平榛脱水素基因的克隆与表达分析  

Cloning and Expression Characteristics of a Novel Dehydrin Gene from Hazelnut(Corylus heterophylla Fisch.)

作　　者：陈新[1,2] 梁丽松[2] 马庆华[2] 赵天田[2] 刘庆忠[1] 王贵禧[2] 

机构地区：[1]山东省果树研究所,山东泰安271000 [2]中国林业科学研究院林业研究所,林木遗传育种国家重点实验室,北京100091

出　　处：《园艺学报》2013年第1期32-40,共9页Acta Horticulturae Sinica

基　　金：国家林业公益性行业科研专项(201304710)

摘　　要：以平榛(Corylus heterophylla Fisch.)花芽为试材,采用 RT-PCR 和 RACE 方法克隆了一个平榛与脱水素基因同源的 cDNA 基因,命名为 ChDHN(GenBank 登录号 HM228389),其全长 639 bp,具有一个 504 bp 的潜在编码区,编码 167 个氨基酸组成的多肽,具有 LEA 类家族成员具有的特征多肽序列,属于 Y4SK2类型 DHN 基因,预测 ChDHN 蛋白质分子量 18.03 kD,预测其理论等电点为 7.28。对ChDHN 的时空表达特性进行了研究,以 Actin 为内参,对 ChDHN 在 4 ℃冷激条件下(0、2、4、8、24和 48 h)的表达模式进行了初步的研究,冷激处理后 ChDHN 表现逐渐上调的表达趋势,24 h 达到最大表达量,48 h 表达量降低;推测 ChDHN 属于植物冷适应调节网络中的应答基因;定量 RT-PCR 分析 ChDHN在不同器官中的表达,在种子中高丰度表达,其次是雄花序和花芽,在树皮中表达最低。用 PCR、酶切和测序鉴定等方法检测已成功构建重组表达载体 pET-32a(+)-DHN,将鉴定完全正确的重组质粒转化大肠杆菌 BL21(DE3),经 SDS-PAGE 分析并经过 Western blotting 鉴定,表明重组蛋白被 IPTG 诱导后高效表达出一条比预测分子量 18.03 kD 大 4 kD 的融合蛋白。A cDNA encoding the dehydrin-like gene homologue was isolated from hazelnut（Corylus heterophylla Fisch.）by RACE-PCR and designated ChDHN（GenBank accession No. HM228389）. Sequence analysis showed that cDNA of ChDHN was 639 bp long and contained a single open reading frame. The predicted ChDHN protein has 167 amino acids with an estimated molecular mass of 18.03 kD and an isoelectric point of 7.28,qRT-PCR analysis showed that the expression of ChDHN was induced by low temperature and peaked at 24 h after exposed to low temperatures of 4 ℃. The transcripts of ChDHN appeared in many hazelnut tissues including male inflorescence,bark,flower bud and seeds,but mostly accumulated in seeds. the prokaryotic expression plasmid of pET-32a（＋）-DHN was sequenced,digested by restricted endonuclease enzyme of SacⅠand EcoRⅠ simultaneously,meanwhile induce it to be expressed in E. coli BL21 by IPTG,SDS-PAGE analysis and Western blot results showed that the recombinant plasmid pET-32a（＋）-His-DHN could be expressed a 4 kD larger molecule mass than predicted molecule mass of fusion protein.

关 键 词：平榛 ChDHN QRT-PCR 原核表达 

分 类 号：S664.4[农业科学—果树学]