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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (6): 1542-1550.doi: 10.3724/SP.J.1006.2023.24126

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

Application of host-induced gene silencing interfering with Sclerotinia sclerotiorum pathogenic gene OAH in Brassica napus resistance to Sclerotinia sclerotiorum

YANG Yi-Dan1(), HE Du1, LIU Jing2, ZHANG Yan1, CHEN Fei-Zhi1, WU Yan-Fei1, DU Xue-Zhu1,*()   

  1. 1School of Life Sciences, Hubei University/State Key Laboratory of Biocatalysis and Enzyme Engineering, Wuhan 430062, Hubei, China
    2Key Laboratory of Biology and Genetics and Breeding of Oil Crops, Ministry of Agriculture and Rural Affairs, Hubei University, Wuhan 430062, Hubei, China
  • Received:2022-05-26 Accepted:2022-10-10 Online:2023-06-12 Published:2022-10-31
  • Contact: *E-mail: duxuezhusk@163.com
  • Supported by:
    Open Project Foundation of Key Laboratory of Oil Crop Biology and Genetics and Breeding of the Ministry of Agriculture and Rural Affairs(KF2022001);National Natural Science Foundation of China(31771837)

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Abstract:

Rapeseed is the largest oil crop in China, and Sclerotinia sclerotiorum is one of the main diseases of rapeseed in China. In this experiment, to study the effect of HIGS-mediated SsOAH gene silencing on the resistance to S. sclerotiorum in Brassica napus, the interference fragment of the key pathogenic gene SS1G_08218 (OAH) in S. sclerotiorum was transformed into Brassica napus by HIGS technique, and the transgenic plants containing siRNA were obtained. The results of disease resistance identification after inoculation with S. sclerotiorum showed that the resistance of SS.OAH.RNAi transgenic rapeseed plants to S. sclerotiorum was enhanced, and the relative expression levels of OAH genes in S. sclerotiorum mycelia of R18, R25, and R36 lines were lower than wild type, indicating that siRNA was successfully expressed in transgenic rapeseed. The lesion size of S. sclerotiorum cultured on the medium supplemented with transgenic rapeseed leaf extract was significantly smaller than wild type, which decreased by 35.29%, 21.98%, and 31.53% respectively. The mycelium grew slowly, the expansion length was shorter, the branches were few, and the growth was abnormal. After inoculating it into normal wild type rapeseed, its pathogenicity decreased significantly. After inoculating the transgenic rapeseed leaves with S. sclerotiorum, it was observed that the mycelium expansion on the leaves was sparse, the growth was blocked, and the infection pad formation was inhibited, suggesting that the interference fragments expressing OAH gene in rapeseed affected the mycelium growth and expansion. Compared with the wild type, the content of oxalic acid in the lesion tissue of transgenic rapeseed was 391 μg g-1 and 446 μg g-1 at 36 hpi and 48 hpi, which decreased 54 μg g-1 and 32 μg g-1, respectively. This study showed that the interference fragment expressing S. sclerotiorum OAH in rapeseed could reduce the accumulation of oxalic acid during S. sclerotiorum infection and enhance the resistance level of transgenic rapeseed to S. sclerotiorum. In this experiment, HIGS technique was used to improve resistance to S. sclerotiorum by transforming interference fragments of OAH gene in rapeseed, and to provide a theoretical basis and germplasm resources for breeding S. sclerotiorum resistant varieties.

Key words: Brassica napus, Sclerotinia sclerotiorum, HIGS, oxalyl acetate acetylhydrolase, oxalic acid

Table 1

Buffer extract preparation"

试剂
Reagent		 相对分子质量
Relative molecular weight (g mol-1)		 浓度
Concentration (mmol L-1)		 配制1000 mL
Formulated 1000 mL
MES 195.24 50 9.762
Triscl 121.14 100 12.114
EDTA 372.24 0.1 0.037,224
NaCl 58.44 30 1.7532

Fig. 1

Relative expression levels of related pathogenic genes in wild rape infected by S. sclerotiorum"

Fig. 2

OAH interference PCR detection of terminal carrier colonies M: DL2000; 1-8: the colony PCR fragments."

Fig. 3

Gel electrophoresis map of positive identification of some SS.OAH.RNAi transgenic plants in T2 generation M: Trans 2K; WT: the negative control."

Fig. 4

Statistical map of lesion area in leaves of T2 generation SS.OAH.RNAi transgenic rapeseed inoculated with bacteria for 36 hours WT: wild Brassica napus; R25, R36, and R18 are T2 generation SS.OAH.RNAi transgenic lines."

Fig. 5

Statistical map of disease spot of T2 generation SS.OAH.RNAi transgenic rapeseed line inoculated with stem for 4 days WT: wild Brassica napus; R25, R36, and R18 are T2 generation SS.OAH.RNAi transgenic lines."

Fig. 6

Relative expression patterns of OAH gene in Sclerotinia sclerotiorum after inoculation of T2 generation SS.OAH.RNAi transgenic rapeseed line WT: wild Brassica napus; R25, R36, and R18 are T2 generation SS.OAH.RNAi transgenic lines."

Fig. 7

Identification of resistance of Sclerotinia sclerotiorum cultured with rape leaf extract WT: wild Brassica napus; R25, R36, and R18 are T2 generation SS.OAH.RNAi transgenic lines. Mock: S. sclerotiorum cultured in PDA medium; A: the statistics of the expanded area of the culture disk after adding PDA to the mycelial extract for 40 hours; B: bar: 1 cm; B1: PDA activated hyphae without added extract; B2: S. sclerotiorum activated by wild-type rape leaf extract; B3-B5 added SS.OAH.RNAi transgenic rape (R18, R25, and R36) leaf extract; B6-B10: corresponding to the mycelial growth of S. sclerotiorum observed under stereomicroscope in Fig. B1-B5 respectively. C: the expansion of S. sclerotiorum hyphae cultured in the medium made of wild-type rape and transgenic rape (R18, R25, and R36) leaf extract 48 hours after inoculation in wild-type rape. Bar: 2 cm."

Fig. 8

Observation on inoculation of rape leaves by cotton blue staining WT: wild Brassica napus; R25, R36, and R18 are T2 generation SS.OAH.RNAi transgenic lines."

Fig. 9

Determination of oxalic acid content in diseased leaf spots of T2 generation SS.OAH.RNAi transgenic rapeseed Different lowercase letters represent statistically significant differences at P < 0.05 based on one-way ANOVA."

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