Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (17): 3251-3260.doi: 10.3864/j.issn.0578-1752.2023.17.002

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

Identification of Target Traits and Genetic Stability of Transgenic Cotton GGK2

LIANG ChengZhen(), ZANG YouYi, MENG ZhiGang, WANG Yuan, MUBASHIR Abbas, HE HaiYan, ZHOU Qi, WEI YunXiao, ZHANG Rui(), GUO SanDui()   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2023-01-17 Accepted:2023-02-23 Online:2023-09-01 Published:2023-09-08
  • Contact: LIANG ChengZhen, ZHANG Rui, GUO SanDui

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Abstract:

【Objective】The objective of this study is to confirm the target traits and genetic stability of transgenic glyphosate- resistant cotton GGK2 and provide technical support for its commercialization. 【Method】T3, T4, and T5 transgenic cotton plants GGK2 were subjected to insertion site-specific PCR, Southern blot, ELISA, bioassays in the laboratory and field, analysis of target herbicide tolerance, and investigation of nutritional constituents. 【Result】The results indicated that the target genes, GR79 EPSPS and GAT, were integrated into the cotton genome as single copies and stably inherited in GGK2 plants. In GGK2 cotton, GR79 EPSPS, GAT, and NPTⅡ proteins were expressed at different stages and in different tissues, with relatively high expression levels in the leaves. At the four-leaf stage, bud stage and boll opening stag, the expression levels in leaves were 128.7-192.4 µg·g-1, 24.4-35.0 µg·g-1, and 17.0-23.9 µg·g-1 fresh weight for GR79 EPSPS, GAT, and NPTⅡ, respectively. In the field, transgenic cotton GGK2 tolerated up to four times the recommended medium dose of glyphosate application. No significant differences were observed in agronomic traits and nutritional constituents compared to the control, Coker312. 【Conclusion】These data demonstrate that transgenic cotton GGK2 is genetically stable and highly resistant to herbicides. Therefore, it can be utilized for breeding high-glyphosate- resistant commercial cotton varieties.

Key words: cotton, GGK2, herbicide tolerance, glyphosate, genetic stability

Fig. 1

PCR analysis of the target genes and flanking sequence in the T3 to T5 generation of transgenic cotton GGK2 The amplified fragment size of primers GR79 EPSPS FP + RP, GAT FP + RP, NPTII FP + RP, RB FP+LB RP, and RB FP+LB RP were 645, 466, 760, 810, 420 bp, respectively. Coker312: The Coker312 cotton was used as negative control; DNA Ladder: DNA marker. The GR79GAT plasmid was used as positive control. T3, T4 and T5 refer to the homozygous lines of GGK2 transgenic cotton T3 to T5 generation. The same as below"

Fig. 2

Southern blot analysis of the target genes in the T3 to T5 generations of transgenic cotton GGK2 A: Schematic representation of the pGR79GAT vector with the position of restriction enzyme and probes. B: Hybridization results of probe 1, genomic DNA was digested by HindⅢ, EcoRⅠ, and BamHⅠ. C: Hybridization results of probe 2, genomic DNA was digested by EcoRⅠ, NheⅠ, and BamHⅠ. D: Hybridization results of probe 3, genomic DNA was digested by HindⅢ, BamHⅠ, and NheⅠ. E: Hybridization results of probe 4, genomic DNA was digested by HindⅢ, EcoRⅠ, and BamHⅠ. F: Hybridization results of probe 5, genomic DNA was digested by HindⅢ, EcoRⅠ, and BamHⅠ"

Fig. 3

Expression analysis of the target genes in the T3 to T5 generation of transgenic cotton GGK2 A: GR79 EPSPS; B: GAT; C: NPTII. Different lowercase letters indicate the significant difference among different transgenic lines (P<0.05). The same as below"

Fig. 4

Expression analysis of the target proteins in the T3 to T5 generation of transgenic cotton GGK2 The expression of GR79 EPSPS (A), GAT (B), and NPTⅡ (C) protein in 4-leaf stage, flowering stage, and boll opening stage. D: ImmunoStrip for the detection of GGK2 cotton in the T3-T5 generations. The arrow indicates the target band. PC: Positive control. NC: Negative control"

Table 1

Glyphosate tolerance analysis of T3 to T5 transgenic cotton GGK2"

