棉蚜ATP合成酶基因AgoATPb的克隆与表达

doi:10.3969/j.issn.1004-1524.2022.02.14

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (2): 329-336.DOI: 10.3969/j.issn.1004-1524.2022.02.14

棉蚜ATP合成酶基因AgoATPb的克隆与表达

杨卫军¹^,²(), 董艳蕾¹, 吴秋芳¹^,², 张美玲¹^,², 韩丽滨¹^,², 张元臣¹^,²^,^*()

1.安阳工学院生物与食品工程学院,河南安阳 455000
2.河南省太行山林业有害生物野外科学观测研究站,河南林州 456550

收稿日期:2021-08-03 出版日期:2022-02-25 发布日期:2022-03-02
作者简介:张元臣,E-mail: zhangyc2011@163.com
杨卫军(1977—),男,河南安阳人,硕士,讲师,主要从事有害生物防治研究。E-mail: 251461879@qq.com
通讯作者: 张元臣
基金资助:
安阳工学院博士启动基金(BSJ2019001);安阳工学院博士启动基金(BSJ2019021);国家自然科学基金(31802007)

Cloning and expression analysis of AgoATPb gene in cotton-melon aphid, Aphis gossypi

YANG Weijun¹^,²(), DONG Yanlei¹, WU Qiufang¹^,², ZHANG Meiling¹^,², HAN Libin¹^,², ZHANG Yuanchen¹^,²^,^*()

1. College of Biological and Food Engineering, Anyang Institude of Technology, Anyang 455000, Henan, China
2. Taihang Mountain, Forest Pests Observation and Research Station of Henan Province, Linzhou 456550, Henan, China

Received:2021-08-03 Online:2022-02-25 Published:2022-03-02
Contact: ZHANG Yuanchen

摘要/Abstract

摘要：

为确定棉蚜(Aphis gossypii)ATP合成酶B亚基基因在棉蚜不同组织和日龄,以及取食不同植物的表达情况,以棉蚜为研究对象,采用RT-PCR和RACE技术获得AgoATPb的全长cDNA序列,通过Expasy、SignalP-4.0 Server等在线工具对其进行了生物信息学分析,同时利用实时荧光定量PCR技术研究了该基因在棉蚜不同组织和不同日龄,以及在不同植物上取食的表达水平。结果表明,棉蚜AgoATPb基因全长cDNA序列为1 247 bp,开放阅读框(ORF)长度为822 bp,编码274个氨基酸,5'端非编码区长128 bp,3'端非编码区长297 bp,理论分子量为31.40 ku,等电点为8.95,无信号肽和跨膜区域。氨基酸序列比对结果表明,棉蚜AgoATPb与其他昆虫同源基因编码蛋白的氨基酸序列一致性为47%~99%。除了不在胚胎表达外,AgoATPb基因在棉蚜其他日龄和不同组织均有表达,且在不同日龄间与不同组织表达水平存在显著差异。取食不同植物后棉蚜AgoATPb基因表达水平存在显著差异,取食棉花表达水平最高,其次是黄瓜和西葫芦,取食甜瓜表达水平最低,推测该基因可能与棉蚜适应寄主植物有关。

关键词: 棉蚜, ATP合成酶B亚基, 生物信息学, 基因克隆, 寄主

Abstract:

To evaluate the effects of tissue, age and host plant on expression of ATP synthase subunit B gene (AgoATPb) of Aphis gossypii, cotton aphid was taken as the research object, full-length cDNA sequence of AgoATPb gene was obtained by RT-PCR and RACE (rapid amplification cDNA ends) technology, and the bioinformatics analysis was carried out using online tools such as Expasy and SignalP-4.0 Server. Expression level of AgoATPb gene was also studied by real-time quantitative PCR technology in different tissues and different day ages of cotton aphid and when feeding on different plants. Full-length cDNA sequence of AgoATPb gene was 1 247 bp, and its open reading frame (ORF) was 822 bp, encoding 273 amino acids. The 5' noncoding region and 3' noncoding region of AgoATPb gene were 128 bp and 297 bp, respectively. Theoretical molecular weight and isoelectric point of amino acid sequence coded by AgoATPb gene were 31.40 ku and 8.95, respectively. There were no signal peptide and no transmembrane region in AgoATPb.Amino acid sequence analysis showed that the sequence identity of AgoATPb with the homologous protein from other insect were between 47% and 99%. Apart from not expressed in the embryonic stage, AgoATPb gene was transcribed throughout all developmental stages and tissues of A. gossypii, but expression levels significantly differed among the developmental stages and among the different tissues. After feeding on different plants, the highest levels of AgoATPb expression were observed on cotton, followed by cucumber and zucchin, and the lowest expression levels was observed on melon. It was speculated that AgoATPb gene might be related to adaptation of cotton aphids to host plants.

