of transgenic lines in RNA and protein levels. Treatments with different doses of glyphosate indicated that the lines of EPS-1, EPS-2, EPS-5, EPS-6, and EPS-7 could tolerate four times of the recommended dose of glyphosate in production. Thus, the novel glyphosate-tolerant rapeseed lines generated in the present study will lay the foundation for the herbicide- tolerance rapeseed breeding in China.

Key words: Brassica napus, I. variabilis EPSPS, transgene, glyphosate, molecular characteristics

Fig. 1

T-DNA region of the binary construct pTGH-1"

Table 1

Primer sequences used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
EPSPS-F ATTAGCGCTAGGGACGTGAG P1 CTCTAGCCAATACGCAAACCGCC
EPSPS-R ATACGCTCCCACATCCTGTC P3 TGTTGTGTGGAATTGTGAGCGGA
35SP-1 GAAACCGGTTACAGGCAAATG SP2 CAGGTGGAGAACGTCGGTACGACT
35SP-2 AGAGGCGGTTTGCGTATT SP3 GGGTTTCGCTCATGTGTTGAGCA
35ST-1 ATAACACATTGCGGACGTTT BnACT-qRTF CTGGAATTGCTGACCGTATGAG
35ST-2 AGCCGGTGCTTGATAACT BnACT-qRTR ATCTGTTGGAAAGTGCTGAGGG
35SP-2 AGAGGCGGTTTGCGTATT EPs-qRTF CAGGGATGGACTCGTTATCAC
p130-35sF CAAGTGGATTGATGTGATAACATG EPs-qRTR GAGGGTCTTCGCAGTCGT
p130-35sR GTAGAGAGAGACTGGTGATTTCAGC EPSPS6 F GGCTTCTTAGTGGGCCTTTC
Iva-EPSPS-F ATTAGCGCTAGGGACGTGAG EPSPS7 F CATCCGCTTTACGACGAAACT
Iva-EPSPS-R ATACGCTCCCACATCCTGTC EPSPS2 F GAACTGAGCATGAATGGCATAAA
P5 ATGTGTGAGTAGTTCCCAGATAAGG

Fig. 2

PCR identification of positive transgenic plants of the I. variabilis EPSPS gene A: PCR identification of the target gene I. variabilis EPSPS by primer pair EPSPS-F/EPSPS-R; B: PCR identification of the CaMV 35S promoter by primer pair 35SP-1/35SP-2; C: PCR identification of the CaMV 35S terminator by primer pair 35ST-1/35ST-2. M: 2 kb DNA marker; P: the positive control of pTGH-1plasmid; N: the negative control of J9707; 1-12: the T0 transgenic plants from EPS-1 to EPS-12."

Fig. 3

Determination of the I. variabilis EPSPS transgenic copy number by Southern blotting analysis in the T0-positive transgenic plants A: detection of Southern blot analysis. The hybrid probe is on the I. variabilis EPSPS gene; the genomic DNA was digested with Hind Ш before electrophoresis. M: DNA marker; 1-31: the positive transgenic plants; 1: EPS-1; 3: EPS-2; 12: EPS-7; 29: EPS-3; 31: EPS-6; N: J9707 (negative control). B: summary of the copy number of transgenic plants."

Fig. 4

Determination of the I. variabilis EPSPS transgenic copy number by Southern blotting analysis in the T2 and T3 positive transgenic plants A: detection of Southern blot with the hybrid probe of CaMV35S promoter and EcoR I restriction enzyme; B: detection of Southern blot with the hybrid probe of CaMV35S promoter and Hind Ш restriction enzyme; C: detection of Southern blot with the hybrid probe of EPSPS gene and EcoR I restriction enzyme; D: detection of Southern blot with the hybrid probe of EPSPS gene and Hind Ш restriction enzyme. M: DNA marker; P: pTGH-1 plasmid (positive control); N: J9707 (negative control); 1-9: the positive transgenic plants, which are a T2 plant of EPS-1, a T2 plant of EPS-2, a T2 plant of EPS-3, a T2 plant of EPS-4, a T2 plant of EPS-5, a T2 plant of EPS-6, a T3 plant of EPS-6, a T2 plant of EPS-7, and a T3 plant of EPS-7, respectively."

Table 2

Glyphosate-resistant segregation analysis of transgenic lines with single T-DNA insertion"

株系编号
Line ID		 总株数
Total plant number		 存活株数
Number of survived plants		 死亡株数
Number of dead plants		 期望分离比
Expected segregated ratio		 χ20.05, 1
EPS-1 50 33 17 3:1 1.71
EPS-2 50 31 19 3:1 3.84
EPS-4 56 39 17 3:1 0.59
EPS-5 49 34 15 3:1 0.55

Fig. 5

Insertion location information of T-DNA of EPS-2, EPS-6, and EPS-7 in the transgenic lines"

Fig. 6

PCR confirmation of the T-DNA insertion in the transgenic lines of EPS-2, EPS-6, and EPS-7 with specific primers A: PCR confirmation of the T-DNA insertion in the transgenic line of EPS-2 with specific primer pair EPSPS2 F/P5. B: PCR confirmation of the T-DNA insertion in the transgenic line of EPSPS6 with specific primer pair EPSPS6 F/P5. C: PCR confirmation of the T-DNA insertion in the transgenic line of EPS-7 with specific primer pair EPSPS7 F/P5. M: 2 kb DNA marker; P: pTGH-1 plasmid (positive control); N: J9707 (negative control); 1: T0-transgenic plant; 2-4: T1-transgenic plant; 5-7: T2-transgenic plant; 8-10: T3-transgenic plant."

Fig. 7

Expression analysis of the transgenic lines of I. variabilis EPSPS A: the RNA expression levels of the target gene in the leaf of T0-positive transgenic lines detected by RT-PCR. M: 3 kb DNA marker; 1-5: the positive transgenic lines of EPS-1, EPS-6, EPS-7, EPS-2, and EPS-5, respectively; N: J9707 (negative control); P: pTGH-1 (positive control). B: the protein expression level of the target gene in the leaves of the T0-positive transgenic lines detected by Western blot. N: J9707 (negative control); 1-6: the positive transgenic lines of EPS-1, EPS-6, EPS-36, EPS-7, EPS-2, and EPS-5, respectively. C: the relative expression of the target gene in different tissues of the T3-positive transgenic lines detected by qRT-PCR. Actin2 (AF111812) in rape is an internal reference gene."

Fig. 8

Phenotypes of T1 transgenic plants of I. variabilis EPSPS under the treatments of glyphosate"

Fig. 9

Phenotypes of T3 transgenic plants of I. variabilis EPSPS under the treatments with different concentrations of glyphosate at seedling stage"

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