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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 1027-1034.doi: 10.3724/SP.J.1006.2022.14066

• 研究简报 • 上一篇    

耐三唑并嘧啶类除草剂花生种质创制与鉴定

刘嘉欣1,2(), 兰玉1,2, 徐倩玉1, 李红叶1, 周新宇3, 赵璇1, 甘毅1, 刘宏波1, 郑月萍1, 詹仪花1, 张刚3, 郑志富1,2,*()   

  1. 1浙江农林大学现代农学院, 浙江杭州 311300
    2浙江农林大学林业与生物技术学院, 浙江杭州 311300
    3文登市佳禾种业有限公司, 山东威海 264400
  • 收稿日期:2021-04-18 接受日期:2021-07-12 出版日期:2022-04-12 网络出版日期:2021-08-11
  • 通讯作者: 郑志富
  • 作者简介:刘嘉欣, E-mail: 252606752@qq.com第一联系人：**同等贡献
  • 基金资助:
    国家自然科学基金项目资助(31871660)

Creation and identification of peanut germplasm tolerant to triazolopyrimidine herbicides

LIU Jia-Xin1,2(), LAN Yu1,2, XU Qian-Yu1, LI Hong-Ye1, ZHOU Xin-Yu3, ZHAO Xuan1, GAN Yi1, LIU Hong-Bo1, ZHENG Yue-Ping1, ZHAN Yi-Hua1, ZHANG Gang3, ZHENG Zhi-Fu1,2,*()   

  1. 1College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    2College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
    3Wendeng Jiahe Seed Corporation, Ltd., Weihai 264400, Shandong, China
  • Received:2021-04-18 Accepted:2021-07-12 Published:2022-04-12 Published online:2021-08-11
  • Contact: ZHENG Zhi-Fu
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31871660)

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摘要：

目前耐除草剂花生的种质资源匮乏, 这制约了花生栽培模式的多元化发展。为创制耐不同除草剂的花生种质资源, 我们利用甲基磺酸乙酯诱变技术创建了一个由55,000多个花生株系组成的诱变群体, 随后选择多种不同除草剂对该群体进行筛选, 获得多个对不同除草剂具有耐性的花生突变体。其中一个突变体在田间叶面喷施处理和多种实验室条件下的除草剂耐受性评估试验中均表现出对唑嘧磺草胺和双氟磺草胺极强的耐性, 但这种耐性性状并未对花生的产量与品质产生不良影响。为明确这种性状是否与除草剂靶标抗性相关联, 我们比较分析了该突变体与野生型中这2种除草剂靶标酶(乙酰羟酸合酶, AHAS)的基因序列及其表达量的差异。分子克隆与序列分析显示, 花生A10与B10染色体上各含一个与拟南芥AtAHAS序列高度相似的基因, 分别命名为AhAHAS1a与AhAHAS1b。花生A08与B08染色体上亦各含一个AHAS基因, 分别命名为AhAHAS2a与AhAHAS2b。然而, 与野生型相比, 突变体中的这些基因并未发生能够致使氨基酸序列发生改变的核苷酸替换。进而发现, AhAHAS基因的表达量在突变体与野生型中没有显著差异。这些结果表明, 该花生突变体的除草剂耐性可能由非靶标抗性机制所致。

关键词: 花生, 甲基磺酸乙酯诱变, 三唑并嘧啶类除草剂, 除草剂耐性, 品质性状

Abstract:

At present, the germplasm resources of herbicide-tolerant peanut (Arachis hypogaea) are scarce, which restrict the diversification of peanut-based cropping system. To create peanut germplasm with tolerance to different herbicides, a mutant population consisting of more than 55,000 peanut lines were generated by ethylmethanesulfonate mutagenesis. We screened this population with a variety of different herbicides to obtain multiple mutants with tolerance to different herbicides. One of these lines displayed strong tolerance to florasulam and flumetsulam in the experiments with foliar herbicide spraying under field conditions as well as in various laboratory evaluation for the herbicide tolerance, while the herbicide-tolerant trait did not have adverse effects on peanut yield and quality. To determine whether this trait was associated with a target-site-based resistance to the herbicides, we compared gene sequences and relative expression levels of these two acetohydroxyacid synthases (AHASs) as the herbicide target enzymes between the mutant and wild type. Molecular cloning and sequence analysis revealed that peanut chromosome A10 and B10 each contained a gene, named as AhAHAS1a and AhAHAS1b, which were highly similar to Arabidopsis AHAS. Peanut chromosome A08 and B08 also each carried an AHAS gene, named as AhAHAS2a and AhAHAS2b, respectively. However, compared with the wild type, the genes in the mutants had no nucleotide substitutions that could alter the amino acid sequences. Furthermore, it was evident that there was no significant difference in the relative expression levels of AhAHAS genes between the mutant and wild type. In summary, these results indicate that the herbicide tolerance of the mutant might be caused by non-target-site-based resistance mechanism.

