西花蓟马几丁质合成酶<i>UAP</i>基因克隆及功能分析

陆承聪; 李恒; 张祥琴; 王谅; 陈艺欣; 田厚军; 林硕; 张洁; 陈勇; 魏辉

doi:10.19303/j.issn.1008-0384.2019.12.009

西花蓟马几丁质合成酶UAP基因克隆及功能分析

doi: 10.19303/j.issn.1008-0384.2019.12.009

陆承聪^1,,
李恒^{1, 2},
张祥琴^{1, 2},
王谅^{1, 2},
陈艺欣¹,
田厚军¹,
林硕¹,
张洁³,
陈勇¹,
魏辉^{1, 2, ,}

1.
福建省农业科学院植物保护研究所/福建省作物有害生物监测与治理重点实验室/农业农村部福州作物有害生物科学观测试验站，福建　福州　350003
2.
闽台作物有害生物生态防控国家重点实验室，福建　福州　350002
3.
云南省农业科学院生物技术与种质资源研究所/云南省农业生物技术重点实验室/农业农村部西南作物基因资源与种质创制重点实验室，云南　昆明　650223

基金项目: 国家重点研发计划项目（2018YFD0201102、2018YFD0201209）；国家自然科学基金项目（31871936、31560499）；福建省科技重大专项（2017NZ0003-1-4）；福建省科技计划公益类专项（2017R1025-9、2018R1025-7、2019R1024-2、2019R1024-3）；福建省农业科学院科技项目（STIT2017-3-2、AGY2018-5、AC2017-6、YC2019003）；云南省应用基础研究计划面上项目（2016FB063）；云南省应用基础研究计划重点项目（2018FA020）；云南省中青年学术技术带头人后备人才项目（2015HB081）

详细信息

作者简介:
陆承聪（1990−），男，硕士，研究方向：昆虫生态与害虫综合治理（E-mail：470258916@qq.com）

通讯作者:
魏辉（1972−），男，博士，研究员，研究方向：昆虫化学生态学（E-mail：weihui@faas.cn）

中图分类号: S 435
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出版历程
- 收稿日期: 2019-10-12
- 修回日期: 2019-11-29
- 刊出日期: 2019-12-01

Molecular Cloning and Functional Analysis of UDP N-acetylglucosamine Pyrophosphorylases Gene in Frankliniella occidentalis

LU Cheng-cong^1
,,
LI Heng^{1, 2},
ZHANG Xiang-qin^{1, 2},
WANG Liang^{1, 2},
CHEN Yi-xin¹,
TIAN Hou-jun¹,
LIN Shuo¹,
ZHANG Jie³,
CHEN Yong¹,
WEI Hui^{1, 2
, ,}

1.
Institute of Plant Protection, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests/Fuzhou Scientific Observing and Experimental Station of Crop Pests of Ministry of Agriculture and Rural Affaris, Fuzhou, Fujian　350003, China
2.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou, Fujian　350002, China
3.
Biotechnology and Germplasm Resource Institute, Yunnan Academy of Agricultural Sciences/Yunnan Provincial Key Laboratory of Agricultural Biotechnology/Key Laboratory for Gene Resources and Germplasm Innovation of Southwest Crops, Ministry of Agriculture and Rural Affairs, Kunming, Yunnan　650223, China

