Cloning and Expression of Bursicon Gene of Myzus persicae (Sulzer)
-
摘要: Bursicon是调节昆虫表皮硬化及展翅的激素类蛋白，它在昆虫表皮硬化和翅伸展过程中起着关键作用。本研究克隆获得桃蚜Bursicon基因（burs-α和burs-β）全长，分别命名为Mpburs-α（GenBank登录号：MF083566）和Mpburs-β（GenBank登录号：MF083567）。Mpburs-α开放阅读框长483 bp，编码160个氨基酸。其相对分子量为17.59 kDa，分子式为C759H1204N210O232S19；理论等电点为7.96，Mpburs-β开放阅读框长417 bp，编码138个氨基酸。其相对分子量为15.45 kDa，分子式为C672H1072N180O212S12；理论等电点为4.84。分子系统进化树分析表明，桃蚜与豌豆蚜Acyrthosiphon pisum和麦双尾蚜Diuraphis noxia具有较近的亲缘关系。RT-qPCR结果表明：Mpburs-α和Mpburs-β在桃蚜各龄期均有表达，以1龄若蚜期表达量最高；成蚜有翅个体中表达量显著高于无翅个体。推测Bursicon在桃蚜翅的形成中发挥着重要作用。研究结果可为续研究Bursicon在桃蚜蜕皮后表皮的骨化、翅的形成方面相关的功能机制奠定基础。Abstract: Bursicon regulates the epidermal sclerosis and wing-spreading in insects. It plays a key role in epidermis hardening of insects after molting. The full-length sequences of the bursicon genes (burs-α and burs-β) of M. persicae (Sulzer) were obtained through cloning for this study, and named Mpburs-α (GenBank accession number:MF083566) and Mpburs-β (GenBank accession number:MF083567), respectively. Mpburs-α contained a 483 bp open reading frame (ORF) and was encoded 160 amino acid residues. Its molecular mass was predicted to be 17.59 kDa with a formula of C759H1204N210O232S19 and an isoelectric point of 7.96. Mpburs-β contained a 417 bp ORF and was encoded 138 amino acids. Its molecular mass to be 15.45 kDa with a formula of C672H1072N180O212S12 and an isoelectric point of 4.8. The phylogenetic tree analysis showed a relatively close genetic relationship of M. persicae with Acyrthosiphon pisum (Harris) and Diuraphis noxia (Mordvilko). The qRT-PCR results indicated that Mpburs-α and Mpburs-β expressed during the entire developmental stage of M. persicae and arrived at a peak expression level in the 1st instar nymph. The expression levels of the two genes in the winged adult aphids were significantly higher than those in the wingless adult aphids suggesting an important role played by bursicon in the wing development for the insect. The finding might lead to further studies on the functions of bursicon in the molting epidermal ossification and wing formation of M. persicae.
-
Key words:
- Myzus persicae(Sulzer) /
- bursicon /
- gene cloning /
- expression analysis
-
图 1 桃蚜burs-α全长序列及氨基酸序列
注：3个糖基化位点的氨基酸用方框标识，3个蛋白激酶磷酸化位点的氨基酸残基用下划波浪线标识，信号肽用下划直线标识。
Figure 1. Full-length amino acid sequence of burs-α in M. persicae
图 2 桃蚜burs-β全长序列及氨基酸序列
注：2个蛋白激酶磷酸化位点的氨基酸残基用下划波浪线标识，信号肽用下划直线标识。
Figure 2. Full-length amino acid sequence of burs-β in M. persicae
图 3 桃蚜与豌豆蚜Bursicon基因三级结构预测
注：A为桃蚜burs-α; B为豌豆蚜burs-α; C为桃蚜burs-β; D为豌豆蚜burs-β。
Figure 3. Tertiary structures of bursicon in M. persicae and A. pisum
图 4 桃蚜burs-α蛋白和其他昆虫的系统发育树
Figure 4. Phylogenetic trees on burs-α amino acid sequence of M. persicae and other insects
图 5 桃蚜burs-β蛋白和其他昆虫的系统发育树
Figure 5. Phylogenetic trees on burs-β amino acid sequence of M. persicae and other insects
图 6 桃蚜不同龄期Mpburs-α基因的表达水平
注:不同字母表示差异显著(P＜0.05)，图 7同。
Figure 6. Expression levels of Mpburs-α in different developmental stages of M. persicae
图 7 桃蚜不同龄期Mpburs-β基因的表达水平
Figure 7. Expression levels of Mpburs-β in different developmental stages of M. persicae
表 1 桃蚜burs-α和burs-β基因扩增及实时荧光定量PCR引物信息
Table 1. Primers used for amplification and qRT-PCR of burs-α and burs-β in M. persicae
