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  

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

作　　者：杨一丹 何督 刘静 张岩 陈飞志 巫燕飞 杜雪竹[1] YANG Yi-Dan;HE Du;LIU Jing;ZHANG Yan;CHEN Fei-Zhi;WU Yan-Fei;DU Xue-Zhu(School of Life Sciences,Hubei University/State Key Laboratory of Biocatalysis and Enzyme Engineering,Wuhan 430062,Hubei,China;Key Laboratory of Biology and Genetics and Breeding of Oil Crops,Ministry of Agriculture and Rural Affairs,Hubei University,Wuhan 430062,Hubei,China)

机构地区：[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

出　　处：《作物学报》2023年第6期1542-1550,共9页Acta Agronomica Sinica

基　　金：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)资助

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391μg g^(-1)、446μg g^(-1),与野生型相比分别减少54μg g^(-1)、32μg 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。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 391μg g^(-1)and 446μg g^(-1)at 36 hpi and 48 hpi,which decreased 54μg g^(-1)and 32μg 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 techni

关 键 词：甘蓝型油菜 核盘菌 HIGS 草酰乙酸乙酰水解酶 草酸 

分 类 号：S435.654[农业科学—农业昆虫与害虫防治]