摘要

摘要: 目的阐明几丁质合成通路中的关键基因UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UDP N-acetylglucosamine pyrophosphorylases, UAP)对西花蓟马Frankliniella occidentalis生长发育的影响。方法基于西花蓟马转录组数据，克隆验证了UAP基因的开放阅读框(Open Reading Frame, ORF)全长序列，命名为FoccUAP。利用RT-qPCR方法分析FoccUAP在各个组织和不同龄期的表达情况。最后通过向西花蓟马蜕皮24 h的2龄若虫显微注射dsUAP，检测沉默效率，观察其对西花蓟马生长发育的影响。结果 FoccUAP基因开放阅读框全长1 545 bp，编码514个氨基酸，预测蛋白分子量为56.738 kDa，具有Glyco - tranf - GTA - type Super family家族的保守结构域，与等翅目、蜚蠊目、直翅目和半翅目昆虫的UAP基因亲缘关系较近。FoccUAP基因在西花蓟马各个组织都有表达，在头部和腹部相对表达量最高，触角、胸部和足相对表达量较低。该基因在虫体生长发育的各个阶段均有表达，其中2龄幼虫、蛹以及羽化后24、48、168、240 h的成虫表达量分别是1龄幼虫的1.30、2.50、1.56、2.0、2.43和2.56倍。RNAi结果表明，注射dsUAP的西花蓟马羽化率（36.3%）和注射后120 h的存活率（5.0%）均显著低于dsGFP对照组，且注射dsUAP的西花蓟马成虫出现翅膀发育不完整、胸腹部皱缩不规则等畸形现象。结论 FoccUAP基因对西花蓟马生长发育具有重要的作用。
- 西花蓟马 /
- UDP-N-乙酰氨基葡萄糖焦磷酸化酶 /
- 几丁质合成 /
- RNAi
Abstract: Objective Effects of UDP N-acetylglucosamine pyrophosphorylases (UAP) on the growth and development of Frankliniella occidentalis were studied. Method Based on a transcriptome dataset, an open reading frame (ORF) of UAP gene of F. occidentalis (FoccUAP) was cloned. Expressions of the FoccUAP in different body tissues and growth stages of the insect were detected by RT-qPCR. Effects of UAP gene on the growth and development were examined by microinjecting dsRAN in the 2^nd instars (eclosion about 24 h) of F. occidentalis. Result The full-length of FoccUAP ORF was 1 545 bp and encoded 514 amino acids with a predicted molecular mass of 56.738 kDa. FoccUAP contained the conserved domain of glyco-tranf-GTA-type super family, which had a close evolutionary relationship with the orthologues in Isoptera, Blattaria, Orthoptera, and Hemiptera. The RT-qPCR results showed that FoccUAP was highly expressed in the head and abdomen, detected in the antenna, thorax and leg, and found in all developmental stages of the insect. The relative transcript levels of FoccUAP in the 2^nd instar, pupa, adult-24 h, adult-48 h, adult-168 h, and adult-240 h were 1.30-, 2.50-, 1.56-, 2.0-, 2.43-, and 2.56-fold, respectively, of those in the 1^st instar. Further examination on the RNAi showed that the injection of dsUAP into the 2^nd instar F. occidentalis significantly decreased the eclosion rate (36.3%) and the survival rate (5.0% in 120 h after microinjection) as compared to dsGFP controls. Moreover, the dsUAP treatment also induced abnormal phenotypes on wings and abdomen–thorax of the thrips. Conclusion UAP played a significant role in the growth and development of F. occidentalis.
- Frankliniella occidentalis /
- UDP N-acetylglucosamine pyrophosphorylases /
- chitin biosynthesis /
- RNAi

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图 1 FoccUAP基因的核苷酸及预测的氨基酸序列

注：下划线部分为信号肽氨基酸序列；双下划线为蛋白保守结构域氨基酸序列；*为终止密码子。

Figure 1. Nucleotide and deduced amino acid sequence of FoccUAP

Note: The signal peptide sequence (MEHQYENLRN) is marked with underline. The conserved amino acid sequences of Glyco-tranf-GTA-type superfamily are marked with double underline. The TAA stop codon is marked with *.

下载: 全尺寸图片幻灯片

图 2 FoccUAP的进化树分析

Figure 2. Phylogenetic tree of FoccUAP

下载: 全尺寸图片幻灯片

图 3 FoccUAP基因在西花蓟马不同发育时期（A）和不同组织（B）的mRNA表达分析

注：数据上不同的字母表示差异显著（Tukey HSD test, P＜0.05）

Figure 3. Relative expression levels of FoccUAP mRNA in different developmental stages (A) and tissues (B) of F. occidentalis

Note: Different lowercase letters means significant difference (Tukey HSD test, P＜0.05).

