亚洲玉米螟两种漆酶基因OfLac1和OfLac2 cDNA 的克隆及基因表达

doi:10.3864/j.issn.0578-1752.2014.07.011

摘要/Abstract

摘要： 【目的】克隆亚洲玉米螟（Ostrinia furnacalis）漆酶-1（OfLac1）和漆酶-2（OfLac2）基因；研究OfLac1和OfLac2在亚洲玉米螟不同发育阶段和不同组织中的表达特异性；分析蜕皮激素处理亚洲玉米螟后OfLac1和OfLac2基因表达量的变化；比较OfLac1和OfLac2表达模式的差别，推测其生理功能。【方法】根据已知的其他昆虫中Lac-1和Lac-2的保守氨基酸序列分别设计引物，以亚洲玉米螟白蛹时期提取的RNA反转录合成的cDNA为模板，通过PCR扩增OfLac1和OfLac2的保守区。然后根据所得的保守区片段分别设计基因特异性引物，采用RACE方法分别克隆OfLac1和OfLac2的3′和5′端序列，通过拼接获得OfLac1和OfLac2的基因全长。收集5龄第4天亚洲玉米螟的表皮、中肠、脂肪体、丝腺、气管、马氏管、精巢7种不同组织材料，通过半定量PCR分析OfLac1和OfLac2在不同组织中的基因表达模式。收集从卵到成虫29个不同生长发育时期的亚洲玉米螟作为材料，采用实时荧光定量PCR分析OfLac1和OfLac2在不同发育时期的基因表达水平。选取5龄第2天亚洲玉米螟幼虫进行蜕皮激素注射，分别在1、4、8、12和24 h后取样，利用实时荧光定量PCR研究OfLac1和OfLac2在蜕皮激素诱导下的基因表达水平。【结果】克隆获得亚洲玉米螟OfLac1和OfLac2的 cDNA序列。OfLac1全长3 065 bp，其中5′非编码区222 bp，3′非编码区440 bp，开放阅读框2 403 bp，共编码800个氨基酸，其编码的蛋白分子质量约为90.6 kD，理论等电点pI为5.34；OfLac2全长3 405 bp，其中5′非编码区162 bp，3′非编码区960 bp，开放阅读框2 283bp，共编码760个氨基酸，其编码的蛋白分子质量约为84.0 kD，理论等电点pI为6.43。通过SignalP分析发现，OfLac1在N端包含一个长为22个氨基酸的信号肽，OfLac2在N端包含一个长为23个氨基酸的信号肽，均为分泌蛋白质。基因表达模式分析表明，在发育过程中，OfLac1主要在成虫中表达，在其他发育时期表达量较低；OfLac2在每个幼虫龄期的末期表达水平上调，在幼虫化蛹过程中的预蛹期表达量最高；在不同组织中，OfLac1在马氏管和中肠中表达量最高，在气管和表皮中的表达量较低，在精巢、丝腺和脂肪体中几乎不表达；OfLac2在中肠和丝腺中表达量最高，在气管和表皮中的表达量较低，在精巢、马氏管和脂肪体中几乎检测不到表达。将蜕皮激素20E注射到亚洲玉米螟体内，OfLac1在注射24 h后表达量明显上调，OfLac2在注射12 h后表达量开始上调，在24 h后上调最明显。【结论】克隆得到亚洲玉米螟两种漆酶OfLac1和OfLac2的cDNA序列。两种漆酶基因的表达模式存在明显差别，但都受蜕皮激素的调控。OfLac2可能参与蜕皮过程表皮褐化。

关键词: 亚洲玉米螟 , 漆酶 , 基因克隆 , 表达分析

Abstract: 【Objective】 The objectives of this study are to clone two genes encoding OfLac1 and OfLac2 from Ostrinia furnacalis, and to predict physiological functions of these two genes based on the stage-specific and tissue-specific expression patterns of the two genes during development as well as their responses to the ecdysone (20E) treatment. 【Method】 The first fragment of OfLac1 or OfLac2 was amplified by RT-PCR using degenerated primers designed according to the conserved amino acid sequences of the other insects’ Lac1 and Lac2, respectively, and cDNAs generated from RNA isolated from O. furnacalis at pupa stage as template. The 3′ and 5′ regions of OfLac1 or OfLac2 were obtained by RACE using gene specific primers designed according to the first fragment of OfLac1 or OfLac2, respectively. Seven different tissues including epidermis, fat body, trachea, midgut, Malpighian tubule, silk gland and testes were collected from the fifth-instar day-4 larvae of O. furnacalis and the expression levels of OfLac1 and OfLac2 in these tissues were analyzed through semi-quantitative PCR. Twenty-nine samples were collected at different growth periods of O. furnacalis from egg to adult and the expression levels of OfLac1 and OfLac2 were analyzed by using real-time PCR. The fifth-instar day-2 larvae of O. furnacalis were treated with ecdysone (20E) and samples were collected at 1, 4, 8, 12 and 24 h post treatment. The expression levels of OfLac1 and OfLac2 were then analyzed by using real-time PCR. 【Result】 The full-length cDNA of OfLac1 and OfLac2 from O. furnacalis were obtained. The cDNA sequence of OfLac1 gene was 3 065 bp, containing a 5′-untranslated region (5′-UTR) of 222 bp and a 3′-untranslated region (3′-UTR) of 440 bp. It contained an ORF of 2 403 bp encoding 800 amino acid residues with a predicted molecular weight of 90.6 kD and an isoeletric point of 5.34. OfLac2 was 3 405 bp in length, containing a 5′-UTR of 162 bp and a 3′-UTR of 960 bp. The ORF of OfLac2 was 2 283 bp, encoding 760 amino acid residues with a predicted molecular weight of 84.0 kD and an isoelectric point of 6.43. OfLac1 was predicted to contain an N-terminal signal peptide with 22 amino acids in length and OfLac2 was predicted to contain an N-terminal signal peptide with 23 amino acids. The expression pattern analysis revealed that OfLac1 was mainly expressed in midgut and Malpighian tubule, and low amount of OfLac1 transcripts could also be detected in trachea and epidermis. During development, OfLac1 was mainly expressed in adult, and the expression level of OfLac1 during larval and pupal stages was low. OfLac2 was mainly expressed in midgut and silk gland, low amount of OfLac2 transcripts could also be detected in trachea and epidermis. During development, OfLac2 was mainly expressed at prepupa stage during the larval-pupal molting. The expression of OfLac2 was slightly up-regulated in the last day of each instar. The expression levels of OfLac1 and OfLac2 were significantly increased at 24 h after 20E treatment. 【Conclusion】 Two genes encoding OfLac1 and OfLac2 were cloned from O. furnacalis. The expression patterns of OfLac1 and OfLac2 were different, but they were both up-regulated by 20E. OfLac2 was related with the cuticle tanning during molting.

Key words: Ostrinia furnacalis , laccase , gene cloning , expression analysis

陈鹏, 屈明博, 杨君, 杨青. 亚洲玉米螟两种漆酶基因OfLac1和OfLac2 cDNA 的克隆及基因表达[J]. 中国农业科学, 2014, 47(7): 1341-1350.

CHEN Peng, QU Ming-Bo, YANG Jun, YANG Qing. Cloning and Expression of Two Laccase Genes OfLac1 and OfLac2 from the Insect Ostrinia furnacalis[J]. Scientia Agricultura Sinica, 2014, 47(7): 1341-1350.

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