Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (17): 3277-3284.doi: 10.3864/j.issn.0578-1752.2023.17.004

• SPECIAL FOCUS: HERBICIDE-TOLERANCE COTTON CREATION BY GENETIC TRANSFORMATION AND GENOME EDITING • Previous Articles     Next Articles

Identification of Molecular Characterizations for Transgenic Cotton R1-3 Line of Glyphosate Tolerance

MA YanBin1(), LI HuanLi1, WEN Jin1, ZHOU XianTing1,2, QIN Xin1, WANG Xia3, WANG XinSheng1, LI YanE1   

  1. 1Institute of Cotton Research, Shanxi Agriculture University, Yuncheng 044000, Shanxi
    2College of Agronomy, Shanxi Agriculture University, Taigu 030800, Shanxi
    3Department of Life Science, Yuncheng University, Yuncheng 044000, Shanxi
  • Received:2023-02-01 Accepted:2023-07-05 Online:2023-09-01 Published:2023-09-08
  • Contact: MA YanBin

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Abstract:

【Objective】 To obtain the transgenic cotton by Agrobacterium-mediated method was the purpose using the new gene G10aroA with high glyphosate tolerance in our laboratory. Meanwhile, it was necessary to provide molecular characteristics of genomic integration of the exogenous gene for breeding utilization in future. 【Method】 The Agrobacterium-mediated method was used for transgenic cotton plants obtained via tissue culture with glyphosate herbicide. Western blot was utilized to detect the expression of exogenous proteins in different organs of transgenic cotton R1-3. The number of loci in cotton genomes were evaluated by Southern blot for detecting integration of the exogenous sequence from the pCAMBIA1300 construct. The flanking sequence near the insertion site was amplified by TAIL-PCR, which the extractions of DNA were cloned and sequenced. The location of the chromosome for the flanking sequences were compared and analyzed on the website of NCBI blast. 【Result】 Regenerated R1-3 cotton plants were successfully obtained by tissue culture depending on glyphosate screening. the specific protein coded by exogenous G10aroA gene could be detected normally via Western blotting in the leaves, bracts, flowers and stems separately, and the size of the exogenous protein around 46 kDa were also observed in this experiment. In addition, the result of Southern-blot confirmed that the exogenous fragment containing G10aroA sequences was single integration in the genome of transgenic cotton R1-3, in which the bands digested severally by KpnⅠ and EcoRⅠ endonucleases were distinctly observed near the position of 6 557 bp and 4 316 bp strips on the nylon membrane respectively. The analysis of flanking sequence alignment for the integration site was predicted to be located on the 11th chromosome of either cotton A or D genome, and the left and right boundaries of the insertion site were further located between 47 525 303 and 47 525 449 of the chromosomes. In addition, the specific identification for the fusion site showed that the target band of approximately 300 bp for testing left border junction, and a specific target band can be amplified about 600 bp for testing the right border fusion site. 【Conclusion】 In this study, we obtained R1-3 transgenic cotton plants that also have exhibited stable genetic characteristics of glyphosate resistance during the process of self-crossing breeding. The protein coded by G10aroA gene was about the size of 46 kDa that could be detected in different tissues of transgenic cotton R1-3 plant. Furthermore, the exogenous fragment including G10aroA gene was identified with a single location by southern blot in the cotton genomes, and the integration site was located at the 11th chromosome. The results of comparative analysis were predicted that a nucleotide sequence about 146 bp length was deleted at the integration of the genome.

Key words: Gossypium hirsutum L., glyphosate-tolerance cotton, copy number, integration site, molecular characterization

Fig. 1

Schematic diagram of the construction for G10aroA gene in pCAMBIA1300 vector"

Table 1

Specific primers of TAIL-PCR and flanking sequence for the integration site of R1-3 transgenic cotton"

引物名称
Primer		 引物序列
Primer sequence (5′-3′)		 目标
Objective
1300-LP1 TTCTCCATAATAATGTGTGAGTAGTTCC 左边界插入位置扩增
Amplification of left border insertion
1300-LP2 TAGGGTTTCGCTCATGTGTTGAGC
1300-LP3 CGAATTAATTCGGCGTTAATTCAGTAC
1300-RP1 CGTCGTTTTACAACGTCGTGACTGG 右边界插入位置扩增
Amplification of right border insertion
1300-RP2 CAGCTGGCGTAATAGCGAAGAGGC
1300-RP3 GAGCAGCTTGAGCTTGGATCAG
35S-正R CTTAGTAGACGAGAGTGTC 左边界融合序列鉴定
Identification of left border integration sequence
SX-左F AGGCGCTACAATCTCAAGATCA
1300-RP2 CAGCTGGCGTAATAGCGAAGAGGC 右边界融合序列鉴定
Identification of right border integration sequence
SX-右R GGTTTCTGGATTCCCTGTTC

Fig. 2

Different stages of regeneration of cotton tissue culture by Agrobacterium-mediated transformation A: Callus induction of cotton hypocotyl explants after agrobacterium infection; B: Callus induced by hypocotyl; C: Callus proliferation; D: Embryogenic callus differentiation; E: The seedlings formed after differentiation; F: Regenerated plants"

Fig. 3

Analysis of exogenous protein expression in different tissues of R1-3 cotton transgenic plants -: Negative control of R15; M: Protein Marker, the size of EPSPS protein bands in R1-3 cotton lines is about 46 kDa"

Fig. 4

Identification of copy number of exogenous gene G10aroA in transgenic cotton The two bands on the left are the hybridization results of R1-3 digested respectively by EcoRⅠ and KpnⅠ endonucleases; -: R15 negative control, digested by endonuclease EcoRⅠ; M: DNA Marker, +: PCAMBIA1300 vector containing exogenous gene G10aroA digested by endonuclease EcoRⅠ, arrows indicate the position of hybrid bands"

Fig. 5

Integration sites, chromosome positions and flanking sequence verification for exogenous sequences in cotton R1-3 plants A: The results of alignment between the TM-1 genome and the flanking sequences of TAIL-PCR amplification for the fusion sites of exogenous sequences in R1-3 transgenic plant, L indicates the insertion position of the left border of the pCAMBIA 1300 Vector, R indicates the insertion position of the right border of the pCAMBIA 1300 Vector; B: Schematic map of exogenous sequences integration in the R15 genome; C: PCR amplification result of flanking sequence for left border integration; D: PCR amplification result of flanking sequence for right border integration"

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