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作物学报 ›› 2024, Vol. 50 ›› Issue (5): 1158-1171.doi: 10.3724/SP.J.1006.2024.34110

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

耐亚磷酸盐除草剂转基因油菜的创建和抗性评价

钟元(), 朱天宇, 戴成, 马朝芝*()   

  1. 华中农业大学作物遗传改良全国重点实验室 / 国家油菜工程技术研究中心 / 洪山实验室, 湖北武汉 430070
  • 收稿日期:2023-07-03 接受日期:2023-10-23 出版日期:2024-05-12 网络出版日期:2023-12-13
  • 通讯作者: 马朝芝, E-mail: yuanbeauty@mail.hzau.edu.cn
  • 作者简介:E-mail: zy14745140090623@163.com
  • 基金资助:
    国家自然科学基金项目(32072105)

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 Published:2024-05-12 Published online:2023-12-13
  • Contact: E-mail: yuanbeauty@mail.hzau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32072105)

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摘要：

在油菜的生产过程中, 大量伴生的杂草严重影响了其产量和品质。由于近年来田间抗除草剂杂草数量的增加, 除草剂的选择正在迅速减少, 这影响了未来农业的可持续发展。植物可以通过正磷酸盐(Pi)转运蛋白吸收亚磷酸盐(Phi), 但Phi不能被代谢为作物的磷肥料, 导致植物生长受到抑制。此前, 从罗尔斯通菌属(Raltsonia sp. strain 4506)中分离出了ptxD (phosphite dehydrogenase)基因, 其139位的酪氨酸突变为谷氨酰胺(Y139Q)的突变蛋白ptxDQ催化Phi转化为Pi的活性显著提高。为了评价ptxDQ/Phi系统对甘蓝型油菜杂草的防治效果, 本研究获得了带有密码子优化的ptxD (Y139Q, ptxDQ)基因的转基因甘蓝型油菜。在以Phi为单一磷元素的条件下, ptxDQ转基因油菜可以正常生长, 体内Pi含量显著提高, 且Pi饥饿响应基因(PT21和PT24等)的表达水平被显著抑制, 说明ptxDQ在油菜体内可以促使Phi转化为Pi。此外, ptxDQ转基因油菜具有比野生型油菜和单子叶杂草(狗尾草)更强的生长竞争优势。综上所述, ptxDQ/Phi是一种有效的油菜磷利用和杂草控制系统, 既可以抑制杂草的生长, 又可以为作物提供充足的Pi营养, 从而提高了农业的可持续性。

关键词: 甘蓝型油菜, 除草剂, 亚磷酸盐, ptxDQ

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

表1

本研究中使用的引物"

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

图1

含有密码子优化ptxDQ的转基因甘蓝型油菜的产生 (a) 甘蓝型油菜Westar在Pi和不同Phi浓度处理7 d后的生长情况。标尺为2 cm。(b) 生长7 d后下胚轴长度的统计情况。(c) 生长7 d后根长的统计情况。(d) 生长7 d后鲜重的统计情况。(e) 植物转化载体p1300-ptxDQ示意图。(f)和(g)以120 μg mL-1 Hygromycin B和0.5 mmol L-1Pi +1.5 mmol L-1Phi为筛选剂进行转化。标尺为2 cm。(h) 油菜再生苗RNA水平的PCR鉴定。(i) 油菜再生苗ptxDQ基因相对表达量分析。数字编号为不同的油菜再生苗单株, M为marker, BnaACT7为油菜内参基因。使用t检验进行显著性检验, *、**、***分别表示在0.05、0.01、0.001概率水平差异显著。"

附图1

不同生态型油菜在Pi和Phi下的水培试验 (a)和(b) 处理2周后不同油菜的生长状况。Pi浓度为1 mmol L-1, Phi浓度为4 mmol L-1。标尺为2 cm。(c) 处理2周后不同油菜根长的统计。(d) 处理2周后不同油菜鲜重的统计。使用t检验进行显著性检验: 当P ≤ 0.05时, 如果用不同的小写字母标注, 则条形图代表的值存在显著差异。"

附图2

ptxDQ蛋白序列分析 PtxDR4506、PtxDR4506 (Y139Q)和PtxDWM88氨基酸序列比对。相同的残基用黑色表示, 相似的残基用灰色表示。"

附图3

潮霉素B和Phi对愈伤组织再生的影响 (a) 以120 μg mL-1 Hygromycin B和1 mmol L-1 Phi为筛选剂进行转化。标尺为2 cm。(b) 油菜再生苗DNA水平的PCR分析。数字编号为不同的油菜再生苗单株, M为marker。(c) 转基因油菜L10株系的根、茎、叶的RT-PCR分析。BnaACT7为油菜内参基因。"

表2

同一载体(pCMBIA1300-PtxDQ)不同筛选剂的转化情况统计"

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

图2

沙培水培法研究Phi对野生型和ptxDQ转基因甘蓝型油菜植株生长的影响 (a)~(c)野生型植株和2个ptxDQ转基因系(L5和L10, T1代)在沙培养(沙 : 蛭石=1:1)中分别生长14 d (a)、56 d (b)和76 d (c), 分别用无P、Pi或Phi营养液处理。(a)~(c)中比例尺分别为3 cm、7 cm、7 cm。(d) 柱状图为不含P、Pi或Phi营养液灌溉2周后野生型植株和2个ptxDQ转基因品系的SPAD值。(e)柱状图为不加P、Pi或Phi营养液灌溉2周后野生型植株和2个ptxDQ转基因品系的Pi含量。使用t检验进行显著性检验: 当P ≤ 0.05时, 如果用不同的小写字母标注, 则条形图代表的值存在显著差异。"

图3

油菜中磷饥饿响应基因的相对表达量分析 (a) PHR的相对表达量; (b) PT1:4的相对表达量; (c) PT21的相对表达量; (d) PT24的相对表达量。使用t检验进行显著性检验: 当P ≤ 0.05时, 如果用不同的小写字母标记, 则条形图代表的值存在显著差异。"

图4

ptxDQ/Phi可抑制田间杂草生长 (a)和(b) Westar、狗尾草和转基因植株在Pi和Phi处理3周后的生长发育情况, Pi浓度为1 mmol L-1, Phi浓度为8 mmol L-1。(a) 标尺为5 cm; (b) 标尺为2 cm。(c) 柱状图为(a)所示43株狗尾草的鲜重。使用t检验进行显著性检验: ***, P < 0.0001。(d) Westar和转基因植株在Pi和Phi处理3周后的鲜重、株高、根长的情况。(e) T1代转基因株系中的ptxDQ特异性条带的PCR分析。1~5, 表示L10的不同转基因植株。使用t检验进行显著性检验: 当P ≤ 0.05时, 如果用不同的小写字母标记, 则条形图代表的值存在显著差异。"

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