类酵母共生菌中两个组氨酸合成基因在褐飞虱发育中的作用

doi:10.16819/j.1001-7216.2016.6026

摘要/Abstract

摘要：

褐飞虱\[Nilaparvata lugens (Stl)\]是我国水稻上的主要害虫，专一性吸食必需氨基酸缺乏的水稻筛管液。利用基因组和转录组数据，人们构建了褐飞虱及其体内类酵母共生菌（YLS）参与组氨酸等必需氨基酸的合成途径。在此基础上，本研究旨在通过基因克隆和RNA干扰研究YLS的His2和His6（分别命名为EdeHis2和EdeHis6）在褐飞虱生长、发育和存活中的作用。同源搜索和系统发育分析表明EdeHis2和EdeHis6均源于YLS基因组，与绿僵菌His2和His6高度同源并在系统发育树中形成一个簇，但在褐飞虱基因组中无同源基因。基因的时空表达分析表明，His2和His6在褐飞虱的各个龄期均表达且呈现一定的波动性，在褐飞虱脂肪体中的表达量均显著高于头、足、体壁和中肠。此外，在褐飞虱头和翅的基因组中未能扩增到目的片段，而在腹部能扩增出目的片段。分别注射外源双链RNA（dsEdeHis2或dsEdeHis6）后的第2、4、6天，EdeHis2的表达量分别显著下调45%~60%，EdeHis6下调27%~55%。dsEdeHis2和dsEdeHis6分别使褐飞虱的死亡率提高了8.3%和9.2%，雌、雄虫的若虫期历期延长了0.43 d、0.33 d和0.65 d、0.36 d，但均未达到显著水平。另外，注射dsEdeHis6的褐飞虱雌雄成虫翅畸形率分别为11%和13%，显著高于对照组。综上所述，源于YLS的EdeHis2和EdeHis6参与褐飞虱组氨酸的合成，与褐飞虱的存活、发育和翅发育相关。

关键词: 褐飞虱, 组氨酸, 类酵母共生菌, His2, His6, RNA干扰

Abstract:

Nilaparvata lugens is a serious phloemfeeding pest of rice in China. Based on genome and transcriptome data of N.lugens and yeastlike symbiont (YLS, also named Entomomyces delphacidicola str. NLU), the major biosynthesis pathways for amino acids in N.lugens were constructed. In this study, we cloned two genes, EdeHis2 and EdeHis6, which catalyze critical steps in histidine biosynthesis pathway, and revealed the negative effects of doublestranded RNA (dsRNA) on the growth, development and survival rate of N.lugens. Homology searches and phylogenetic analysis showed that EdeHis2 and EdeHis6 origin from YLS genome, share high similarities with that of Metarhizium acridum and formed a clad in the phylogenetic tree, whereas no His2 or His6like genes was found in N.lugens genome. Temporal expression profiles of EdeHis2 and EdeHis6 showed that both genes were ubiquitously but unevenly expressed among the different life stages, and the spatial expression pattern showed they have higher expression levels in the fat body rather than head, leg, integument and midgut. Furthermore, no target products were amplified in head and wing genomic DNA of N. lugens, rather than that in abdomen genomic DNA. At two, four and six days after dsEdeHis2 or dsEdeHis6 injection, the mRNA abundance of target genes was decreased by 45%-60% (EdeHis2)or 27%-55% (EdeHis6), comparing with blank control.Downregulation of EdeHis2 or dsEdeHis6 slightly increased the mortality by 8.3% or 9.2%, and delayed the nymphal duration of male and female by 0.43 and 065 day and 0.33 and 0.36 day, respectively. Moreover, both male (11%) and female adult (13%) showed wing deformation after injection of dsEdeHis6, higher than that in the blank control. In conclusion, EdeHis2 and EdeHis6 that origin from YLS were involved in histidine biosynthesis pathway, contributed to the survivor, development and wing formation of N. lugens.

Key words: Nilaparvata lugens, yeastlike symbiont, histidine, His2, His6, RNAi

中图分类号:

唐耀华1,2,万品俊2,郝培应1,傅强2,*,俞晓平1,*. 类酵母共生菌中两个组氨酸合成基因在褐飞虱发育中的作用[J]. 中国水稻科学 2016,30(4): 406-416. , DOI: 10.16819/j.1001-7216.2016.6026 .

TANG Yaohua1,2, WAN Pinjun2, HAO Peiying1, FU Qiang2, *, YU Xiaoping1, *. Roles of Two Genes Involved in Histidine Biosynthetic Pathway in YeastLike Symbiont in Development of Nilaparvata lugens (Stl)[J]. Chinese Journal of Rice Science 2016,30(4): 406-416., DOI: 10.16819/j.1001-7216.2016.6026 .

