›› 2006, Vol. 49 ›› Issue (6): 930-937.

• 研究论文 • 上一篇    下一篇

家蚕羧酸酯酶基因克隆及差异表达

高贵田,陈克平*, 姚勤, 王林玲,陈慧卿   

  1. (江苏大学生命科学研究院,江苏镇江212013)
  • 出版日期:2006-12-29 发布日期:2006-12-20
  • 通讯作者: 陈克平

Cloning and differential expression of Bombyx mori carboxylesterase gene

GAO Gui-Tian, CHEN Ke-Ping, YAO Qin, WANG Lin-Ling, CHEN Hui-Qing   

  1. (Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013, China)
  • Online:2006-12-29 Published:2006-12-20
  • Contact: CHEN Ke-Ping

摘要: 家蚕浓核病毒 (Bombyx mori densonucleosis virus,BmDNV)是蚕业生产上危害比较严重的一类病毒。用完全抗浓核病中国镇江株(BmDNV-Z)的家蚕品系秋丰、感性品系华八及以华八为轮回亲本回交8代和自交8代构建的近等基因系BC8为材料,采用mRNA荧光差显技术首次分离克隆了家蚕羧酸酯酶(B. mori carboxylesteraseBmCarE)基因全长cDNA,并用实时荧光定量PCR检测了添毒后12 h36 h72 h BmCarE在感、抗BmDNV-Z家蚕品系中肠内的表达差异。结果表明: 1)添毒后12 h不同品系家蚕中肠BmCarE表达差异最大,抗性品系BC8和秋丰分别是感性品系华八的17.714倍和3.602倍,三者彼此间的差异达到极显著水平;(2)同一品系添毒后12 h与添清水后12 h BmCarE表达也有较大差异, BC8添毒是BC8添清水的15.08, 秋丰添毒是秋丰添清水的3.39, 差异达到极显著水平,而华八添毒和添清水的BmCarE表达量均低,二者差异不显著;(3)同一品系添毒后不同时间BmCarE表达也有较大差异, BC8和秋丰添毒后12 h BmCarE表达量最高,显著高于各自添毒后36 h72 h表达水平,而添毒后36 h72 h表达无显著差异;华八添毒后12 h36 h72 hBmCarE表达无显著差异。上述结果提示羧酸酯酶基因可能与家蚕抗浓核病毒有一定关系。

关键词: 家蚕, 羧酸酯酶基因, 表达差异, 近等基因系, RTPCR

Abstract: Total RNA was isolated from the fully resistant silkworm (Bombyx mori) strain Qiufeng against BmDNV-Z, the highly susceptible silkworm strain Huaba and near-isogenic line BC8 which was bred by using Qiufeng to cross with Huaba followed by successive backcrossing to Huaba till the eighth generation, respectively, and then subjected to fluorescent differential display reverse transcription PCR (DDRT-PCR). A full-length cDNA of B. mori carboxylesterase was obtained. Its expression in the midgut of the susceptible strain and the resistant strain against BmDNV-Z was analyzed by real-time fluorescence quantitative PCR at 12 h, 36 h and 72 h after administration with BmDNV-Z. The results showed that: (1) cDNA quantity of carboxylesterase in the midguts of the resistant strains reached the peak 12 h after infection. The cDNA quantity in the resistant strains BC8 and Qiufeng were 17.714 and 3.602 times respectively that in the susceptible strain Huaba. (2) Within the same strain, there existed a remarkable difference in the detected carboxylesterase cDNA quantity at 12 h between the infected group and the control group. The expression in the resistant strains BC8and Qiufeng were 15.08 and 3.39 times respectively that of the control group. On the other hand, the expression of carboxylesterase was quite low in the susceptible strain Huaba of both the infected group and the control group. (3) The expression of carboxylesterase varied at different time after infection. Expression in the resistant strains BC8 and Qiufeng reached its peaks 12 h after infection, remarkably higher than those at 36 h and 72 h. There was no significant difference of the expression in the susceptible strain Huaba infected for 12 h, 36 h and 72 h. The results suggested that the expression of carboxylesterase in B. mori might be related to its resistance against DNV.

 

Key words: Bombyx mori, carboxylesterase gene, differential expression, near-isogenic line, RT-PCR