小立碗藓PpLBD20基因敲除载体的构建及功能研究  

Construction and protoplast transformation of PpLBD20 knockout vector

作　　者：闫慧清 敖选真 刘露 周琴 孙贵连 黄绒 YAN Huiqing;AO Xuanzhen;LIU Lu;ZHOU Qin;SUN Guilian;HUANG Rong(School of Life Sciences,Guizhou Normal University,Guiyang 550025)

机构地区：[1]贵州师范大学生命科学学院,贵阳550025

出　　处：《安徽农业大学学报》2021年第1期15-20,共6页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(32060587);贵州师范大学2017年度学术新苗培养及创新探索专项项目(黔科合平台人才[2017]5726号);贵州师范大学2020年全国大学生生命科学竞赛计划项目(55092)共同资助。

摘　　要：LBD (Lateral organ boundaries domain)是植物中特有的基因家族。前期研究发现灰霉菌处理小立碗藓导致配子体中的PpLBD20上调表达,但PpLBD20的功能尚不明确。因此提取小立碗藓DNA,PCR扩增PpLBD20基因的上下游片段,依次插入PTN182载体,利用同源重组原理构建敲除表达载体,酶切和测序验证插入序列的正确性。通过工作浓度为20%的PGE6000介导小立碗藓原生质体转化,筛选鉴定得到敲除PpLBD20后的突变体植株,观察到敲除后小立碗藓不形成茎叶体结构且配子体成丝状。结果为深入探究PpLBD20在小立碗藓的形态建成调控病原菌侵染的抗性作用奠定基础。Lateral organ boundaries domain(LBD) is a kind of the plant-specific gene family. Previously, we identified that Pp LBD20 was significantly induced in Pyscomitrella patens with Botrytis cinerea infection, but the function of the Pp LBD20 is still not known. Here, we extracted the genomic DNA from P. patens, and amplified the upstream and downstream homologous fragments of PpLBD20 by PCR and respectively inserted into the PTN182 vector. The knockout vector was constructed using homologous recombination and was verified by enzyme digestion and sequencing. The constructed vector was transformed into protoplast by addition of polyethylene glycol 6000 with the concentration of 20%, and the transformants were selected and identified. We observed that the knockout P. patens gametocyte did not show the leaf-like structure and formed the filamentous branches. The current finding will facilitate to further explore the PpLBD20 in regulating P. patens resistance to B. cinerea.

关 键 词：LBD基因 载体构建 小立碗藓 配子体 

分 类 号：Q782[生物学—分子生物学]