genecloningandsequenceanalysisingofantifreeze-应用昆虫学报.doc

荒漠甲虫光滑鳖甲和小胸鳖甲抗冻蛋白基因及其3(-UTR的序列分析* 侯小娟 梁娜 邱立明 马纪** （新疆大学生命科学与技术学院 新疆生物资源基因工程重点实验室 新疆大学 乌鲁木齐830046） 摘要：拟步甲科昆虫光滑鳖甲(Anatolica polita Kaszab)和小胸鳖甲（Microdera punctipennis Kaszab）分别是新疆准噶尔盆地荒漠地区的广布种和特有种。它们的生活习性有很大差异，光滑鳖甲白天活动，且活动范围广，而小胸鳖甲喜阴避光夜晚活动，活动范围小，但在两者体内都发现有多拷贝的抗冻蛋白基因，并且抗冻蛋白基因在夏季也有表达。为了比较这两种昆虫的抗冻蛋白序列以及不同时期表达的抗冻蛋白序列的差异情况，本研究分别以光滑鳖甲和小胸鳖甲的过冬和夏季成虫为材料，根据已克隆的抗冻蛋白基因序列设计通用引物，扩增新的抗冻蛋白基因及其3’-非翻译区（3’-UTR）。序列分析结果表明，无论是冬季还是夏季表达的抗冻蛋白都具有拟步甲科昆虫抗冻蛋白的特点，含有数量的 “TCTXSXXCXXAX” 12个氨基酸重复序列antifreeze proteins genes and their 3’-UTRs from desert insects Microdera punctipennies and Anatolica polita HOU Xiao-Juan LIANG Na QIU Li-Ming MA Ji＊＊ ( Xinjiang Key Laboratory of Biological Resources and Genetic EngineeringCollege of Life Science and Technology, Xinjiang University, Urumqi 830046, China) *资助项目：国家自然科学基金、新疆生物资源基因工程重点实验室开放课题XJDX0201-201105； 新疆大学博士启动基金BS090129。 **通讯作者：majiuci@ Abstract Anatolica polita Kaszab and Microdera punctipennis Kaszab are wide species and endemic species respectively in Junggar Basin of Xinjiang. They have quite different life habit in that A.polita is daily active in a wide area while M. punctipennies is light avoiding and night active in a relatively narrow area. However, both of them produce multiple copies of antifreeze protein genes (afps) which are also expressed in hot summer. To study if there are any differences of AFPs between these two insects and between those expressed in winter and summer, afp cDNAs were cloned both from A. polita and M. punctipennis collected in summer and winter respectively with universal primers designed according to previously cloned afp cDNAs. 3’-UTR sequences of antifreeze protein genes from these two species were also cloned. Sequence analysis results showed that the AFPs from both insects in summer and winter all have different numbers of TCTXSXXCXXAX motifs, the feature of antifreeze proteins in tenebrionid insects, but A. polita AFPs were more varied in their N-terminal ends and in the much conserved sites