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

doi:10.3969/j.issn.1004-1524.2022.02.14

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (2): 329-336.DOI: 10.3969/j.issn.1004-1524.2022.02.14

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S435.622⁺.1

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.

Figures/Tables 7

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

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.

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.

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.

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.

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

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

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

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