摘要
【目的】克隆小菜蛾几丁质合成酶基因PxUAP并阐明其功能。【方法】基于小菜蛾基因组信息,利用RT-PCR克隆小菜蛾PxUAP基因的cDNA序列,并应用荧光定量PCR和RNA干扰技术分析该基因的表达模式及功能。【结果】PxUAP(GenBank登录号:OR659549)开放阅读框长度为1467 bp,预测编码488个氨基酸,蛋白分子质量为54.985 ku,理论等电点为6.12;与金凤蝶UAP(KPJ20218.1)同源蛋白亲缘关系较近,氨基酸同源性为80.33%。发育表达谱结果表明,PxUAP在小菜蛾各个发育阶段均有表达,在雄成虫期的表达量最高;组织表达谱结果表明,PxUAP在雌成虫足和翅膀的表达量最高。RNAi结果表明,与注射ds GFP对照组相比,注射ds PxUAP显著降低了靶标基因和几丁质合成酶PxCHSA基因的表达量,且PxCHSB基因在24和48 h的表达量也显著下降;注射ds PxUAP的小菜蛾大部分无法化蛹,蛹壳出现皱缩等畸形现象,并在注射后168 h全部死亡。【结论】PxUAP是小菜蛾几丁质合成酶通路中的关键调控基因,对小菜蛾生长发育起至关重要的作用。
【Aim】This study aimed to clone the chitin synthetase gene PxUAP and elucidate its function in Plutella xylostella.【Method】Based on the genomic information of diamondback moth,the full-length cDNA sequence of PxUAP was amplified using RT-PCR,and the expression pattern and function of the gene were analyzed using quantitative real-time PCR and RNA interference(RNAi).【Result】PxUAP(GenBank number:OR659549)contains an open reading frame of 1467 bp that encodes 488 amino acids with a predicted size of 54.985 ku and a theoretical isoelectric point of 6.12.Phylogenetic analysis demonstrated that the amino acid sequences of PxUAP and Papilio machaon(KPJ20218.1)UAP clustered into a single clade with 80.33%similarity.The developmental expression profile revealed that PxUAP was expressed at various developmental stages of P.xylostella,with the highest expression level in the male adult stage,and the tissue expression profile showed that the expression of PxUAP was highest in the legs and wings of female adults.After RNAi,the expression levels of PxUAP and PxCHSA in the ds PxUAP injection group were significantly downregulated compared with those in the control group(ds GFP injection).At 24 and 48 h after RNAi,the expression level of PxCHSB was also significantly lower in the ds PxUAP treatment than in the ds GFP control group.In addition,most of P.xylostella could not pupate and died because of pupal shell shrinkage after RNAi,and the survival rate decreased to 0 after injection of ds PxUAP at 168 h.【Conclusion】PxUAP is a key regulatory gene in the chitin synthase pathway of diamondback moth and plays a crucial role in the growth and development of P.xylostella.
作者
李恒
田厚军
林硕
陈艺欣
魏辉
陈勇
LI Heng;TIAN Houjun;LIN Shuo;CHEN Yixin;WEI Hui;CHEN Yong(Institute of Plant Protection,Fujian Academy of Agricultural Sciences/Fuzhou Scientific Observing and Experimental Station of Crop Pests of Ministry of Agriculture,Fuzhou,Fujian 350013,China)
出处
《生物安全学报（中英文）》
CSCD
北大核心
2024年第4期415-422,共8页
Journal of biosafety
基金
国家自然科学基金项目(32072425)
福建省自然科学基金项目(2022J01457)
福建省农业科学院科技项目(GJYS202205、XTCXGC2021011、XTCXGC2021017)。