Key words: cotton aphid, ATP synthase subunit B, bioinformatics, gene cloning, host

中图分类号:

S435.622⁺.1

杨卫军, 董艳蕾, 吴秋芳, 张美玲, 韩丽滨, 张元臣. 棉蚜ATP合成酶基因AgoATPb的克隆与表达[J]. 浙江农业学报, 2022, 34(2): 329-336.

YANG Weijun, DONG Yanlei, WU Qiufang, ZHANG Meiling, HAN Libin, ZHANG Yuanchen. Cloning and expression analysis of AgoATPb gene in cotton-melon aphid, Aphis gossypi[J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 329-336.

图/表 7

表1 棉蚜AgoATPb基因克隆与qRT-PCR引物

Table 1 Primers used in cloning and qRT-PCR of AgoATPb gene

用途Purpose	引物 Primers	引物序列Primer sequences(5'-3')
部分AgoATPb序列的克隆	ATPb-1F	ATGTTATCCAGATTGGCTC
Cloning of partial AgoATPb gene	ATPb-1R	CAGTACGTGTCAAGTTAGCC
AgoATPb基因5'扩增	ATPb-5'GST	CAAGATCACGTTCGGGTCCGTCATAAG
5' RACE of AgoATPb gene	ATPb-5'NGST	CAGTGGTGCTACTCTGAACAGTTCTG
AgoATPb基因3'扩增	ATPb-3'GST	CATGAGTTCCCTTATGTGTTGGCTAC
3' RACE of AgoATPb gene	ATPb-3'NGST	CGCGAACGTGCTCTTCAAGCCTACAAC
AgoATPb基因的qRT-PCR	ATPb-2F	TGACTTGACCGACTACTTGATG
qRT-PCR for AgoATPb	ATPb-2R	TCCAAAGCGACATAGCACAA
内参基因	β-actin-F	TGACTTGACCGACTACTTGATG
Reference gene(β-actin)	β-actin-R	TCCAAAGCGACATAGCACAA

图1 AgoATPb基因全长cDNA序列与氨基酸序列红色代表为起始密码子;*代表终止密码子;黑色横线部分为加尾信号(AATAAA)。

Fig.1 Nucleotide and deduced amino acid sequences of AgoATPb from Aphis gossypii Red letter, * and the horizontal line were start codon, stop codon and tail signal (AATAAA), respectively.

图2 棉蚜AgoATPb和其他物种ATP合成酶B亚基氨基酸序列的系统发育树采用邻接法,自举检验1 000次;图中标尺为遗传距离。

Fig.2 Phylogenetic tree based on amino acid of ATP synthase subunit B from A. gossypii and other insects Neighbor Joining method with 1 000 bootstrap replicates. The scale bar represented the genetic distance.

图3 AgoATPb与其他昆虫ATP合成酶B亚基氨基酸序列的多重联配图黑色横线部分代表ATP合成酶B亚基的典型结构域。黑色阴影为氨基酸具有100%一致性,灰色表示一致性在50%以上,白色表示一致性在50%以下。AaeATPb,埃及伊蚊;CquATPb,致倦库蚊;GmeATPb,大蜡螟;SfrATPb,草地贪夜蛾;AgoATPb,棉蚜;MpeATPb,桃蚜;ApiATPb,豌豆蚜。

Fig.3 Amino acid sequence alignment of AgoATPb with ATP synthase subunit B from other insects The black lines represented typical domain of ATP synthase. Amino acids with 100% identity were in black box, those with over 50% identity in grey box and with below 50% identity in white box. AaeATPb, Aedes aegypti; CquATPb, Culex quinquefasciatus; GmeATPb, Galleria mellonella; SfrATPb, Spodoptera frugiperda; AgoATPb, Aphis gossypii; MpeATPb, Myzus persicae; ApiATPb, Acyrthosiphon pisum.

