Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (10): 2009-2019.doi: 10.3864/j.issn.0578-1752.2020.10.008

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Expression Analysis of the Halloween Gene Family in Agasicles hygrophila

LIU YiRan,ZHANG Hong,JIN JiSu,ZHOU ZhongShi,GUO JianYing()   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2019-09-27 Accepted:2019-10-29 Online:2020-05-16 Published:2020-05-22
  • Contact: JianYing GUO E-mail:guojianying@caas.cn

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Abstract:

【Background】Agasicles hygrophila is an effective host-specific biological control agent for the worldwide malignant invasive weed Alternanthera philoxeroides. The population dynamics of A. hygrophila fluctuates during the year. In Hunan Province of China, the population of A. hygrophila decreases dramatically in summer leading to a significant reduction in the control of A. philoxeroides. The Halloween gene family is involved in the synthesis of ecdysone, which affects the growth and reproduction of insects, and then has a certain effect on population dynamics.【Objective】The objective of this study is to reveal the expression patterns of the Halloween gene family at different developmental stages and in different tissues of A. hygrophila, and to provide a theoretical basis for further exploring the roles of Halloween gene family in A. hygrophila and a new idea for the propagation of biological prevention means.【Method】The ovarian transcriptome data were screened with the combination of bioinformatics analysis and Halloween family genes were cloned. Conserved domains were analyzed through NCBI, MEGA6.0 was used for multiple sequence alignment and phylogenetic tree was constructed. Meanwhile, the transcript levels of the six Halloween family genes in A. hygrophila at different developmental stages (egg, larva, pupa, female adult) and in different tissues (head, thorax, midgut, ovary, fatbody) were analyzed by qRT-PCR.【Result】Six members of the Halloween gene family were identified and cloned, namely AhCYP302A1, AhCYP306A1, AhCYP307A1, AhCYP307A2, AhCYP314A1 and AhCYP315A1, respectively. All of them belonged to the P450 superfamily and had certain conservatism. Among them, AhCYP306A1, AhCYP307A1, AhCYP307A2 were grouped into CYP2 group and the other three genes belonged to mitochondrial group. Meanwhile, all of AhCYP306A1, AhCYP314A1, AhCYP315A1 had one transmembrane region at the 5′ end. The six Halloween genes were expressed with different levels at different developmental stages and in different tissues of A. hygrophila. The expression level of AhCYP302A1 was the highest on the 6th to 7th day of pupae stage, but it was lower at the adult stage. The expression level of AhCYP306A1 fluctuated throughout the life cycle of A. hygrophila and it was the highest on the 10th day of larvae stage. Higher expressions of AhCYP307A2 were detected at the late larvae and early pupae stages of A. hygrophila. In brief, these three genes were highly expressed at larvae and pupae stages of A. hygrophila. AhCYP307A1 was highly expressed at the egg stage, and its expression showed fluctuation and decline in subsequent developmental stages. While the expression levels of AhCYP314A1 and AhCYP315A1 were higher in adults and eggs than those in larvae and pupae. Among head, thorax, midgut, ovary and fatbody of A. hygrophila, most of the six Halloween genes were highly expressed in the ovary except AhCYP307A1. AhCYP307A1 had significantly high expression in the head of female adults.【Conclusion】Six Halloween genes were cloned in A. hygrophila, and their expression levels differed at different developmental stages and in different tissues of the insect. By comparing their expression patterns, it is speculated that the Halloween gene family may play important roles in the larval development and adult reproduction of A. hygrophila by affecting its molting and ovary development.

Key words: Agasicles hygrophila, Alternanthera philoxeroides, ecdysone, Halloween gene family, expression analysis, qRT-PCR

Table 1

Complete or partial ORF primers for the cloning of the Halloween genes in A. hygrophila"

基因
Gene		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
AhCYP302A1 CACAGTTTTGGAAAAATACC TTATCTTCTAATAAACTC
AhCYP306A1 ATGATGATTACTTATCTAGTGG CTAACCTGCTTGAAAAGGCA
AhCYP307A1 GCACATACATTTCCACGAGC TTATCTTTGTGCAAATTT
AhCYP307A2 ATGCTCGCGCTCGTTGTG TCAAGATCTGGGAGTTAGC
AhCYP314A1 ATGATAGACGAAAGCCTGTAC CTAAACTCTTTCTCTAA
AhCYP315A1 ATGTTTTTTCACGTAAAACAATT TTAATCGATTTTACGAAACACC

Table 2

Primers used to detect the expression level of the Halloween genes in A. hygrophila"

基因
Gene		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
AhCYP302A1 GATGAGAGCGATGTAACTGAGG TCGGGTATGAATTTGTCTGGAG
AhCYP306A1 CGTAAGCCATCCATAGAAG AGGTCTGATTCGCATAGT
AhCYP307A1 ACTTTGGCGTTTTGTGATTGG TGGGTTTCTGTCAAGATAACGG
AhCYP307A2 ACTTGCAGAGGACGCG GGAGGGGAATTGTAGTCC
AhCYP314A1 GACTTCTTGTTCGTTGGTTC TGTAGATGGTCTCTTCGCTA
AhCYP315A1
Actin		 TCGGTGGTTCTAACTATAAG
AGGTATTGCCGACAGAAT		 AGGACAACAGTATCATCTAAT
TGCTGGAAGGTGGATAAG

Fig. 1

Domain architecture of A. hygrophila Halloween gene family"

Fig. 2

Phylogenetic tree of the Halloween gene family based on amino acid sequence (neighbor-joining)"

Fig. 3

Relative expression of the Halloween gene family at different developmental stages of A. hygrophila"

Fig. 4

Relative expression of the Halloween gene family in different tissues of A. hygrophila"

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