扩增区域 引物名称 序列(5′-3′) 中间片段 Mpburs-αF GTCTACCATCAACCAGGAAT Mpburs-αR GCGGCATAGTTGGACATT Mpburs-βF ACCCAGAAGAATGTGAGAC Mpburs-βR GGTCAATCTTACGCCATC 5′末端扩增 5′Mpburs-αR1: GCGATACCGAAGCTTCTCGTTC 5′Mpburs-αR2: CTGCTGCAGCGACCTGTACATG 5′Mpburs-αR3: GGCGTTACTTGGCAGTCGTCGC 5′Mpburs-βR1: GCAATGTGTAAGAGCGATCACTC 5′Mpburs-βR2: CCACTAGGAGTAATGACACTAG 5′Mpburs-βR3: CTTGTTCACTGCTATGTCACC 3′末端扩增 3′Mpburs-αF1 TGGTCGTGACCGCTAGGTCTAGC 3′Mpburs-αF2 GAACGAGAAGCTTCGGTATCGC 3′Mpburs-αF3 CACTGGAATGCATGTGCCGC 3’ Mpburs-βF1 GAACTTGGACGGCTTCAGAGGAC 3′Mpburs-βF2 GGAGCGTGCAATTCACAAGTAC 3′Mpburs-βF3 GAGAGAGAGAGTGATCGCTC 荧光定量 yg-Mpburs-αF GTCATCCATGTTCTACAATAC yg-Mpburs-αR CTTCTCGTTCACCACTCTC yg-Mpburs-βF TGGTGACATAGCAGTGAAC yg-Mpburs-βR GGATCGTAGCAATGTGTAAG 内参基因 actin-F TGGTATCGTCTTGGATTCTG actin-R TTAGGTAGTCGGTGAGATCA 
下载: 导出CSV
-
[1] 秦焕菊, 王桂芬.我国烟田蚜虫与病毒病害[J].中国烟草学报, 1996, 3(3):75-78. http://www.cqvip.com/QK/85595X/199602/3001131597.html [2] DEVONSHIRE A L, FIELD L M, FOSTER S P, et al. The evolution of insecticide resistance in the peach-potato aphid, Myzus persicae[J]. Philosophical Transactions of the Royal Society B:Biological Sciences, 1998, 353(1376):1677-1684. doi: 10.1098/rstb.1998.0318 [3] AN S, DONG S, WANG Q, et al. Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish[J]. PLoS One, 2012, 7(3):200-206. https://www.ncbi.nlm.nih.gov/pubmed/22470576 [4] 朱斌, 刘孝明, 杜孟芳, 等.昆虫鞣化激素研究进展[J].昆虫学报, 2013, 56(12):1469-1479. http://www.insect.org.cn/CN/abstract/abstract13826.shtml [5] DAVIS M M, O'KEEFE S L, PRIMROSE D A, et al. A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster[J]. Development, 2007, 134(24):4395-4404. doi: 10.1242/dev.009902 [6] 王荫长.昆虫生理学[M].北京:中国农业出版社, 2004:7-15. [7] FRAENKEL G, HSIAO C. Bursicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects[J]. Journal of Insect Physiology, 1965, 11(5):513-556. doi: 10.1016/0022-1910(65)90137-X [8] COTTRELL C B. The imaginal ecdysis of blowflies. Detection of the blood-borne darkening factor and determination of some of its properties[J]. Journal of Experimental Biology, 1962, 39(3):413-430. http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.550.5527 [9] COTTRELL C B. The Imaginal Ecdysis of Blowflies. The Control of Cuticuar Hardening and Darkening[J]. Journal of Experimental Biology, 1962, 39(3):395-412. http://jeb.biologists.org/content/39/3/395.short [10] PEABODY N C, DIAO F, LUAN H, et al. Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death[J]. Journal of Neuroscience, 2008, 28(53):14379-14391. doi: 10.1523/JNEUROSCI.2842-08.2008 [11] AN S, WANG S, GILBERT L I, et al. Global identification of bursicon-regulated genes in Drosophila melanogaster[J]. BMC genomics, 2008, 9(1):424-437. doi: 10.1186/1471-2164-9-424 [12] ARAKANE Y, LI B, MUTHUKRISHNAN S, et al. Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum[J]. Mechanisms of development, 2008, 125(11):984-995. https://www.ncbi.nlm.nih.gov/pubmed/18835439 [13] DAI L, DEWEY E M, ZITNAN D, et al. Identification, developmental expression, and functions of bursicon in the tobacco hawkmoth, Manduca sexta[J]. Journal of Comparative Neurology, 2008, 506(5):759-774. doi: 10.1002/(ISSN)1096-9861 [14] VAN LOY T, VAN HIEL M B, VANDERSMISSEN H P, et al. Evolutionary conservation of bursicon in the animal kingdom[J]. General and comparative endocrinology, 2007, 