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作物学报 ›› 2023, Vol. 49 ›› Issue (6): 1542-1550.doi: 10.3724/SP.J.1006.2023.24126

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

寄主诱导的基因沉默干扰核盘菌致病基因OAH在甘蓝型油菜抗菌核病中的应用

杨一丹1(), 何督1, 刘静2, 张岩1, 陈飞志1, 巫燕飞1, 杜雪竹1,*()   

  1. 1湖北大学生命科学学院/省部共建生物催化与酶工程国家重点实验室, 湖北武汉 430062
    2农业农村部油料作物生物学与遗传育种重点实验室, 湖北武汉 430062
  • 收稿日期:2022-05-26 接受日期:2022-10-10 出版日期:2023-06-12 网络出版日期:2022-10-31
  • 通讯作者: *杜雪竹, E-mail: duxuezhusk@163.com
  • 作者简介:E-mail: duxuezhusk@163.com
  • 基金资助:
    农业农村部油料作物生物学与遗传育种重点实验室开放课题基金(KF2022001);国家自然科学基金项目(31771837)

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 Published:2023-06-12 Published online: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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摘要：

油菜是我国第一大油料作物, 菌核病是我国油菜面临的主要病害之一。本试验利用HIGS技术将核盘菌中关键致病基因SS1G_08218 (OAH)的干扰片段转化到甘蓝型油菜中, 获得含有siRNA的转基因油菜植株, 研究HIGS介导的SsOAH基因沉默对油菜抗菌核病的影响。接种核盘菌后的抗病鉴定结果表明, SS.OAH.RNAi转基因油菜植株对核盘菌抗性增强; R18、R25和R36株系叶片接菌后病斑部位核盘菌菌丝内的OAH基因表达量均低于野生型, 证明siRNA在转基因油菜中成功表达; 在添加了转基因油菜叶片提取物的培养基上培养的核盘菌扩展面积显著小于野生型, 分别降低35.29%、21.98%、31.53%, 且菌丝生长显著迟缓, 扩展长度较短, 分支少, 生长过程中发生断裂, 生长异常, 将其接种于正常的野生型油菜后, 其致病力明显下降; 对转基因油菜叶片接种核盘菌后观察发现, 叶片上的菌丝扩展较为稀疏, 生长受阻, 侵染垫形成受到抑制, 暗示在油菜中表达OAH 基因的干扰片段影响了菌丝生长和扩展; 进一步检测其病斑组织中的草酸含量发现, 接菌36 h、48 h后转基因油菜病斑组织中的草酸含量为391 μg g-1、446 μg g-1, 与野生型相比分别减少54 μg g-1、32 μg g-1。这一研究表明在油菜中表达核盘菌OAH的干扰片段能够降低核盘菌侵染时草酸的积累, 从而增强转基因油菜对核盘菌的抗性水平。本试验利用HIGS技术研究了干扰核盘菌关键致病基因OAH增强油菜对核盘菌的抗性, 为选育油菜抗菌核病品种提供理论基础和种质资源。

关键词: 甘蓝型油菜, 核盘菌, HIGS, 草酰乙酸乙酰水解酶, 草酸

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

表1

Buffer提取液配制"

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

图1

核盘菌侵染野生型油菜后相关致病基因的表达情况"

图2

OAH干扰终载体菌落PCR检测 M: DL2000; 1~8: 菌落PCR片段。"

图3

T2代SS.OAH.RNAi转基因植株部分阳性鉴定凝胶电泳图 M: Trans 2K; WT: 阴性对照。"

图4

T2代SS.OAH.RNAi 转基因油菜株系离体叶片接菌36 h病斑面积统计图 WT: 野生甘蓝型油菜; R25、R36、R18为T2代SS.OAH.RNAi转基因株系材料。"

图5

T2代SS.OAH.RNAi转基因油菜株系茎秆接菌4 d病斑统计图 WT: 野生甘蓝型油菜; R25、R36、R18为T2代SS.OAH.RNAi转基因株系材料。"

图6

T2代SS.OAH.RNAi转基因油菜叶片接菌后病斑部位核盘菌菌丝内OAH基因的表达模式分析 WT: 野生甘蓝型油菜; R25、R36、R18为T2代SS.OAH.RNAi转基因株系材料。"

图7

油菜叶片提取物培养核盘菌抗性鉴定 WT: 野生甘蓝型油菜; R25、R36、R18为T2代SS.OAH.RNAi转基因株系材料。Mock: PDA培养基培养的核盘菌; A:添加提取物的PDA培养基培养核盘菌40 h后的菌丝扩展面积统计; B: 标尺为1 cm; B1: 无添加萃取液的PDA活化的菌丝; B2: 添加了野生型油菜叶片萃取液活化的核盘菌; B3~B5为添加了SS.OAH.RNAi转基因油菜(R18、R25、R36)叶片萃取液活化的核盘菌; B6~B10: 分别对应图B1~B5在体视显微镜下观察到的核盘菌菌丝生长情况; C: 野生型油菜与转基因油菜(R18、R25、R36)叶片提取物制成的培养基培养的核盘菌菌丝接种于野生型油菜后48 h的病斑扩展情况, 标尺为2 cm。"

图8

棉蓝染色观察油菜叶片接菌情况 WT: 野生甘蓝型油菜; R25、R36、R18为T2代SS.OAH.RNAi转基因株系材料。"

图9

T2代SS.OAH.RNAi转基因植株叶片病斑部位草酸含量测定 字母表示具有统计学意义的差异值, 基于P < 0.05的单因素方差分析。"

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