Key words: Arachis hypogaea, ethylmethanesulfonate mutagenesis, triazolopyrimidines, herbicide tolerance, quality trait

表1

本研究所用引物序列"

引物
Primer		 引物序列
Primer sequence (5'-3')		 目的基因
Target gene
LY1/2FP ATGGCTGCCACTGCTTCCAAAC A10或B10染色体的AHAS
AHAS on chromosome A10 or B10
LY1RP TCAATATTTTGTTCTGCCATCGCCT
LY2RP CAATATTTTGTTCTGCCATCACCT
XQY1FP CATTCTCCACCGTATCTCCATC A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY1RP TACATGCCACTGTCTGGTTACTACT
LY31FP ACGAAGCATGCTTACCTTGTTCTTG A10或B10染色体的AHAS
AHAS on chromosome A10 and B10
LY31RP AACATAACTGGTTGATCCCAATTGG
XQY2FP AGCTTGAGGCTTTCGCGAGT A08或B08染色体的AHAS
AHAS on chromosome A08 and B08
XQY2RP CCCTTTTCCTCCAAGATCCCA
XQY4FP CAGGATTTGCCGGTGATGATG β-肌动蛋白
β-Actin
XQY4RP TCTGTTGGCCTTCGGGTTGAG

图1

除草剂叶面喷施对野生型与突变体花生幼苗生长的影响 在三叶期对花生进行除草剂处理, 麦喜除草剂(450 mL hm-2)使用剂量为常规大田用量的3倍, 处理4周后照相。A: 野生型(‘山花15’); B: M7 耐除草剂突变体(H2-20-1)。"

图2

除草剂叶面喷施对成熟期野生型与突变体花生生长的影响 在三叶期对花生进行除草剂处理, 麦喜除草剂(450 mL hm-2)使用剂量为常规大田用量的3倍, 成熟期时照相。A: 野生型(‘山花15’); B: M7 耐除草剂突变体(H2-20-1)。"

图3

除草剂浸种处理对突变体H2-20-1和野生型‘山花15’根系生长的影响 唑嘧磺草胺(Flu)和双氟磺草胺(Flo)的浓度分别为9.90 g hm-2与7.50 g hm-2, 除草剂使用浓度为大田常用剂量的2倍, 处理15 d后照相。WT: 野生型花生。"

图4

沙培条件下除草剂对突变体H2-20-1和野生型‘山花15’根系生长的影响 除草剂溶于水后与细沙混合。唑嘧磺草胺(Flu)和双氟磺草胺(Flo)的浓度分别为9.90 g hm-2与7.50 g hm-2, 除草剂使用浓度为大田常用剂量的2倍, 处理15 d后照相。WT: 野生型花生。"

图5

野生型与突变体H2-20-1花生成熟种子的油脂含量 2批不同种植年份(2020和2019)的种子用于分析。WT: 野生型。t-test统计分析显示, 在连续2个年份, 突变体花生种子油脂含量和野生型相比没有显著差异。"

图6

野生型与突变体H2-20-1花生成熟种子中的脂肪酸组分 两批不同种植年份(2020和2019)的种子用于分析。WT: 野生型。星号(*)表示与WT相比, 在P < 0.05 (t-test)水平上的统计显著性差异。"

图7

野生型与突变体H2-20-1花生成熟种子中的氨基酸组分 WT: 野生型。星号(*)表示与WT相比, 在P < 0.05 (t-test)水平上的统计显著性差异。"

表2

花生突变体H2-20-1与野生型AHAS1 基因序列的相似度"

AHAS1a (MT) AHAS1a (WT) AHAS1b (MT) AHAS1b (WT)
AHAS1a (MT) 100 99 99
AHAS1a (WT) 99 99
AHAS1b (MT) 100
AHAS1b (WT)

表3

花生突变体H2-20-1与野生型中AHAS2 基因序列的相似度"

AHAS2a (MT) AHAS2a (WT) AHAS2b (MT) AHAS2b (WT)
AHAS2a (MT) 100 99 99
AHAS2a (WT) 99 99
AHAS2b (MT) 100
AHAS2b (WT)

图8

野生型花生不同发育时期不同组织中AHAS基因表达的差异分析 以幼苗期茎中的基因表达量为1。DAF: 开花后天数。"

图9

野生型花生与突变体H2-20-1中AHAS基因表达的差异分析 以野生型叶中AHAS2的表达量为1。t-test统计分析显示, 突变体花生AHAS基因表达水平和野生型相比没有显著差异。"

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