小桐子低温诱导型启动子JcDnaJ20p的克隆及烟草转化功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0620

摘要/Abstract

摘要：

低温转录组数据显示,DnaJ20是小桐子低温诱导基因。为鉴定该基因启动子的低温诱导活性,基于PCR技术从小桐子叶片基因组DNA中克隆到DnaJ20基因（JcDna20）启动子序列,命名为JcDnaJ20p,利用重组技术构建了JcDnaJ20p启动子驱动GUS标记基因的植物双元表达载体pCambia1381Z-JcDnaJ20p-GUS,并通过农杆菌介导转化烟草进行功能解析。结果表明,克隆的小桐子DnaJ20基因启动子序列长度2 023 bp,序列分析显示该启动子中具有真核生物典型的核心启动子区元件TATA-box和CAAT-box,另外,还鉴定到激素如脱落酸、赤霉素响应元件与植物抗逆性相关如低温、干旱胁迫响应元件。以转化空质粒pCambia1381Z-GUS与35S启动子驱动pCambia1381Z-35S-GUS的烟草为对照,对转化pCambia1381Z-JcDnaJ20p-GUS的烟草叶片分别进行15、4℃低温处理24 h,通过GUS组织化学染色表明,JcDnaJ20p在低温处理下能够提高GUS基因的表达量,具有低温诱导启动子活性。

关键词: 小桐子, 低温诱导, 启动子, DnaJ20, 基因克隆, 功能鉴定

Abstract:

DnaJ20 is a cold-induced gene based on Jatropha curcas cold transcriptome data. In order to identify the cold-induced activity of this gene,the promoter sequence of DnaJ20 gene（JcDna20）from the genomic DNA of Jatropha curcas leaf was cloned based on PCR technology,and designated as JcDnaJ20p.,then,a plant binary expression vector pCambia1381Z-JcDnaJ20p-GUS was constructed by fusing JcDnaJ20p promoter with GUS reporter gene,and transferred into tobacco（Nicotiana tabacum）by Agrobacterium tumefaciens system. The results showed that the sequence length of DnaJ20 gene promoter was 2 023 bp. Sequence analysis found that JcDnaJ20p had the typical eukaryotic promoter core regions（TATA-box and CAAT box）. In addition,hormonal responsive elements（abscisic acid and gibberellin）and resistance-related responsive elements（low temperature and drought）were identified in the sequence of JcDnaj20p. Compared to plants transformed with empty vector of pCambia1381Z-GUS and pCambia1381Z-35S-GUS,histochemical staining of the transgenic tobacco with pCambia1381Z-JcDnaJ20p-GUS showed that the expression of GUS gene increased under 15 and 4℃ cold treatments for 24 h,indicating that JcDnaJ20p was an efficient cold-inducible promoter.

Key words: Jatropha curcas, cold induction, promoter, DnaJ20, gene cloning, functional identification

王海波, 郭俊云. 小桐子低温诱导型启动子JcDnaJ20p的克隆及烟草转化功能鉴定[J]. 生物技术通报, 2021, 37(2): 24-31.

WANG Hai-bo, GUO Jun-yun. Molecular Cloning of Cold-induced JcDnaJ20p Promoter from Jatropha curcas and Its Functional Identification in Transgenic Tobacco[J]. Biotechnology Bulletin, 2021, 37(2): 24-31.

图/表 4

图1 小桐子JcDnaJ20p启动子的克隆 A：启动子PCR扩增;B：菌落PCR验证;C：表达载体Sal I与Hind III双酶切验证;D：重组质粒pCambia1381Z-JcDnaJ20p-GUS;M：DNA Marker

图2 小桐子JcDnaJ20p启动子与GUS基因融合植物表达载体的构建

图3 小桐子JcDnaJ20p启动子顺式作用元件的分析下划线表示JcDnaJ20基因起始密码子,方框表示光响应元件（ATC-motif,核心序列AGTAATCT;GT1-box,核心序列CCTTAA;Gap-box,核心序列CAAATGAA;TCCC-motif,核心序列TCTCCCT;TCT-motif,核心序列TCTTAC）。1-脱落酸ABA响应元件（ABRE,核心序列ACGTG）;2-赤霉素响应元件（P-box,核心序列CAAAAGG）;3-MYB与MYC转录因子结合位点（CCAAT-box,核心序列CAACGG）;4-低温、干旱响应元件（DRE,核心序列ACCGAGA）

图4 转基因烟草叶片的GUS组织化学染色

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