Preliminary Study On The Function Of Cinnamyl Alcohol Dehydrogenase 1 In Physcomitrella Patens Under The Stress Of Botrytis Cinerea

Physcomitrella patens is a non-vascular plants which does not have true root and leaf differentiation,the grooming vascular system,only have rhizoid and mimicry leaves.Lignin is a major component of vascular plants and the important feature to adapt to the external environment.Cinnamyl-alcohol dehydrogenase(CAD)is a key enzyme in lignin metabolism pathway.It catalyzes the final reaction of lignin monomer synthesis,reduction of three cinnamyl aldehydes to the three cinnamyl alcohols.Physcomitrella patens is a non vascular plant,it has been controversial for the synthesis of lignin,Previous research reported that lignin formation was analyzed under the stress of Botrytis cinerea using HPLC.The results showed that it has synthesized the lignin like material.Meanwhile,they analized the expression of key genes in the lignin pathway under the stress of Botrytis cinerea by transcriptome and RT-PCR,the results showed that CAD1 appeared differential expression.There have been reported on the function of CAD in other plants,However,the function of CAD in Physcomitrella patens under biological stress has been not reported yet.In this study,reverse genetics technology was used to study the function of CAD1 under the stress of Botrytis cinerea.The aim was to reveal the response mechanism of genes related to organisms of Physcomitrella patens under Botrytis cinerea stress,and provide a theoretical basis for the resistance mechanism and evolution of the early landlocked plants.The results of the study are as follows:1.The high activity and large numbers of protoplasts were obtained under the condition of 1% collapse enzyme and treated 30 min,and the regeneration rate reached over 90%.Stable transformants were obtained when PEG concentration was 20%.Through the optimization of the above conditions,the PEG mediated protoplast transformation system has been established.The study of this experiment laid the foundation for the identification of further gene function.2.CAD1-PTFH15.3 overexpression vector and CAD1-GFP-PTN182 knocked vector were constructed and transformed into the protoplasts by PEG-mediated genetic transformation method.Through the two resistance screening and the molecular level identification,we obtained knockout and overexpression transformatic plants respectively.3.The wild-type and transformed plants were infected with Botrytis cinerea,then the infection behavior and mycelial formation rate were observed under fluorescence microscope.The results showed that the mycelial formation rates of the wild-type and knockout transformants reached 65.5% and 65.9%,and the over-expressing transformed strains reached 49.3%.The mycelial formation rate of the over-expressing transformed strains was lower than the wild type plant.It indicated that CAD1 may participate in the defense reaction by synthetic lignin under the stress of Botrytis cinerea.The mycelial formation rates of wild-type and knockout-type transformants were similar,because of other homologous genes of CAD were involved in functional compensation probably.