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
通讯作者: 张元臣
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
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

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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

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