摘要
为揭示沙葱萤叶甲Galeruca daurica己糖激酶基因的功能,根据本课题组组装的沙葱萤叶甲转录组测序数据,应用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)克隆获得沙葱萤叶甲己糖激酶基因的cDNA全长序列,分析其编码蛋白的氨基酸序列与其它昆虫同源蛋白氨基酸序列之间的系统进化关系,采用实时荧光定量技术(real-time quantitative polymerase chain reaction,qPCR)测定不同发育阶段和不同温度下沙葱萤叶甲己糖激酶基因的相对表达量以及对RNA干扰的响应。结果表明,沙葱萤叶甲己糖激酶基因的cDNA全长为1699 bp,开放阅读框长为1479 bp,编码492个氨基酸;沙葱萤叶甲己糖激酶氨基酸序列与玉米根萤叶甲Diabrotica virgifera virgifera己糖激酶氨基酸序列一致性最高,为65.42%。己糖激酶基因在沙葱萤叶甲不同发育阶段均有表达,成虫滞育期间相对表达量维持在低水平,而滞育结束后相对表达量急剧上升达最高值。在0~40℃范围内,随着温度增加,己糖激酶基因在沙葱萤叶甲成虫体内相对表达量呈先升高后下降的趋势。采用RNA干扰技术沉默沙葱萤叶甲成虫己糖激酶基因2 d后,与对照相比,己糖激酶基因相对表达量下调90%以上,4 d后仍下调40%以上;该基因干扰后12 d内,沙葱萤叶甲成虫存活率下降了25%以上。表明己糖激酶基因可能在沙葱萤叶甲生长发育和滞育过程中起着重要作用。
In order to investigate the functions of the hexokinase gene in leaf beetle Galeruca daurica(GdHK),the full-length cDNA of GdHK gene was cloned by RACE based on the transcriptome data of G.daurica assembled in laboratory,and the phylogenetic relationships among its amino acid sequence and those of other insects’homologous proteins were analyzed using the current software.Real-time quantitative polymerase chain reaction(qPCR)was used to determine the relative expression levels of GdHK gene at different developmental stages and in adults under different temperatures,and RNA interference(RNAi)effects.The results showed that the full-length cDNA of the GdHK gene was 1699 bp,which contained a 1479 bp open reading frame(ORF)encoding 492 amino acids.Homology and phylogenetic analyses showed that the amino acid sequence of GdHK had the highest identity(65.42%)with that of Diabrotica virgifera virgifera HK.The GdHK gene was expressed at all developmental stages of G.daleruca,and the expression kept at the lowest level during the adult diapause but rose abruptly to the highest level after diapause terminated.The relative expression level of GdHK gene began to increase initially,but then decreased with the temperature increasing from 0℃to 40℃.Compared with the control,the relative expression level of GdHK gene reduced by over 90%two days after RNAi was performed in the adults and decreased by more than 40%four days after RNAi,and the adult survivorship declined by over 25%in 12 days after RNAi.These results suggested that GdHK gene may play an important role in the growth,development and diapause of G.daleruca.
作者
马红悦
李玲
李艳艳
谭瑶
庞保平
MA Hongyue;LI Ling;LI Yanyan;TAN Yao;PANG Baoping(Research Center for Grassland Entomology,Inner Mongolia Agricultural University,Hohhot 010020,Inner Mongolia Autonomous Region,China)
出处
《植物保护学报》
CAS
CSCD
北大核心
2020年第6期1210-1218,共9页
Journal of Plant Protection
基金
国家自然科学基金(31760517)。
关键词
沙葱萤叶甲
己糖激酶
基因克隆
表达量
RNA干扰
滞育
Galeruca daurica
hexokinase
gene cloning
expression profiling
RNA interference
diapause