153(1):59-63. https://www.sciencedirect.com/science/article/pii/S001664800600387X [15] WILCOCKSON D C, WEBSTER S G. Identification and developmental expression of mRNAs encoding putative insect cuticle hardening hormone, bursicon in the green shore crab Carcinus maenas[J]. General and comparative endocri-nology, 2008, 156(1):113-125. doi: 10.1016/j.ygcen.2007.12.003 [16] RICHARDS S, GIBBS R A, WEINSTOCK G M, et al. The genome of the model beetle and pest Tribolium castaneum[J]. Nature, 2008, 452(7190):949-955. doi: 10.1038/nature06784 [17] DEWEY E M, MCNABB S L, EWER J, et al. Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading[J]. Current biology, 2004, 14(13):1208-1213. doi: 10.1016/j.cub.2004.06.051 [18] LUO C W, DEWEY E M, SUDO S, et al. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(8):2820-2825. doi: 10.1073/pnas.0409916102 [19] MENDIVE F M, VAN LOY T, CLAEYSEN S, et al. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2[J]. FEBS letters, 2005, 579(10):2171-2176. doi: 10.1016/j.febslet.2005.03.006 [20] 刘孝明, 燕赛英, 孙成贤, 等.禾谷缢管蚜鞣化激素基因克隆及功能分析[J].昆虫学报, 2015, 58(2):103-114. http://www.oalib.com/paper/4229663 [21] 杨科, 许益鹏, 董胜张, 等.灰飞虱Bursico基因的克隆, 序列分析及在不同发育阶段的表达[J].昆虫学报, 2012, 55(11):1255-1263. http://www.oalib.com/paper/4225688 [22] 马小丽, 何玮毅, 尤燕春, 等.小菜蛾鞣化激素基因克隆及其功能分析[J].昆虫学报, 2013, 56(10):1101-1109. http://www.insect.org.cn/CN/abstract/abstract13785.shtml [23] COSTA C P, ELIAS-NETO M, FALCON T, et al. RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera[J]. PloS one, 2016, 11(12):e0167421. doi: 10.1371/journal.pone.0167421 [24] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262 [25] VITT U A, HSU S Y, HSUEH A J W. Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules[J]. Molecular endocri-nology, 2001, 15(5):681-694. doi: 10.1210/mend.15.5.0639 [26] HUANG J, ZHANG Y, LI M, et al. RNA interference-mediated silencing of the bursicon gene induces defects in wing expansion of silkworm[J]. FEBS letters, 2007, 581(4):697-701. doi: 10.1016/j.febslet.2007.01.034 [27] WANG S, AN S, SONG Q. Transcriptional expression of bursicon and novel bursicon-regulated genes in the house fly Musca domestica[J]. Archives of insect biochemistry and physiology, 2008, 68(2):100-112. doi: 10.1002/(ISSN)1520-6327 [28] LOVEALL B J, DEITCHER D L. The essential role of bursicon during Drosophila development[J]. BMC developmental biology, 2010, 10(1):92-108. doi: 10.1186/1471-213X-10-92 [29] ISHAAYA I, PALLI S R, HOROWITZ A R. Advanced technologies for managing insect pests[M]. Springer Science & Business Media. 2012, 90-93. [30] KIMURA K I, KODAMA A, HAYASAKA Y, et al. Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster[J]. Development, 2004, 131(7):1597-1606. doi: 10.1242/dev.01049 [31] BAI H, PALLI S R. Functional characterization of bursicon receptor and genome-wide analysis for identification of genes affected by bursicon receptor RNAi[J]. Developmental biology, 2010, 344(1):248-258. doi: 10.1016/j.ydbio.2010.05.003 [32] HONEGGER H W, EST VEZ-LAO T Y, HILLYER J F. Bursicon-expressing neurons undergo apoptosis after adult ecdysis in the mosquito Anopheles gambiae[J]. Journal of insect physiology, 2011, 57(7):1017-1022. doi: 10.1016/j.jinsphys.2011.04.019 -
点击查看大图
图(7) / 表(1)
计量
- 文章访问数: 1069
- HTML全文浏览量: 153
- PDF下载量: 100
- 被引次数: 0
