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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (5): 1158-1171.doi: 10.3724/SP.J.1006.2024.34110

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

Developing and resistance assessing of phosphite-tolerant herbicide transgenic Brassica napus L.

ZHONG Yuan(), ZHU Tian-Yu, DAI Cheng, MA Chao-Zhi*()   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University / National Engineering Research Center of Rapeseed / Hongshan Laboratory, Wuhan 430070, Hubei, China
  • Received:2023-07-03 Accepted:2023-10-23 Online:2024-05-12 Published:2023-12-13
  • Contact: E-mail: yuanbeauty@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32072105)

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Abstract:

During the production of Brassica napus, the yield and quality of the crop are seriously affected by a large number of accompanying weeds. Herbicide control options are rapidly diminishing due to the recent increase in the number of herbicide-resistant weeds in fields, which affects the sustainable development of agriculture in the future. Plants can take up phosphite (Phi) via orthophosphate (Pi) transporters, but the Phi cannot be metabolized and used as a phosphorus fertilizer for crops, resulting in plant growth inhibition. Previously, a ptxD gene isolated from Ralstonia sp. 4506, and the mutant protein ptxDQ at position 139, which had tyrosine mutation to glutamine (Y139Q), significantly improved the conversion activity of Phi to Pi. To evaluate the efficacy of ptxDQ/Phi-based weed control system in Brassica napus, we generated transgenic B. napus plants with a codon-optimized ptxD (Y139Q, ptxDQ) gene. Ectopic expression of ptxDQ confered the ability to convert Phi to Pi, resulting in improving plant growth in the presence of Phi. Pi-starvation-induced genes (e.g. PT21 and PT24) were suppressed in the ptxDQ transgenic lines by supplying Phi, indicating that ptxDQ can transform Phi into Pi in rapeseed. In addition, ptxDQ transgenic Brassica napus had a greater competitive growth advantage over wild-type Brassica napus and monocotyledonous weeds (Setaria glauca). In conclusion, the ptxDQ/Phi system provided an effective alternative for suppressing the weed growth while providing adequate Pi nutrition to the crops, which in turn improved the sustainability of agriculture.

Key words: Brassica napus, herbicide, phosphite, ptxDQ

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 Tm (℃) 产物扩增大小
Product length (bp)
Y139Q-M-Sal I-F TCCATCGATAGTACTGTCGACATGAAGCCTAAAGTTGTTTTGACTC 66 1050
Y139Q-M-Kpn I-R GCTCACCCCGGGAGCGGTACCAGCAGCTTTAACACCTGGG 75
ptxDQ-F ATGAAGCCTAAAGTTGTTTTGACTCAT 56 1009
ptxDQ-R AGCAGCTTTAACACCTGGGT 57
ptxDQ-DNA-F GTTTTGACTCATTGGGTTCATCCAGA 59 993
ptxDQ-DNA-R AGCAGCTTTAACACCTGGGT 58
ptxDQ-qRT-F CGATTCTGCTTTCCTTGAAGAGTG 59 216
ptxDQ-qRT-R CCAGATCTGATTTGTCTATCTCCCTC 59
BnACT7-RT-F CTATCCTCCGTCTCGATCTCGC 61.8 173
BnACT7-RT-R CTTAGCCGTCTCCAGCTCTTGC 63.2
RT-PCR PT21 F ATCGGCCGCAACAGGTAAGG 61.5 181
RT-PCR PT21 R AGGGACAAGGAAGGTGAAGAG 59.4
RT-PCR PT1;4-F GTACCGGCGGAGATCTTCCCAGC 65.4 324
RT-PCR PT1;4-R CTACACAATGGGGACCGTTC 61.2
RT-PCR PT24 F CCTGAAACTGCTCGTTACACC 58.8 109
RT-PCR PT24 R CCTCTGCTCTTTCCTCCATCTC 58.6
RT-PCR PHR F ACTGAGGCTCTGCGACTTCA 60.2 360
RT-PCR PHR R TCTTGTTCGGATTTGGATGGTGAAT 59.5

Fig. 1

Generation of transgenic B. napus harboring a codon-optimized ptxDQ (a): the growth of Brassica napus Westar treated with Pi and Phi at different concentrations for 7 days. Scale bar: 2 cm. (b): statistics of hypocotyl length after 7 days of growth. (c): statistics on root length after 7 days of growth. (d): Statistics on fresh weight after 7 days of growth. (e): Schematic diagram of the plant transformation vector p1300-ptxDQ. (f) and (g): 120 μg mL-1 hygromycin B and 0.5 mmol L-1 Pi + 1.5 mmol L-1 Phi were used as the screening agents for transformation. Scale bar: 2 cm. (h): PCR identification of RNA levels in Brassica napus seedlings. (i): the relative expression level of ptxDQ gene in Brassica napus regenerated seedlings. The numbers are different Brassica napus regenerated seedling monocots, M is marker, and BnaACT7 is Brassica napus internal gene. The significance is analyzed by t-test. *, **, and *** mean significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

