小立碗藓PpCAD4基因敲除载体的构建及功能研究

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。

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

Lignin is an important component of plants against the external environment. Cinnamyl-alcohol dehydrogenase (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 cinerea 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 development 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.