Identification and expression analysis of the halloween gene family in Agasicles hygrophila.
Identification and expression analysis of the halloween gene family in Agasicles hygrophila.
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DOI：
10.3864/j.issn.0578-1752.2020.10.008
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发表时间：
2020-01-01
影响因子：
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通讯作者：
Guo, J. Y.
中科院分区：
文献类型：
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作者：
Liu, Yi-ran;Zhang, Hong;Guo, J. Y.
背景资料：喜湿棒蚧是一种有效的寄主专一性生物防治剂，可有效防治世界性恶性入侵杂草空心莲子草。研究了该虫的种群动态。喜湿植物在一年中有波动。在中国湖南省，A.在夏季，喜湿螨的数量急剧减少，导致对喜湿螨的控制显著减少。空心莲子草Halloween基因家族参与蜕皮激素的合成，影响昆虫的生长和繁殖，进而对种群动态产生一定的影响。目的：本研究的目的是揭示万圣节基因家族在不同发育阶段和不同组织中的表达模式。为进一步探讨Halloween基因家族在喜湿草中的作用提供理论依据。提出了一种新的生物防治繁殖手段。方法：结合生物信息学分析筛选卵巢转录组数据，克隆Halloween家族基因。通过NCBI分析保守结构域，MEGA 6. 0进行多重序列比对，构建系统发育树。同时，对A.通过qRT-PCR分析不同发育阶段（卵、幼虫、蛹、雌性成虫）和不同组织（头、胸、中肠、卵巢、脂肪体）中的嗜湿单胞菌。测试结果：通过对Halloween基因家族6个成员的克隆和鉴定，分别命名为AhCYP 302 A1、AhCYP 306 A1、AhCYP 307 A1、AhCYP 307 A2、AhCYP 314 A1和AhCYP 315 A1。它们都属于P450超家族，具有一定的保守性。其中AhCYP 306 A1、AhCYP 307 A1、AhCYP 307 A2属于CYP 2基因组，其余3个基因属于线粒体基因组。AhCYP 306 A1、AhCYP 314 A1、AhCYP 315 A1在5'端均存在一个跨膜区。6个万圣节基因在不同发育阶段和不同组织中均有不同程度的表达。喜湿植物AhCYP 302 A1在蛹期6 ~ 7天表达量最高，成虫期表达量较低。AhCYP 306 A1在A.在幼虫期第10天最高。AhCYP 307 A2基因在家蚕幼虫晚期和蛹早期表达量较高。喜湿植物总之，这三个基因在棉铃虫幼虫期和蛹期都有高表达。喜湿植物AhCYP 307 A1在卵期高表达，在随后的发育阶段表达呈波动和下降趋势。AhCYP 314 A1和AhCYP 315 A1在成虫和卵中的表达量高于幼虫和蛹。在头、胸、中肠、卵巢和脂肪体中。除AhCYP 307 A1外，6个Halloween基因中的大多数在卵巢中高表达。AhCYP 307 A1在雌性成虫头部有显著高表达。结论：克隆了6个万圣节基因。在不同发育阶段和不同组织中的表达水平不同。通过比较它们的表达模式，推测Halloween基因家族可能在中华绒螯蟹的幼虫发育和成虫繁殖中起重要作用。湿生植物通过影响其蜕皮和卵巢发育而影响其生长发育。
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.