下载: 全尺寸图片幻灯片

图 4 注射dsUAP对西花蓟马死亡率及生长发育的影响

注：*或者不同的字母表示差异显著（Tukey HSD test, P＜0.05）

Figure 4. Effects of dsUAP on growth and mortality rate of F. occidentalis

Note：* or different lowercase letters means significant difference (Tukey HSD test, P＜0.05).

下载: 全尺寸图片幻灯片

表 1 引物信息

Table 1. Primers used

引物名称 Primer name	序列 Sequence
FoccUAP-ORF-F	ATGGAGCACCAGTACGAAAACCTC
FoccUAP-ORF-R	TTAGTGAACACCATTTGATATCCCA
dsFoccUAP-F	TAATACGACTCACTATAGGGTACAAATGCTACCTGCACTGAAAGTAAGCT
dsFoccUAP-R	TAATACGACTCACTATAGGGTGGCAACATCATCCTGGATTCTCC
qFoccUAP-F	TCCGTTTACTGATAGTCTTGTTGTCTGGG
qFoccUAP-R	GCTGTGCGTGGGCAATCCTTTT
qβ-actin-F	CACCACCGCTGAGCGTGAAATCG
qβ-actin-R	GTGATGACCTGACCGTCGGGAAGC
dsGFP-F	TAATACGACTCACTATAGGGCGAGGAGCTGTTCACCGG
dsGFP-R	TAATACGACTCACTATAGGGTCCTCGATGTTGTGGCGG

下载: 导出CSV

参考文献(22)

