橘小实蝇蜕皮激素合成通路基因鉴定分析及饥饿对幼虫发育的影响

【目的】蜕皮激素(ecdysone)是昆虫体内一种重要的激素,参与调控昆虫的生长发育,并且能对环境胁迫进行响应。研究旨在明确橘小实蝇(Bactrocera dorsalis Hendel)蜕皮激素合成通路基因(ecdysone synthesis pathway gene)的分布和特定条件下的表达特性,为进一步开展橘小实蝇变态发育与抗逆胁迫机制的研究提供理论支持。【方法】采用RT-PCR和RACE技术克隆蜕皮激素合成通路基因Bd Cyp302a1、Bd Cyp315a1和Bd Cyp314a1,利用实时定量PCR(q PCR)技术检测该合成通路基因Bd Nvd、Bd Cyp306a1、Bd Cyp302a1、Bd Cyp315a1和Bd Cyp314a1在幼虫不同发育阶段(幼虫1—8日龄)、不同组织(前胸腺复合体、脂肪体、中肠、马氏管、表皮和气管)和饥饿条件下的表达模式,明确饥饿对幼虫发育的影响。【结果】克隆获得了3个蜕皮激素合成通路基因序列的开放阅读框,分别为Bd Cyp302a1(Gen Bank登录号:JQ027284)、Bd Cyp315a1(Gen Bank登录号:KC515377)和Bd Cyp314a1(Gen Bank登录号:JQ229645),且上述序列均具备P450典型结构域:Helix-C/I/K、PERF基序、血红蛋白结合位点以及脯氨酸/甘氨酸富集区,其氨基酸序列较为保守。q PCR检测结果显示蜕皮激素合成通路基因Bd Nvd、Bd Cyp306a1和Bd Cyp314a1幼虫末龄阶段表达量显著升高,其最高表达量分别是最低点的7.33、10.89和7.82倍,但在幼虫前4 d表达量差异不显著。而Bd Cyp302a1和Bd Cyp315a1在整个幼虫阶段表达量保持相对稳定。对不同组织的研究发现,蜕皮激素合成通路基因在所选的6个组织中均有表达,其中Bd Nvd、Bd Cyp306a1和Bd Cyp315a1在幼虫前胸腺表达量最高,在其他组织中表达量差异不显著;Bd Cyp302a1在各组织中的表达量由高到低为脂肪体前胸腺表皮/马氏管气管/中肠,该基因在脂肪体的表达量是中肠的30倍;Bd Cyp314a1在中肠、马氏管和脂肪体内的表达量依次降低,但均极显著高于其他3个组织。橘小实蝇幼虫在饥饿处理12 h后即出现化蛹现象,其化蛹比例随饥饿时间延长而上升。对蛹宽和蛹长的测量中发现,饥饿导致蛹个体显著缩小,但未影响其存活率。同时,饥饿导致蜕皮激素合成通路基因Bd Nvd、Bd Cyp302a1和Bd Cyp314a1表达量在饥饿处理6 h后出现显著上调,并在处理48 h后出现显著下调;但饥饿处理并未影响Bd Cyp315a1的表达水平。【结论】蜕皮激素合成通路基因在橘小实蝇幼虫组织的表达具有差异性,并参与介导幼虫-蛹的变态过程和对营养胁迫的响应。

Identification of Ecdysone Synthesis Pathway Genes and Analysis on the Impact of Food Deprivation on Larvae Development of Bactrocera dorsalis Hendel

【Objective】 As one of the most important endohormones, ecdysone plays an important role in regulating the development of insects and responding to environmental factors. The objective of this study is to investigate the expression profiles of ecdysone synthesis pathway genes in Bactrocera dorsalis Hendel in various tissues and different conditions, providing theoretical knowledge to understand the mechanisms of metamorphosis and the stress response. 【Method】RT-PCR and RACE technologies were applied to clone the ecdysone synthesis pathway genes BdCyp302a1, BdCyp315a1 and BdCyp314a1. And quantitative PCR (qPCR) was carried to evaluate the mRNA expression patterns of ecdysone synthesis pathway genes BdNvd, BdCyp306a1, BdCyp302a1, BdCyp315a1 and BdCyp314a1 at different larval developmental stages (1- to 8-day-old larvae), tissues (prothoracic gland mixture, fat body, midgut, Malpighian tubes, integument and trachea), and starvation condition. Further, the impacts of food deprivation on larvae development were also examined in this research.【Result】The open reading frames (ORF) of BdCyp302a1 (GenBank accession number: JQ027284), BdCyp315a1 (GenBank accession number: KC515377) and BdCyp314a1 (GenBank accession number: JQ229645) were finally obtained. The analysis indicated that the protein sequences were highly conserved, which harbored typical and P450 motifs, such as Helix-C/I/K, PERF motifs, heme-binding domains and prolin/glycine rich domain. The results of qPCR showed that the expression levels of BdNvd, BdCyp306a1 and BdCyp314a1 were stable in the first four days at the larval stage, but significantly elevated in the last two days, which were up-regulated for 7.33, 10.89 and 7.82 folds comparing to the lowest level, respectively. And the expression levels BdCyp302a1 and BdCyp315a1 were stable at the larval stage. The expression profiles showed that all of the ecdysone synthesis pathway genes were traceable in the selected tissues. The highest relative expression levels of BdNvd, BdCyp306a1 and BdCyp315a1 were observed in the prothoracic glands, and no significant differences among other tissues. The expression levels of BdCyp302a1 followed the precedence order: fat body > prothoracic glands > integument/Malpighian tubes > trachea/midgut. The expression level of BdCyp302a1 in the fat body was 30 folds greater than that in midgut. And the expression abundances of BdCyp314a1 were extremely higher in the midgut, Malpighian tubes and fat body than in other tissues. Starvation could shorten the larval duration by prepupation. The larvae began to pupate in 6 h after food deprivation and the pupation rate increased with time extended. And the measurements indicated that the length and width of pupa were affected by starvation, which were significantly decreased in the treated group. However, no significant difference was detected in the survival rate. The expression levels of BdNvd, BdCyp302a1 and BdCyp314a1 were significantly elevated at 6 h post food deprivation, and down-regulated at 48 h. However, the expression levels of BdCyp315a1 were not relevant to nutrition condition.【Conclusion】Ecdysone synthesis pathway genes were transcribed differently in larval tissues, and played a vital role in mediating larva-pupa metamorphosis and nutrition stress.