莲草直胸跳甲Halloween基因家族鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2020.10.008

摘要/Abstract

摘要：

【背景】莲草直胸跳甲（Agasicles hygrophila）是世界恶性入侵杂草空心莲子草（Alternanthera philoxeroides）的专一性生防天敌;莲草直胸跳甲种群动态呈季节性波动趋势,在我国湖南地区其夏季种群数量骤减,导致对空心莲子草的防控效果大大降低。而Halloween基因家族参与昆虫蜕皮激素的合成,影响昆虫生长和繁殖,进而对昆虫种群动态具有一定影响。【目的】揭示Halloween基因家族在莲草直胸跳甲不同发育时期和不同组织中的表达模式,为进一步探究Halloween基因家族在莲草直胸跳甲中的功能打下基础,进而为生防天敌的扩繁提供新思路。【方法】通过筛选莲草直胸跳甲卵巢转录组数据并结合生物信息学分析,找到并克隆其体内Halloween基因家族成员;使用NCBI在线分析其保守结构域,利用MEGA6.0对Halloween家族基因进行多序列比对并构建系统发育树;通过实时荧光定量PCR（qRT-PCR）技术检测各基因在莲草直胸跳甲不同发育时期（卵、幼虫、蛹、雌成虫）和雌成虫不同组织（头、胸、中肠、卵巢、脂肪体）中的表达水平。【结果】莲草直胸跳甲中存在6个Halloween基因家族成员,分别为AhCYP302A1、AhCYP306A1、AhCYP307A1、AhCYP307A2、AhCYP314A1和AhCYP315A1;分析发现其均属于P450超家族且具有一定保守性,其中AhCYP306A1、AhCYP307A1、AhCYP307A2聚为一支,属于P450超家族中CYP2集团,AhCYP302A1、AhCYP314A1、AhCYP315A1聚为一支,属于线粒体集团;同时,AhCYP306A1、AhCYP314A1、AhCYP315A1在5′端存在跨膜区域。6个Halloween基因在莲草直胸跳甲整个生活史及各个组织中均有不同程度的表达。其中,AhCYP302A1在蛹期第6、7天表达量最高,成虫期表达量整体较低;AhCYP306A1在整个生活史中波动表达,以幼虫期第10天表达量最高;AhCYP307A2在末龄幼虫和化蛹初期的表达量较高;以上3个基因在幼虫期和蛹期的表达量较高。AhCYP307A1在卵期高表达,此后各个时期呈波动下降;AhCYP314A1、AhCYP315A1在成虫期和卵期的表达量相对高于其他两个时期。在头、胸、中肠、卵巢、脂肪体5种组织中,除AhCYP307A1外,Halloween家族其他基因在卵巢中表达量均较高;AhCYP307A1在雌成虫头部显著性高表达。【结论】克隆并鉴定得到6个莲草直胸跳甲Halloween基因家族成员,其在不同发育时期及不同组织中的表达具有一定差异。通过其表达模式推测Halloween基因家族在莲草直胸跳甲幼虫发育及成虫繁殖过程中可能发挥重要作用,对其幼虫蜕皮与成虫卵巢发育均有一定影响。

关键词: 莲草直胸跳甲, 空心莲子草, 蜕皮激素, Halloween基因家族, 表达分析, 实时荧光定量PCR

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

刘亦然,张虹,靳继苏,周忠实,郭建英. 莲草直胸跳甲Halloween基因家族鉴定及表达分析[J]. 中国农业科学, 2020, 53(10): 2009-2019.

LIU YiRan,ZHANG Hong,JIN JiSu,ZHOU ZhongShi,GUO JianYing. Identification and Expression Analysis of the Halloween Gene Family in Agasicles hygrophila[J]. Scientia Agricultura Sinica, 2020, 53(10): 2009-2019.

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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

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