小立碗藓PpCAD4基因敲除载体的构建及功能研究  

Construction of PpCAD4 gene knockout vector and function study of PpCAD4 in Physcomitrium patens

作　　者：田旭 闫慧清 刘露 谢丽 成亮 姜山[1] TIAN Xu;YAN Huiqing;LIU Lu;XIE Li;CHENG Liang;JIANG Shan(School of Life Science,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》2023年第3期423-428,共6页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(32060587,32060611);贵州省自然科学基金重点项目(ZK[2023]ZD026)共同资助。

摘　　要：木质素是植物抵御外界环境的重要成分,肉桂醇脱氢酶(cinnamic alcohol dehydrogenase,CAD)是木质素合成途径中的关键酶和限速酶之一。前期数据表明,在灰霉菌侵染下,PpCAD4基因上调表达,但PpCAD4功能尚未明确。本次研究利用同源重组原理构建PpCAD4.1-PTN182-PpCAD4.2敲除载体,从而破坏PpCAD4的alcohol dehydrogenase GroES-like domain(ADH_N)和zinc-binding dehydrogenase(ADH_zinc_N)结构域。利用PGE 6000介导小立碗藓原生质体转化,筛选并鉴定得到敲除PpCAD4突变体植株。qRT-PCR表明,敲除型植株中PpCAD4的表达量相对野生型减少了84%。通过对配子体的形态观察发现,PpCAD4突变株通过增加配子体中拟叶的数量,使得植株生物量增加,这表明CAD4在早期陆生植物的生长发育中起着重要作用。用灰霉菌侵染小立碗藓发现,0.5 d后野生型配子体菌丝入侵率为15%,cad4-ko菌丝入侵率为40%。侵染1 d后,Ppcad4-ko配子体菌丝侵入率是野生型的2倍。表明PpCAD4能够增加小立碗藓对真菌病原菌的抗性。Lignin is an important component of plants against the external environment.Cinnamyl-alcohol de-hydrogenase(CAD)is one of the key enzymes and rate-limiting enzymes in the pathway of lignin synthesis.The previous data showed that the expression of PpCAD4 in Physcomitrium patens was upregulated after Botrytis ciner-ea assault,however,the function of PpCAD4 remained obscure in P.patens.In this study,knockout PpCAD4.1-PTN182-PpCAD4.2 vector was constructed in terms of homologous recombination,which could disrupt the alcohol dehydrogenase GroES-like(ADH_N)and zinc-binding dehydrogenase(ADH_zinc_N)domains encoded by PpCAD4.Subsequently,the Ppcad4 mutant plant was obtained by protoplast transformation mediated by PGE 6000 and identified by PCR.Compared with wild-type(WT),qRT-PCR analysis indicated that the expression of PpCAD4 in Ppcad4-ko mutant plant was reduced by 84%.Interestingly,the number of phyllids and plant biom ass in Ppcad4-ko mutant plant were increased,suggesting that PpCAD4 plays an important role in the growth and devel-opment in the early terrestrial plants.The rate of infected hyphae was 15%in WT,whereas the rate was 40%in the Ppcad4-ko plant at 0.5 dpi(days post inoculated).The rate of infected hyphae of Ppcad4-ko was 2 times of WT at 1 dpi.The result indicated that PpCAD4 can increase the resistance of P.patens against B.cinerea.

关 键 词：木质素 肉桂醇脱氢酶 小立碗藓 PpCAD4 基因敲除 灰霉菌 

分 类 号：Q782[生物学—分子生物学]