棉花花粉中高效转录U6启动子的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2015.19.002

摘要/Abstract

摘要： 【目的】以棉花品种新海16基因组DNA为模板，克隆海岛棉（Gossypium barbadense L.）GbU6启动子，筛选出能在棉花生殖细胞中（花粉）高效转录的GbU6启动子，为利用基因组编辑技术进行棉花分子育种奠定重要基础。【方法】采用两轮PCR方法克隆完整的GbU6启动子。根据网上公布的棉花基因组数据库序列，利用Primer 5.0软件设计5对引物，进行第一轮能覆盖完整GbU6启动子的PCR扩增，产物克隆测序正确后，再利用transfer PCR法将GbU6启动子精确亚克隆到CRISPR/Cas9基因组编辑载体中；第二轮扩增产物测序正确后，运用DNAMAN软件对克隆成功的5个GbU6启动子序列中具有转录功能的必要元件进行分析；然后以pBI101质粒DNA为模板，PCR扩增并克隆GUS报告基因，经酶切鉴定、测序正确后，用BbsⅠ酶切GUS，连入经同样酶切后的5个GbU6启动子对应的CRISPR/Cas9基因组编辑载体中，转化、酶切鉴定，获得对应的5种GbU6::GUS的表达载体。将含有CaMV35S启动子驱动的GUS表达载体作为棉花花粉瞬时转化的阳性对照，以上述6种表达载体DNA为模板，采用高保真酶的PCR扩增法获得高浓度DNA片段，利用基因枪轰击法将5种GbU6::GUS和阳性对照的DNA片段分别转化棉花花粉，并进行GUS染色，最后用体式显微镜观察染色情况。每个启动子的基因枪转化重复3次，最后根据染色深浅筛选出在棉花生殖细胞中高效转录的GbU6启动子。【结果】经两轮PCR扩增后获得5种GbU6启动子，启动子长度分别为1 166、1 119、1 134、1 214和1 176 bp，并构建获得了相应的5种CRISPR/Cas9基因组编辑载体；对拟南芥和克隆的5个GbU6启动子序列进行序列比对，结果表明，GbU6启动子区与拟南芥U6启动子一样，也含有比较保守的-60 bp位置的USE元件和-30 bp位置的TATA框，而且这两个元件之间的距离也非常固定；用GbU6::GUS的DNA片段瞬时转化棉花花粉，发现基因枪瞬时转化的3次重复结果显示克隆得到的5个GbU6启动子有4个能驱动GUS在棉花花粉中表达，棉花花粉被染成蓝色。其中GbU6-5P::GUS的染色相对较深，接近于CaMV35S启动子。【结论】成功克隆了棉花生殖细胞中高效转录的GbU6启动子，为构建棉花CRISPR/Cas9基因组编辑载体系统提供了有效的启动子。

关键词: 棉花, GbU6启动子, 克隆, 花粉, 瞬时表达

Abstract: 【Objective】In order to take advantage of the genome editing technology in cotton molecular breeding, U6 promoters were cloned from cotton variety Xinhai16 (Gossypium barbadense L.) and then a GbU6 promoter was selected with high transcription activity in cotton germ cells (pollen), which will lay an important foundation for cotton molecular breeding.【Method】Two rounds of PCR were adopted to clone accurately the full-length of GbU6 promoter without redundant sequence at 3’ end, five pairs of primers were designed using software Primer Premier 5.0. The first round PCR was carried out to obtain DNA fragments which cover five full-length GbU6 promoters, respectively. After confirming by sequencing, five GbU6 promoters were subcloned precisely into CRISPR/Cas9 genome editing vector by the second round of PCR (transfer PCR). After right sequencing of the transfer PCR products, the necessary element for transcriptional function of 5 kinds of GbU6 promoter was analyzed using DNAMAN software. Then GUS report gene was cloned using pBI101 plasmid as template. After confirming by sequencing, GUS gene was cloned into CRISPR/Cas9 genome editing vectors carrying five GbU6 promoters, respectively, by BbsⅠdigestion and ligation, which create five kinds of fusion expression vectors of GbU6::GUS. DNA fragments of CaMV35S promoter-driven GUS and fiveGbU6::GUS were obtained through High fidelity PCR and then were transferred into cotton pollen using a particle gun. Results were observed using a stereomicroscope after GUS staining. Each transformation was repeated three times. 【Result】Five different GbU6 promoters were cloned after two rounds of PCR and they contain 1 166 bp, 1 119 bp, 1 134 bp, 1 214 bp and 1 176 bp, respectively. Construction of the corresponding five CRISPR/Cas9 genome editing vector with five different GbU6 promoters were done. After sequence comparation of U6 promoter between Arabidopsis and cotton, results showed that cotton U6 promoter contained the conserved -60 USE motif and -30 TATA box and the distance between two elements also was fixed, just like Arabidopsis U6 promoter. The results of transient transformation showed that four cloned GbU6 promoters could drive the expression of GUS gene in cotton pollen which were stained into blue. Among the four promoters, the GbU6-5P::GUS exhibited deeper blue, similar to the CaMV35S promoter.【Conclusion】GbU6 promoters with high-level transcription in cotton pollen were cloned, thus providing an efficient promoter for genome editing technology of CRISPR/Cas9 in cotton.

Key words: cotton;GbU6 promoter, cloning, pollen, transient expression

雷建峰，伍娟，陈晓俊，於添平，倪志勇，李月，张巨松，刘晓东. 棉花花粉中高效转录U6启动子的克隆及功能分析[J]. 中国农业科学, 2015, 48(19): 3794-3802.

LEI Jian-feng, WU Juan, CHEN Xiao-jun, YU Tian-ping, NI Zhi-yong, LI Yue, ZHANG Ju-song, LIU Xiao-dong(College of Agronomy, Xinjiang Agricultural University/Research Center of Cotton Engineering, Mini. Cloning and Functional Analysis of Cotton U6 Promoter with High Transcription Activity in Cotton Pollen[J]. Scientia Agricultura Sinica, 2015, 48(19): 3794-3802.

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