Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (10): 2118-2131.doi: 10.3864/j.issn.0578-1752.2021.10.008

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning, Preparation of Antibody and Response Induced by 20-Hydroxyecdysone of Target of Rapamycin in Apolygus lucorum

TAN YongAn1(),ZHAO XuDong2,JIANG YiPing1,ZHAO Jing1,XIAO LiuBin1(),HAO DeJun2()   

  1. 1Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
    2Co-Innovation Center for the Sustainable Forestry in Southern China/College of Forestry, Nanjing Forestry University, Nanjing 210037
  • Received:2020-07-31 Accepted:2020-09-07 Online:2021-05-16 Published:2021-05-24
  • Contact: LiuBin XIAO,DeJun HAO E-mail:kellytan001@163.com;xlb@jaas.ac.cn;dejunhao@163.com

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Abstract:

【Objective】The objective of this study is to clone the target of rapamycin gene (TOR) of Apolygus lucorum (AlTOR), analyze the expression profile of AlTOR as well as its expression patterns after injection of 20-hydroxyecdysone (20E) and its inhibitor U73122, obtain the recombinant protein and antibody against AlTOR protein, and to lay the foundation for the further study of AlTOR protein function.【Method】The full-length of AlTOR was cloned by RACE method. Using qRT-PCR technique, the expression pattern of AlTOR was determined at different developmental stages and different tissues in A. lucorum, as well as its expression patterns after injection of 20E and U73122. To construct its prokaryotic expression pCzn1-AlTOR, T vector containing the target gene was digested with the restriction enzyme EcoR I and Xho I and subcloned. The recombinant plasmid containing target gene was specifically expressed after induction by IPTG. The target recombinant protein has over expressed and has been purified by using Ni-NTA agarose. The polyclonal antibody was obtained by rabbit immunity, cell fusion and ascites preparation, and Western blot was used to detect the specificity of antibody.【Result】The open reading frame of AlTOR encodes 435 amino acids. AlTOR protein has the typical domains including the SIN1 domain, highly conserved CRIM domain and PH domain. qRT-PCR results showed that AlTOR was expressed in 1-day-old to 16-day-old of A. lucorum, in which the expression level was the highest at 1-day-old. AlTOR was expressed in all tissues of A. lucorum female adults, and the fat body had the highest expression level. 20E could induce the expression of AlTOR at different day-old nymphs, and could also induce the up-regulated expression of AlTOR in the head, wing, ovary and fat body of the female adults, which was increased by 200.00%, 118.89%, 20.53% and 60.82%, respectively, while U73122 significantly suppressed AlTOR expression in the midgut and ovary. The vector containing AlTOR was digested and linked to the vector pCzn1. The recombinant plasmid pCzn1-AlTOR expressed the target recombinant protein of 36 kD after induction by IPTG. The 36 kD target protein from the strain containing AlTOR was obtained by the Ni-NTA agarose and used as the antigen, and one cell line polyclonal antibody could specifically bind to the AlTOR recombinant protein and the total protein of the 3rd instar nymph of A. lucorum, indicating that the prepared AlTOR polyclonal antibody has good specificity.【Conclusion】The expression of AlTOR shows the developmental stage- and tissue-specificity. After the induction of 20E and its inhibitor, AlTOR shows the opposite responses. The obtained polyclonal antibody against AlTOR recombinant protein is highly specific.

Key words: Apolygus lucorum, target of rapamycin (TOR), gene cloning, 20-hydroxyecdysone (20E), polyclonal antibody, expression

Table 1

Primer sequences used in this study"

引物名称/目的
Primer name/Purpose		 序列
Primer sequence (5′ to 3′)
克隆Cloning
5′-AlTOR-1 AACGACTGGGCAAGAA
5′-AlTOR-2 CAAGACGTTTGGGTAGCA
3′-AlTOR-1 AGAGACTCAGTCGCTCACTATCAAGA
3′-AlTOR-2 CATTGAGACCACACCCCAGACACA
AlTOR-F ATGGTGGAAAAATATGCTCG
AlTOR-R GCTAGCCAGTATACAACG
表达谱分析qRT-PCR
RT-AlTOR-F GGGGTCGTCAACTGGCAGAA
RT-AlTOR-R CCTCCTTCTTCTTGGGTGCGA
Al-β-Actin-F ACCTGTACGCCAACACCGT
Al-β-Actin-R TGGAGAGAGAGGCGAGGAT

Fig. 1

PCR products of 5′, 3′ end and full sequences from AlTOR"

Fig. 2

Nucleotide and deduced amino acid sequences of AlTOR Yellow shaded amino acids represent SIN1 domain of N-terminus, grey shaded amino acids represent highly conserved CRIM domain, and red shaded amino acids represent PH domain of the C-terminus associated with lipid-membrane binding"

Fig. 3

Amino acid sequence alignment of AlTOR and other Hemiptera insects"

Fig. 4

Phylogenetic tree based on amino acid sequence of AlTOR and other insects Using the Bootstrap method for analysis, 1 000 bootstrap replicates and values representing the Bootstrap estimate"

Fig. 5

Relative expression levels of AlTOR at different stages of A. lucorum Date in the figure are means±SE, and different letters above bars indicate significant difference (P<0.05). The same as below"

Fig. 6

Relative expression levels of AlTOR in different tissues of A. lucorum female adult"

Fig. 7

Relative expression levels of AlTOR at different stages of A. lucorum under different treatments"

Fig. 8

Relative expression levels of AlTOR in different tissues of A. lucorum female adult under different treatments"

Fig. 9

Expression SDS-PAGE analysis induced by IPTG (A) of pCzn1-AlTOR and SDS-PAGE of purified pCzn1-AlTOR fusion protein (B)"

Fig. 10

Western blot analysis of antibody against the expressed AlTOR protein"

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