处理时间
Time		 性状
Phenotype		 喷施物
Spray		 柯字棉312
Coker312		 GGK2
T3 T4 T5
喷施后2周
Two weeks after spraying		 株高
Plant height (cm)		 草甘膦Glyphosate / 26.74±1.40 27.44±1.74 28.12±1.41
清水Water 27.34±2.29 27.11±1.08 26.42±1.84 26.72±1.22
叶片受害率
Leaf damage rate (%)		 草甘膦Glyphosate 100.00±0.00 0.96±0.10 0.71±0.23 0.82±0.07
清水Water 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00
喷施后4周
Four weeks after spraying		 株高
Plant height (cm)		 草甘膦Glyphosate / 46.88±2.54 47.03±1.22 47.23±3.18
清水Water 47.60±1.92 47.27±0.53 46.93±1.52 48.03±1.39
叶片受害率
Leaf damage rate (%)		 草甘膦Glyphosate 100.00±0.00 1.21±0.26 0.83±0.03 1.13±0.11
清水Water 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00
/：全部死亡 /: All dead

Table 2

Nutritional composition analysis of T3 to T5 transgenic cotton GGK2"

名称Name 水分
Water content (g/100 g)		 灰分
Ash content (g/100 g)		 蛋白质
Protein content (g/100 g)		 脂肪
Fat content (g/100 g)
T3 T4 T5 T3 T4 T5 T3 T4 T5 T3 T4 T5
GGK2 9.10 8.57 8.84 4.10 4.20 4.10 20.80 20.80 23.90 17.30 16.60 18.20
柯字棉312
Coker312		 8.15 8.77 8.37 4.00 4.10 4.20 22.10 21.10 23.60 17.20 17.10 18.10
粗纤维
Coarse fiber content (g/100 g)		 维生素E
<BOLD>V</BOLD>itamin E content (mg/100 g)		 维生素B2
<BOLD>V</BOLD>itamin B2 content (mg/100 g)		 肉豆蔻酸
Myristic chemicing (%)
T3 T4 T5 T3 T4 T5 T3 T4 T5 T3 T4 T5
GGK2 14.40 14.60 17.80 8.48 7.84 7.09 5.96×10-2 5.85×10-2 5.68×10-2 0.59 0.60 0.82
柯字棉312
Coker312		 15.50 17.00 15.70 9.71 10.30 13.20 5.75×10-2 5.57×10-2 5.65×10-2 0.69 0.69 0.70
棕榈酸
Palmitic acid (%)		 棕榈油酸
Palmitoleic acid (%)		 十七烷酸
Heptacosanoic acid (%)		 硬脂酸
Stearine acid (%)
T3 T4 T5 T3 T4 T5 T3 T4 T5 T3 T4 T5
GGK2 21.30 21.40 25.40 0.64 0.64 0.75 7.39×10-2 7.56×10-2 8.17×10-2 2.20 2.22 2.56
柯字棉312
Coker312		 22.10 22.00 22.10 0.61 0.61 0.61 7.91×10-2 7.80×10-2 7.85×10-2 2.35 2.41 2.37
油酸
Oleic acid (%)		 亚油酸
Linoleic acid (%)		 十九烷酸
Nonadecanoic acid (%)		 亚麻酸
Linolenic acid (%)
T3 T4 T5 T3 T4 T5 T3 T4 T5 T3 T4 T5
GGK2 15.70 15.30 14.60 57.50 57.80 52.60 0.23 0.24 0.18 0.23 0.22 0.16
柯字棉312
Coker312		 14.70 14.70 14.60 57.50 57.50 57.60 0.25 0.28 0.26 0.18 0.19 0.18
花生酸
Arachic acid (%)		 山嵛酸
Behenic acid (%)		 棉酚
Gossypol (mg·kg-1)		 植酸酶
Phytase (g·kg-1)
T3 T4 T5 T3 T4 T5 T3 T4 T5 T3 T4 T5
GGK2 0.20 0.19 0.26 0.13 0.13 0.13 5.42×103 5.16×103 5.45×103 28.50 29.10 29.10
柯字棉312
Coker312		 0.23 0.22 0.22 0.14 0.14 0.14 5.41×103 5.25×103 5.39×103 30.00 26.40 27.70
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