Fig. S1

Hydroponics experiments of different ecotypes of Brassica napus under Pi and Phi (a) and (b) growth of different Brassica napus after 2 weeks of treatment. The Pi concentration is 1 mmol L-1 and the Phi concentration is 4 mmol L-1. Scale bar: 2 cm. (c) statistics of different Brassica napus root length after 2 weeks of treatment. (D) statistics of different Brassica napus fresh weight after 2 weeks of treatment. The significance is analyzed at P ≤ 0.05 by t-test. The values represented by the bars chart are significantly different if marked with different lowercase."

Fig. S2

Protein sequence analysis of ptxDQ Alignment of PtxDR4506, PtxDR4506 (Y139Q), and PtxDWM88 amino acid sequence. The identical residues were shaded by black, and the similar residues were shaded by gray."

Fig. S3

Callus regeneration with Hygromycin B or Phi selection on shoot initiation media (a) 120 μg mL-1 Hygromycin B and 1 mmol L-1 Phi were used as screening agents for transformation. Scale bar: 2 cm. (b) PCR identification of Brassica napus seedlings at DNA level. The numbers are different Brassica napus regenerated seedling monocots, M is marker. (c) RT- PCR analysis of roots, stems, and leaves of L10 strain of transgenic rapeseed. BnaACT7 is Brassica napus internal gene."

Table 2

Statistics of transformation of different screening agents in the same vector pCMBIA1300-PtxDQ"

筛选抗性
Resistance		 实验重复Replicate 外植体数
Number of explants		 愈伤数Number of calluses 外植体转化效率
Callus conversion rate (%)		 再生苗数
Number of green seedlings		 绿苗比例
Green seedling rate (%)		 阳性转基因
株系比例
Transformation
efficiency (%)
Pi+ Phi 1 403 364 90.32 7 1.73 0
2 313 255 81.47 2 0.64 0
3 235 194 82.55 2 0.85 0
平均值Average 317 271 84.78 3.6 1.07 0
Hyg B 1 342 100 29.24 11 3.22 100
2 216 106 49.07 9 4.17 100
3 257 108 42.02 10 3.89 100
平均值Average 272 105 40.11 10 3.76 100

Fig. 2

Sand culture hydroponics study to evaluate the effect of Phi on the growth of Westar and ptxDQ transgenic B. napus plants (a)-(c): the image shows the Westar plants and two ptxDQ transgenic lines (L5 and L10, T1 generation) grown in sand culture (sand : vermiculite = 1:1) for 14-day (a), 56-day (b) and 76-day (c) by irrigating no P, Pi, or Phi nutrient solutions. In (a)-(c), the scale bar was 3, 7, and 7 cm, respectively. (d): bar graph shows the SPAD values of Westar and two ptxDQ transgenic lines irrigated by no P, Pi, or Phi nutrient solutions for two weeks. (e): bar graph shows the Pi contents of Westar and two ptxDQ transgenic lines irrigated by no P, Pi, or Phi nutrient solutions for two weeks. Different lowercase letter indicates significant difference at P ≤ 0.05 by t-test."

Fig. 3

Relative expression analysis of phosphorus starvation response genes in Brassica napus (a): the relative expression level of PHR; (b): the relative expression level of PT1:4; (c): the relative expression levels of PT21; (d): the relative expression of PT24. Different lowercase letter indicates significant difference at P ≤ 0.05 by t-test."

Fig. 4

ptxDQ/Phi suppresses weed growth in the field (a) and (b): growth and development of Westar, Setaria, and transgenic plants after 3 weeks of Pi and Phi treatment, Pi concentration was 1 mmol L-1, Phi concentration was 8 mmol L-1, (a) Scale bar: 5 cm; (b) Scale bar: 2 cm. (c): bar graph shows fresh weight of Setaria glauca representing in (a). The significance is analyzed by t-test: ***: P < 0.0001. (d): fresh weight, plant height, and root length of Westar and transgenic plants after 3 weeks of Pi and Phi treatment. (e): ptxDQ specific band in T1 transgenic lines by PCR.1-5, indicated different transgenic plants of L10. Different lowercase letter indicates significant difference at P ≤ 0.05 by t-test. "

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