图4 AgoATPb基因在4日龄棉蚜不同部位的表达图中的数值用平均值±标准误表示,柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.4 AgoATPb expression patterns in different parts of 4th day-old A. gossypii Values in the figure were represented by x -±s. Data on the bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图5 AgoATPb基因在胚胎和不同日龄棉蚜中表达水平

Fig.5 Expression level of AgoATPb in different day-age and embryo of Aphis gossypii

图6 不同寄主植物上4日龄棉蚜体内AgoATPb基因的表达量

Fig.6 Expression levels of AgoATPb in Aphis gossypii of 4 day-old reared on different host plants

参考文献 28

[1]	ZHANG Y C, LEI H X, MIAO N H, et al. Comparative transcriptional analysis of the host-specialized aphids Aphis gossypii(Hemiptera: Aphididae)[J]. Journal of Economic Entomology, 2017, 110(2):702-710. DOI URL
[2]	雒珺瑜, 张帅, 任相亮, 等. 近十年我国棉花虫害研究进展[J]. 棉花学报, 2017, 29(S1):100-112.
	LUO J Y, ZHANG S, REN X L, et al. Research progress of cotton insect pests in China in recent ten years[J]. Cotton Science, 2017, 29(S1):100-112.(in Chinese with English abstract)
[3]	PRICE T, VALVERDE R, SINGH R, et al. First report of cotton leafroll dwarf virus in Louisiana[J]. Plant Health Progress, 2020, 21(2):142-143. DOI URL
[4]	WANG Z J, LIANG C R, SHANG Z Y, et al. Insecticide resistance and resistance mechanisms in the melon aphid, Aphis gossypii, in Shandong, China[J]. Pesticide Biochemistry and Physiology, 2021, 172:104768. DOI URL
[5]	芦屹, 王佩玲, 刘冰, 等. 新疆棉花主栽品种的抗蚜性及其机制研究[J]. 棉花学报, 2009, 21(1):57-63.
	LU Y, WANG P L, LIU B, et al. Resistance and relevant mechanism to Aphis gossypii glover of main cotton varieties in Xinjiang[J]. Cotton Science, 2009, 21(1):57-63.(in Chinese with English abstract)
[6]	杨田甜, 杜海荣, 陈刚, 等. 植物化感作用的研究现状及其在农业生产中的应用[J]. 浙江农业学报, 2012, 24(2):343-348.
	YANG T T, DU H R, CHEN G, et al. Current research on plant allelopathy and its application in agricultural production[J]. Acta Agriculturae Zhejiangensis, 2012, 24(2):343-348.(in Chinese with English abstract)
[7]	JUNGE W, NELSON N. ATP synthase[J]. Annual Review of Biochemistry, 2015, 84(1):631-657. DOI URL
[8]	HE J Y, FORD H C, CARROLL J, et al. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(13):3409-3414.
[9]	KAWASAKI I, HANAZAWA M, GENGYO-ANDO K, et al. ASB-1, a germline-specific isoform of mitochondrial ATP synthase b subunit, is required to maintain the rate of germline development in Caenorhabditis elegans[J]. Mechanisms of Development, 2007, 124(3):237-251. DOI URL
[10]	SAWYER E M, BRUNNER E C, HWANG Y, et al. Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila[J]. BMC Cell Biology, 2017, 18(1):16. DOI URL
[11]	HAHN A, VONCK J, MILLS D J, et al. Structure, mechanism, and regulation of the chloroplast ATP synthase[J]. Science, 2018, 360(6389):1-10.
[12]	PARIS M, MELODELIMA C, COISSAC E, et al. Transcription profiling of resistance to Bti toxins in the mosquito Aedes aegypti using next-generation sequencing[J]. Journal of Invertebrate Pathology, 2012, 109(2):201-208. DOI URL
[13]	王卫杰, 刘新颖, 方福瑾, 等. 淡色库蚊ATP合酶B亚基基因克隆和序列分析及其与溴氰菊酯抗性的关系[J]. 昆虫学报, 2017, 60(8):900-905.
