棉铃虫 JNKs 基因的克隆及表达分析

c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)是MAPK家族(mitogen-activated protein kinases, 丝裂原活化蛋白激酶)的重要成员, 具有参与昆虫抗逆反应等多种功能。为明确JNK在棉铃虫 Helicoverpa armigera 中的表达特性及其对Bt杀虫蛋白的应激与免疫反应, 本研究通过PCR克隆得到2个棉铃虫 JNK 基因 HaJNK1 和 HaJNK2; 生物信息学分析结果显示: HaJNK1和 HaJNK2基因开放阅读框分别为1 191、1 143 bp, 分别编码397、381个氨基酸。系统进化树分析结果表明棉铃虫 HaJNK1 与黏虫 Mythimna separata 聚为一支, 亲缘关系较近, HaJNK2与 家蚕 Bombyx mori 聚为一支, 同源性较高。利用实时荧光定量PCR技术分析 HaJNK1 与 HaJNK2 在棉铃虫不同发育时期、不同组织中的表达量, 发现 HaJNK1 与 HaJNK2 在卵中表达量最高, 其次是雌成虫; HaJNK1 在性腺中表达量最高, 其次是唾液腺; HaJNK2 在头部表达量最高, 其次是性腺。取食Cry1Ac的4龄棉铃虫幼虫的中肠组织中, HaJNK1 与 HaJNK2 的表达量均显著升高。推测 HaJNKs 基因可能参与棉铃虫抵御Bt杀虫蛋白伤害的应激和抗逆反应。

Cloning and expression analysis of JNKs gene in cotton bollworm Helicoverpa armigera

c-Jun N-terminal kinase (JNK) is an important member of the MAPK family (mitogen-activated protein kinases), which has various functions such as participating in insect anti-stress response. In order to clarify the expression characteristics of HaJNK and its role in defense against Bt insecticidal protein in cotton bollworm Helicoverpa armigera, we cloned two HaJNK gene coding sequences HaJNK 1 and HaJNK 2 by PCR amplification. Bioinformatics analysis showed that the open reading frames of HaJNK 1 and HaJNK 2 genes were 1 191 bp and 1 143 bp, encoding 397 and 381 amino acids, respectively. Phylogenetic tree analysis showed that HaJNK 1 of H. armigera had high homology with that of Mythimna separata and HaJNK 2 of H. armigera is clustered with that of Bombyx mori. Quantitative real-time PCR was used to analyze the expression levels of HaJNK 1 and HaJNK 2 in different developmental stages and tissues. It was found that the expression levels of HaJNK 1 and HaJNK 2 were the highest in the stage of egg, following by female adults. The highest expression of HaJNK 1 was observed in gonads followed by salivary glands, and the highest expression of HaJNK 2 was in head followed by gonads. For the fourth instar larvae, after cotton bollworm fed on Cry1Ac, the expression levels of HaJNK 1 and HaJNK 2 in midgut were significantly increased. It was speculated that the HaJNKs of H. armigera might be involved in emergency response and antistress response against Bt insecticidal protein.