参考文献

[1]Wang Y, Chen J, Zhu Y C, et al. Susceptibility to neonicotinoids and risk of resistance development in the brown planthopper, Nilaparvata lugens (Stl) (Homoptera: Delphacidae). Pest Manag Sci, 2008, 64(12): 12781284.
[2]Bottrell D G, Schoenly K G. Resurrecting the ghost of green revolutions past: The brown planthopper as a recurring threat to highyielding rice production in tropical Asia. J AsiaPacif Entomol, 2012, 15(1): 122140.
[3]傅强，张志涛，胡萃，等. 高温处理后褐飞虱体内共生酵母菌和氨基酸需求的变化. 昆虫学报，2001，44(4)：534540.
Fu Q, Zhang Z T, Hu C, et al. The effects of high temperature on both yeastlike symbionts and amino acid requirements of Nilaparvata lugens. Acta Entomol Sin, 2001, 44(4): 534540.(in Chinese with English abstract)
[4]Chen Y H, Bernal C C, Tan J, et al. Planthopper "adaptation" to resistant rice varieties: Changes in amino acid composition over time. J Insect Physiol, 2011, 57(10): 13751384.
[5]王国超，傅强，赖凤香，等. 褐飞虱体内类酵母共生菌与氨基酸营养的关系. 昆虫学报，2005，48(4): 483490.
Wang G C, Fu Q, Lai F X, et al. Relationship between yeastlike symbiotes and amino acid requirements in the rice brown planthopper, Nilaparvata lugens (Stl) ( Homoptera: Delphacidae).Acta Entomol Sin, 2005, 48(4): 483490.(in Chinese with English abstract)
[6]Wan P J, Yang L, Yuan S Y, et al. RNA interferenceaided knockdown of a putative saccharopine dehydrogenase leads to abnormal ecdysis in the brown planthopper, Nilaparvata lugens (Stl) (Hemiptera: Delphacidae). Bull Entomol Res, 2015, 105(4): 390398.
[7]Xue J, Zhou X, Zhang CX, et al. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genom Biol, 2014, 15(521): 119.
[8]Wan P J, Yang L, Wang W X, et al. Constructing the major biosynthesis pathways for amino acids in the brown planthopper, Nilaparvata lugens Stl (Hemiptera: Delphacidae), based on the transcriptome data. Insect Mol Biol, 2014, 23(2): 152164.
[9]傅强. 褐飞虱全纯人工饲料继代饲养技术及营养生理学研究.杭州：浙江大学，1999.
Fu Q. Continous rearing and nutitional physiology of the brown planthopper, Nilaparvata lugens (Stl) on chemically defined diets. Hangzhou：Zhejiang University, 1999.(in Chinese with English abstract)
[10]Fan H W, Noda H, Xie H Q, et al. Genomic analysis of an Ascomycete fungus from the rice planthopper reveals how it adapts to an endosymbiotic lifestyle. Genome Biol Evol, 2015, 7(9): 1334.
[11]Larkin M A, Blackshields G, Brown N P, et al. Clustal W and Clustal X version 2.0. Bioinformatics, 2007, 23(21): 29472958.
[12]Tamura K, Peterson D, Peterson N, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 2011, 28(10): 27312739.
[13]王渭霞，罗举，赖凤香，等. 水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析. 昆虫学报，2010，53(6): 647654.
Wang W X, Luo J, Lai F X, et al. Identification and phylogenetic analysis of symbiotic bacteria Arsenophonusfrom the rice brown planthopper, Nilaparvata lugens (Stl) ( Homoptera: Delphacidae).Acta Entomol Sin, 2010, 53(6): 647654.(in Chinese withEnglishabstract)
[14]Wang W X, Li K L, Chen Y, et al. Identification and function analysis of enolase gene NlEno1 from Nilaparvata lugens (Stl) (Hemiptera:Delphacidae). J Insect Sci, 2015, 15(1): 19.
[15]Li K L, Wan P J, Wang W X, et al. Ran involved in the development and reproduction is a potential target for RNAinterferencebased pest management in Nilaparvata lugens. PLoS ONE, 2015, 10(11): e0142142.
[16]Livak K J, Schmittgen T D. Analysis of relative gene expression data using realtime quantitative PCR and the 2(T)(Delta Delta C) method. Methods, 2001, 25(4): 402408.
[17]Yuan M, Lu Y, Zhu X, et al. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reversetranscription quantitative PCR. PLoS ONE, 2014, 9(1): e86503.
[18]Tang Q Y, Zhang C X. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci, 2013, 20(2): 254260.
[19]Cheng D J, Hou R F. Histological observations on transovarial transmission of a yeastlike symbiote in Nilaparvata lugens Stl (Homoptera, Delphacidae). Tissue Cell, 2001, 33(3): 273279.
[20]Dong S, Pang K, Bai X, et al. Identification of two species of yeastlike symbiotes in the brown planthopper, Nilaparvata lugens. Curr Microbiol, 2011, 62(4): 11331138.
[21]Fu Q, Zhang Z, Hu C, et al. The effects of high temperature on both yeastlike symbionts and amino acid requirements of Nilaparvata lugens. Acta Entomol Sin, 2001, 44(4): 534540.
[22]Wilkinson T L, Ishikawa H. On the functional significance of symbiotic microorganisms in the Homoptera: A comparative study of Acyrthosiphon pisum and Nilaparvata lugens. Physiol Entomol, 2001, 26(1): 8693.
[23]Wan P J, Yuan S Y, Tang Y H, et al. Pathways of amino acid degradation in Nilaparvata lugens (Stl) with special reference to lysineketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH). PLoS ONE, 2015, 10(5): e0127789.
[24]Hinnebusch A G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Revi, 1988, 52(2): 248273.
[25]Koslowsky S, Riegler H, Bergmuller E, et al. Higher biomass accumulation by increasing phosphoribosylpyrophosphate synthetase activity in Arabidopsis thaliana and Nicotiana tabacum. Plant Biotechnol J, 2008, 6(3): 281294.
[26]Zhang Y, Morar M, Ealick S E. Structural biology of the purine biosynthetic pathway. Cellul Mol Life Sci, 2008, 65(23): 36993724.
[27]Ingle R A. Histidine biosynthesis. The Arabidopsis book / American Society of Plant Biologists, 2011, 115.
[28]Stepansky A, Leustek T. Histidine biosynthesis in plants. Amino Acids, 2006, 30(2): 127142.