	WANG W J, LIU X Y, FANG F J, et al. Cloning and sequence analysis of ATP synthase B subunit gene and its association with deltamethrin resistance in Culex pipiens Pallens (DIPTERA: CULICIDAE)[J]. Acta Entomologica Sinica, 2017, 60(8):900-905.(in Chinese with English abstract)
[14]	CHEN Y N, WU C H, ZHENG Y, et al. Knockdown of ATPsyn-b caused larval growth defect and male infertility in Drosophila[J]. Archives of Insect Biochemistry and Physiology, 2015, 88(2):144-154. DOI URL
[15]	冯娅琳, 郝培应, 俞飞飞, 等. 褐飞虱ATP合酶b亚基基因ATPSb的克隆与功能分析[J]. 昆虫学报, 2018, 61(5):519-526.
	FENG Y L, HAO P Y, YU F F, et al. Molecular cloning and function analysis of ATP synthase b subunit gene ATPSb in the brown planthopper, Nilaparvata lugens(Hemiptera: Delphacidae)[J]. Acta Entomologica Sinica, 2018, 61(5):519-526.(in Chinese with English abstract)
[16]	MUMMERY-WIDMER J L, YAMAZAKI M, STOEGER T, et al. Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi[J]. Nature, 2009, 458(7241):987-992. DOI URL
[17]	CAROLAN J C, CARAGEA D, REARDON K T, et al. Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach[J]. Journal of Proteome Research, 2011, 10(4):1505-1518. DOI URL
[18]	BOULAIN H, LEGEAI F, GUY E, et al. Fast evolution and lineage-specific gene family expansions of aphid salivary effectors driven by interactions with host-plants[J]. Genome Biology and Evolution, 2018, 10(6):1554-1572. DOI URL
[19]	WU Z Z, QU M Q, CHEN M S, et al. Proteomic and transcriptomic analyses of saliva and salivary glands from the Asian Citrus psyllid, Diaphorina citri[J]. Journal of Proteomics, 2021, 238:104136. DOI URL
[20]	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 URL
[21]	YANAI I, KORBEL J O, BOUE S, et al. Similar gene expression profiles do not imply similar tissue functions[J]. Trends in Genetics, 2006, 22(3):132-138. DOI URL
[22]	ARRESE E L, SOULAGES J L. Insect fat body: energy, metabolism, and regulation[J]. Annual Review of Entomology, 2010, 55:207-225. DOI URL
[23]	LI S, YU X Q, FENG Q L. Fat body biology in the last decade[J]. Annual Review of Entomology, 2019, 64(1):315-333. DOI URL
[24]	严盈, 刘万学, 万方浩. 唾液成分在刺吸式昆虫与植物关系中的作用[J]. 昆虫学报, 2008, 51(5):537-544.
	YAN Y, LIU W X, WAN F H. Roles of salivary components in piercing-sucking insect-plant interactions[J]. Acta Entomologica Sinica, 2008, 51(5):537-544.(in Chinese with English abstract)
[25]	LIN P A, CHEN Y T, CHAVERRA-RODRIGUEZ D, et al. Silencing the alarm: an insect salivary enzyme closes plant stomata and inhibits volatile release[J]. The New Phytologist, 2021, 230(2):793-803. DOI URL
[26]	HEIDEL-FISCHER H M, FREITAK D, JANZ N, et al. Phylogenetic relatedness and host plant growth form influence gene expression of the polyphagous comma butterfly (Polygonia calbum)[J]. BMC Genomics, 2009, 10:506. DOI URL
[27]	MACK K L, NACHMAN M W. Gene regulation and speciation[J]. Trends in Genetics, 2017, 33(1):68-80. DOI URL
[28]	ROMERO I G, RUVINSKY I, GILAD Y. Comparative studies of gene expression and the evolution of gene regulation[J]. Nature Reviews Genetics, 2012, 13(7):505-516. DOI URL

棉蚜ATP合成酶基因AgoATPb的克隆与表达

Cloning and expression analysis of AgoATPb gene in cotton-melon aphid, Aphis gossypi

