基于宿主诱导的基因沉默技术创制抗拟轮枝镰孢玉米自交系

Fusarium verticillioides Resistant Maize Inbred Line Development Using Host-Induced Gene Silencing Technology

【目的】拟轮枝镰孢(Fusarium verticillioide)是一种主要的病原真菌,侵染玉米可以导致穗粒腐病、茎腐病、苗期根腐病及引起种子腐烂。由拟轮枝镰孢引起的病害不仅影响玉米的产量和品质,而且其病原菌代谢过程中产生的伏马菌素等多种真菌毒素严重威胁了人畜安全。通过宿主诱导的基因沉默(host-induced gene silencing,HIGS)技术创制抗拟轮枝镰孢的玉米种质,为玉米抗病育种提供新的优异抗源。【方法】通过同源克隆方法克隆可能与拟轮枝镰孢生长发育相关的关键基因,并通过体外转录获得相应的dsRNA片段;将不同基因的dsRNA与拟轮枝镰孢的分生孢子悬浮液预混后,用于后续的体外RNA沉默试验;对感病玉米自交系西502的种子进行消毒与接种,在培养皿中28℃避光培养48 h,调查种子的发病程度;在混有dsRNA的孢子悬浮液中加入葡萄糖,25℃培养24 h后,在显微镜下观察孢子萌发与菌丝生长情况;将三叶期的西502幼苗转移至预混dsRNA的孢子悬浮液中进行培养,7 d后观察苗期根腐病发病状况;通过种子鉴定与苗期鉴定体系,逐步筛选具有显著抑制效果的沉默靶标基因;合成筛选出的重点靶标基因片段,构建沉默载体并转化感病玉米自交系西502;对转基因株系的种子接种鉴定,验证转化玉米株系的抗性;提取接种后转基因种子的总RNA,对拟轮枝镰孢的靶标基因进行荧光定量分析,确定HIGS株系的沉默效果。【结果】从拟轮枝镰孢中克隆出18个与其生长发育相关的候选基因;通过种子接种鉴定,发现11个候选基因被沉默后,种子的发病等级极显著降低;进一步筛选出6个沉默后影响拟轮枝镰孢的孢子萌发和菌丝生长的候选靶标基因deo、Ras2、Dpdc、Hsp90、Frp1和Atg15;通过苗期接种鉴定,最后筛选出3个在体外具有显著抑制效果的沉默靶标基因deo、Atg15和Frp1;进而将3个靶标基因的特异区段人工融合成一段序列并构建沉默载体,获得转基因植株;鉴定发现转基因植株的T2代种子对拟轮枝镰孢的抗性显著增强,且3个靶标基因的表达量均显著下降。【结论】拟轮枝镰孢基因deo、Atg15、Frp1与其生长发育密切相关,且沉默后能够显著提升玉米对拟轮枝镰孢的抗性。

【Objective】Fusarium verticillioides(F.verticillioides)is a common pathogen,which can cause ear rot,stalk rot,seedling blight,and seed rot in maize.These diseases caused by Fusarium verticillioides not only affected the yield and quality of maize,but also seriously threatened to the safety of human and livestock by a variety of fungal toxins such as fumonisin which produced during the metabolic process of the pathogen.So far,there is no report about the major resistance gene cloned and utilized for Fusarium verticillioides in maize.Using host-induced gene silencing technology provides a new strategy for resistance breeding in maize.【Method】Key genes associated with the Fusarium verticillioides development were cloned using homologous gene sequence method,and the dsRNA were produced by in-vitro transcription(IVT)assay.The dsRNA for different genes was premixed with suspension spores of Fusarium verticillioides used for RNA silencing experiment in vitro.For investigate the degree of disease,the seeds of the susceptible inbred line Xi502 were sterilized and inoculated,and then were cultured in a petri dish at 28℃in the dark for 48 h.For investigate the incidence of the seeds after inoculation,glucose was added to the spore suspension mixed with dsRNA,then spore germination and mycelia growth were observed under the microscope after 25℃culture for 24 h.The Xi502 seedlings of the trifoliate stage were transferred to the spore suspension with premixed dsRNA for culture,and the incidence of seedlings blight was observed after 7 days.In order to select the target gene for HIGS,combine the seed morphological observation result after inoculation and seedling inoculation result.Then,the silent vector about these key target genes were constructed and transferred into the susceptible inbred line Xi502.The transgenic seeds were evaluated by artificial inoculation.The total RNA of the transgenic seeds after inoculation was extracted,and the relative expression of target genes in F.verticillioide was analyzed by qRT-PCR to determine the silencing effect of HIGS line.【Result】Eighteen candidate genes related to growth were cloned by homologous cloning method in Fusarium verticillioides.It was found that the disease level of seeds was significantly reduced after 11 candidate genes silencing by seed inoculation experiments.Furthermore,six of the 11 candidate target genes,deo,Ras2,Dpdc,Hsp90,Frp1,and Atg15,were found that response to the spore germination and mycelium growth after gene silencing.Finally,based on the results of seedling inoculation,3 silencing target genes deo,Atg15 and Frp1 with significant inhibitory effect in vitro were selected.Then the silencing vector was constructed by combine three specific segments from the three target genes,transgenic plants were obtained.It was found that the resistance level was highly increased in T2-generation seeds compared to the none-transgenic plants.As well as the expression levels of all the three target genes were significantly decreased in Fusarium verticillioides.【Conclusion】Three genes,deo,Atg15 and Frp1,are important for development of Fusarium verticillioides.By constructing transgenic HIGS plants for target gene deo,Atg15 and Frp1,the increase the resistance to Fusarium verticillioides in maize.