[1]	BAILEY S F. The distribution of injurious thrips in the United States [J]. Journal of Economic Entomology, 1940, 33: 133−136. doi: 10.1093/jee/33.1.133
[2]	KIRK W D J, TERRY L I. The spread of the western flower thrips Frankliniella occidentalis (Pergande) [J]. Agricultural and Forest Entomology, 2003, 5(4): 301−310. doi: 10.1046/j.1461-9563.2003.00192.x
[3]	王海鸿, 雷仲仁, 李雪, 等. 西藏发现重要外来入侵害虫—西花蓟马 [J]. 植物保护, 2013, 39(1):181−183. doi: 10.3969/j.issn.0529-1542.2013.01.039 WANG H H, LEI Z R, LI X, et al. An important invasive pest, Frankliniella occidentalis, inspected in Tibet [J]. Plant Protection, 2013, 39(1): 181−183.(in Chinese) doi: 10.3969/j.issn.0529-1542.2013.01.039
[4]	程峻峰, 万方浩, 郭建英, 等. 外来有害入侵生物--西花蓟马 [J]. 中国生物防治, 2005, 21(2):74−79. CHENG J F, WAN F H, GUO J Y, et al. Newly invaded insect pest, Frankliniella occidentalis (Thysanoptera: Thripidae) [J]. Chinese Journal of Biological Control, 2005, 21(2): 74−79.(in Chinese)
[5]	MESCE K A, FAHRBACH S E. Integration of endocrine signals that regulate insect ecdysis [J]. Frontiers in Neuroendocrinology, 2002, 23(2): 179−199. doi: 10.1006/frne.2002.0228
[6]	COHEN E. Chitin synthesis and inhibition: a revisit [J]. Pest Management Science, 2001, 57(10): 946−950. doi: 10.1002/ps.363
[7]	CANDY DJ, KILBY BA. Studies on chitin synthesis in the desert locust [J]. Journal of Experimental Biological, 1962, 39: 129−140.
[8]	GLASER L, BROWN D H. The synthesis of chitin in cell-free extracts of Neurosporacrassa [J]. Journal of Biological Chemistry, 1957, 228: 729−742.
[9]	张文庆, 陈晓菲, 唐斌, 等. 昆虫几丁质合成及其调控研究前沿 [J]. 应用昆虫学报, 2011, 48(3):475−479. doi: 10.7679/j.issn.2095-1353.2011.084 ZHANG W Q, CHEN X F, TANG B, et al. Insect chitin biosynthesis and its regulation [J]. Chinese Journal of Entomology, 2011, 48(3): 475−479.(in Chinese) doi: 10.7679/j.issn.2095-1353.2011.084
[10]	刘晓健, 孙亚文, 崔淼, 等. 飞蝗海藻糖酶基因的分子特性及功能 [J]. 中国农业科学, 2016, 49(22):4375−4386. doi: 10.3864/j.issn.0578-1752.2016.22.010 LIU X J, SUN Y W, CUI M, et al. Molecular characteristics and functional analysis of trehalase genes in Locusta migratoria [J]. Scientia Agricultura Sinica, 2016, 49(22): 4375−4386.(in Chinese) doi: 10.3864/j.issn.0578-1752.2016.22.010
[11]	TONNING A, HELMS S, SCHWARZ H, et al. Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in drosophila [J]. Development, 2006, 133(2): 331−341.
[12]	ARAKANE Y, BAGUINON MC, JASRAPURIA S, et al. Both UDP-N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival and fecundity [J]. Insect Biochemistry and Molecular Biology, 2011, 4l(1): 42−50.
[13]	LIU X J, LI F, LI D Q, et al. Molecular and functional analysis of UDP-N-acetylglucosamine pyrophosphorylases from the migratory locust, Locusta migratoria [J]. PLoS One, 2013, 8(8): e71970. doi: 10.1371/journal.pone.0071970
[14]	陈洁, 陈宏鑫, 姚琼, 等. 甜菜夜蛾UAP的克隆、时空表达及RNAi研究 [J]. 中国农业科学, 2014, 47(7):1351−1361. doi: 10.3864/j.issn.0578-1752.2014.07.012 CHEN J, CHEN H X, YAO Q, et al. Molecular cloning, expression patterns and RNAi of UDP-N-acetylglucosamine pyrophosphorylase in Spodoptera exigua [J]. Scientia Agricultura Sinica, 2014, 47(7): 1351−1361.(in Chinese) doi: 10.3864/j.issn.0578-1752.2014.07.012
[15]	MIO T, YABE T, ARISAWA M, et al. The eukaryotic UDP-N- acetylglucosamine pyrophosphorylases gene cloning, protein expression, and catalytic mechanism [J]. Journal of Biological Chemistry, 1998, 273(23): 14392−14397. doi: 10.1074/jbc.273.23.14392
[16]	WANG-GILLAM A, PASTUSZAK I, STEWART M, et al. Identification and modification of the uridine-binding site of the UDP-GalNAc (GlcNAc) pyrophosphorylase [J]. Journal of Biological Chemistry, 2000, 275(2): 1433−1438. doi: 10.1074/jbc.275.2.1433
[17]	ARAKANE Y, BAGUINON M C, JASRAPURIA S, et al. Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneumare critical for molting, survical and fecundity [J]. Insect Biochemistry and Molecular Biology, 2011, 41(1): 42−50. doi: 10.1016/j.ibmb.2010.09.011
[18]	ARAKANE Y, MUTHUKRISHNAN S, Kramer K J, et al. The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix [J]. Insect Molecular Biology, 2005, 14(5): 453−463. doi: 10.1111/j.1365-2583.2005.00576.x
[19]	SCHIMMELPFENG K, STRUNK M, STORK T, et al. Mummy encodes an UDP-N-acetylglucosamine-dipohosphorylase and is required during Drosophila dorsal closure and nervous system development [J]. Mechanisms of Development, 2006, 123(6): 487−499. doi: 10.1016/j.mod.2006.03.004
[20]	SHEU K F. FREY E A UDP-glucose pyrophosphorylase stereochemical course of the reaction of glucose 1-phosphate with uridine-5’[1-Thiotriphosphate] [J]. Journal of Biological Chemistry, 1978, 253(10): 3378−3380.
[21]	STROMINGER J L, SMITH M S. The preparation of uridine diphospho acetylgalactosamine [J]. Journal of Biological Chemistry, 1959, 234(7): 1828−1829.
[22]	RAFAELA S G, ALEX G T, MARIA GBK. Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa [J]. Anais da Academia Brasileira de Ciencias, 2012, 84(2): 469−486. doi: 10.1590/S0001-37652012000200016