Inheritance and Molecular Characterization of a Novel Mutated AHAS Gene Responsible for the Resistance of AHAS-Inhibiting Herbicides in Rapeseed (Brassica napus L.)

Abstract

The use of herbicides is an effective and economic way to control weeds, but their availability for rapeseed is limited due to the shortage of herbicide-resistant cultivars in China. The single-point mutation in the acetohydroxyacid synthase (AHAS) gene can lead to AHAS-inhibiting herbicide resistance. In this study, the inheritance and molecular characterization of the tribenuron-methyl (TBM)-resistant rapeseed (Brassica napus L.) mutant, K5, are performed. Results indicated that TBM-resistance of K5 was controlled by one dominant allele at a single nuclear gene locus. The novel substitution of cytosine with thymine at position 544 in BnAHAS1 was identified in K5, leading to the alteration of proline with serine at position 182 in BnAHAS1. The TBM-resistance of K5 was approximately 100 times that of its wild-type ZS9, and K5 also showed cross-resistance to bensufuron-methyl and monosulfuron-ester sodium. The BnAHAS1^544T transgenic Arabidopsis exhibited higher TBM-resistance than that of its wild-type, which confirmed that BnAHAS1^544T was responsible for the herbicide resistance of K5. Simultaneously, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS1^544T. In addition, a method for the fast screening of TBM-resistant plants at the cotyledon stage was developed. Our research identified and molecularly characterized one novel mutative AHAS allele in B. napus and laid a foundation for developing herbicide-resistant rapeseed cultivars.

Keywords: acetohydroxyacid synthase; herbicide resistance; rapeseed (Brassica napus L.); single-point mutation.

Figure 1

Resistance of the rapeseed mutant line K5 to tribenuron-methyl (TBM). (a) Untreated wild-type ZS9 plants. (b) Untreated line K5 plants. (c) ZS9 plants treated with 0.6 g a.i. ha⁻¹ TBM. (d) K5 plants treated with 0.6 g a.i. ha⁻¹ TBM.

Figure 2

Activity assay of AHAS of ZS9 and K5 after spraying with three different herbicides. (a–c) Relative AHAS activity of rapeseed ZS9 and the mutant K5 after spraying with tribenuron-methyl (TBM) (a), bensufuron-methyl (BSM) (b) and monosulfuron-ester sodium (MES) (c). (d) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of AHAS enzyme extracted from fresh leaves of herbicide-free K5 and ZS9. In the absence of herbicides, no significant difference was detected in the AHAS average specific activity (U/mg protein/h) between K5 (0.075 ± 0.013, n = 4) and ZS9 (0.098 ± 0.011, n = 4).

Figure 3

Mutant position of BnAHAS1^544T in the mutant K5. Primer nucleotide sequences for amplifying BnAHAS1 are indicated by the forward and reverse arrows. The boxes represent the initiation or the stop codons of the BnAHAS1^544T gene. The star indicates the position (544^th of the BnAHAS1^544T coding sequence in K5) of the single base substitution from C–T. The amino acid is substituted from proline (P) to serine (S).

Figure 4

Development and verification of co-dominant AS–PCR markers. (a) Distinguishing susceptive and resistant parents and their F_1s. M, marker; ZS9, Zhongshuang No.9, the susceptive line and K5, the mutant resistant line. (b) Verification of AS-PCR markers in 30 susceptive accessions. M, marker; K5, the mutant resistant line and lanes 1–30, 30 rapeseed susceptive accessions (Table S4).

Figure 5

Tribenuron-methyl (TBM) resistance in transgenic Arabidopsis plants expressing the mutant allele BnAHAS1^544T. (a) Phenotype of wild-type (WT) and transgenic Arabidopsis line (TA1) after spraying with 0.3 g a.i. ha⁻¹ of TBM at the 4–6 leaf stage. (b) Expression analysis of the BnAHAS1^544T gene in WT and TA1 by semiquantitative PCR. AtUBC21, ubiquitin-conjugating enzyme 21. (c) Fresh weight inhibition of WT and TA1-1 two weeks after spraying with different rates of TBM.

Figure 6

Phenotype of ZS9 and K5 at the cotyledon stage after irrigating with tribenuron-methyl (TBM) solution. Seeds were sown in the pots, and each pot was immediately irrigated with 300 mL TBM solution at different rates. Photographs were taken two weeks after seed sowing. ZS9, Zhongshuang No.9 and K5, the mutant line.