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈亚飞, 史肖肖, 余水生, 叶碧欢, 宋其岩, 沈建军, 陈友吾. 浙江省遂昌九龙山国家级自然保护区虫生真菌资源调查[J]. 浙江农业学报, 2025, 37(9): 1891-1904.
[2]	缪百灵, 陈娟娟, 李亮杰, 楚宗丽, 董向向. 浙江红花油茶CchABCG5基因的功能[J]. 浙江农业学报, 2025, 37(7): 1407-1416.
[3]	狄延翠, 嵇泽琳, 王媛媛, 娄世浩, 张涛, 国志信, 申顺善, 朴凤植, 杜南山, 董晓星, 董韩. 番茄SlMYB52基因鉴定、亚细胞定位及表达分析[J]. 浙江农业学报, 2025, 37(4): 808-819.
[4]	张美莹, 莫倩, 齐秀双, 佟宁宁, 孔凡, 刘政安, 吕长平, 彭丽平. 牡丹PoLPAT2基因的克隆及表达分析[J]. 浙江农业学报, 2025, 37(2): 321-328.
[5]	崔博文, 张思懿, 王佳玲, 王竞红, 蔺吉祥, 杨青杰. 宽叶苔草WRKY家族成员生物信息学分析与耐旱基因挖掘[J]. 浙江农业学报, 2025, 37(10): 2087-2103.
[6]	何晓婵, 周小军, 朱浩, 龚婉, 张敬泽, 朱丽燕. 莴苣盘梗霉菌自然寄主专化性的分化及其对莴苣的侵染性[J]. 浙江农业学报, 2025, 37(1): 126-133.
[7]	孙培媛, 冉彬, 王佳蕊, 李洪有. 苦荞FtDELLA基因的克隆与表达分析[J]. 浙江农业学报, 2024, 36(8): 1709-1718.
[8]	朱贵爽, 李艳肖, 张安宁, 孙浩楠, 徐兴源, 李志刚, 向殿军. 蓖麻GeBP转录因子的全基因组鉴定与GeBP2基因的克隆、表达分析[J]. 浙江农业学报, 2024, 36(8): 1731-1740.
[9]	蒋文骏, 舒红锁, 陈正满, 任典挺, 杨党, 田荣江, 杜照奎. 秋茄KoWRKY43基因克隆、表达与生物信息学分析[J]. 浙江农业学报, 2024, 36(8): 1832-1843.
[10]	李亚萍, 金福来, 黄宗贵, 张涛, 段晓婧, 姜武, 陶正明, 陈家栋. 铁皮石斛糖苷水解酶GH3基因家族鉴定及表达模式分析[J]. 浙江农业学报, 2024, 36(4): 790-799.
[11]	张露荷, 王多锋, 张德, 张广忠, 赵通, 吕斌燕, 张洋军, 李毅. 枣树novel-miR16靶基因ZjTCP4鉴定及生物信息学分析[J]. 浙江农业学报, 2024, 36(3): 534-543.
[12]	张余, 金明伟, 任丽, 章毅颖, 赵洪, 刘昆, 邓姗, 褚云霞, 李寿国, 张靖立, 黄静艳, 陈海荣. 辣椒CaERF70的表达特征和转录自激活活性分析[J]. 浙江农业学报, 2024, 36(10): 2247-2256.
[13]	宋传生, 康晓飞, 樊庆忠, 王俊刚, 石雪, 张子汝, 谭青青, 曾小娇, 刘芳, 李英赛, 侯常跃. 枣疯病植原体胸苷激酶基因的克隆、序列分析与原核表达[J]. 浙江农业学报, 2023, 35(8): 1763-1772.
[14]	张丽, 王媛媛, 王瑞, 刘丽霞. 牦牛DRA基因克隆测序及生物信息学分析[J]. 浙江农业学报, 2023, 35(7): 1564-1570.
[15]	刘光瑞, 宗渊, 李云, 曹东, 刘宝龙, 包雪梅, 李建民. 当归转录因子AsMYB44的克隆与功能研究[J]. 浙江农业学报, 2023, 35(6): 1253-1264.