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

Lignin is one of the important products of plant phenylpropane metabolism.It has important biological functions such as enhancing the mechanical strength of plants,facilitating long-distance water transport,and resisting the invasion of poor external environments.It plays an important role in plants from aquatic plants to land.The evolution of living plants plays a vital role.Physcomitrella patens(P.patens)is a representative of early-landing plants and contains the entire set of lignin synthesis genes except F5 H,but it is still unclear whether bryophytes can synthesize lignin.Cinnamy alcohol dehydrogenase(Cinnamy1-alcoho1 dehydrogenase,CAD)is the final reaction enzyme of the lignin synthesis pathway,and hydroxycinnamaldehyde is reduced to the corresponding three cinnamyl alcohols under its action.Preliminary experimental analysis indicated that lignin-like compounds may be induced to synthesize after the infection of Botrytis cinerea.In this paper,the function of PpCAD1 gene was studied,and the transcription levels of key genes in the lignin synthesis pathway of three strains(WT,CAD1-KO,and CAD1-OE)of PpCAD1 under the stress of Botrytis cinerea were investigated.In view of the fact that Physcomitrella patens is an ideal material for studying gene function and its special evolutionary status,the results of this study lay a good foundation for understanding the effect of PpCAD1 transcription level of Physcomitrella patens on the whole ligninpathway and preliminary exploration of the gene function of PpCAD1.1.The CAD1 overexpression vector and the gene knockout vector were constructed respectively,and transform the vectors into Protoplasts of P.patens via PEG-mediated genetic transformation.The molecular identification results indicate that the transformants containing the knockout vector(CAD1-KO)and the overexpression vector(CAD1-OE)have been successfully obtained.qRT-PCR showed that the expression level of CAD1 was only 0.0018 times the normal expression level in CAD1-KO,and the expression level of CAD1 in CAD1-OE was up to15.27 times the normal expression level.2.A preliminary study on the changes of CAD enzyme activity of WT,CAD1-KO and CAD1-OE before and after Botrytis cinerea inoculation showed that the activity of CAD enzyme in CAD1-OE was higher than that of WT(1.92 nmol/min/g FW).Compared with CAD1-KO(1.48 nmol/min/g FW),it is 2.81 nmol/min/g FW,indicating that the expression level of CAD1 may affect the activity of CAD enzyme;on the2 day of inoculation,the WT CAD enzyme activity is 1.78 times higher than before inoculation The CAD enzyme activity of CAD1-KO was increased by about 1.52 times compared with that before inoculation,and the CAD enzyme activity of CAD1-OE was increased by about 1.27 times compared with before inoculation,indicating that infection with Botrytis cinerea can cause the CAD of WT,CAD1-KO and CAD1-OE increasedenzyme activity.3.The total phenol content of WT,CAD1-KO and CAD1-OE before and after Botrytis cinerea inoculation was preliminarily determined.The results showed that the total phenol content of WT,CAD1-KO and CAD1-OE were 0.513 mg/g 0.332 mg/g and 0.835 mg/g,when they were not infected with Botrytis cinerea,indicating that CAD1 is involved in the accumulation of phenolic substances;the total phenolic content of WT is1.840 mg/g at 3 days after inoculation,which is 2.61 times higher than that before inoculation,CAD1-KO total phenol The content is 1.548 mg/g,which is 3.66 times higher than before inoculation,and the total phenol content of CAD1-OE is 3.019 mg/g,which is 2.61 times higher than before inoculation,indicating that the accumulation of phenolic substances in WT,CAD1-KO and CAD1-OE plants could be induced after inoculation.4.qRT-PCR was used to analyze the transcription level expression of key genes in the lignin pathway of P.patens during B.cinerea infection.The results show that: after CAD1 is knocked out,the remaining three family members of CAD will not compensate for it;changes in the expression level of CAD1 will change the lignin synthesis genes of C4H1,C4H2,CCoAOMT2,CCR2,CCR3,CCR5,COMT2 expression level,and cause most lignin synthesis genes in CAD1-OE to have different gene expression patterns from WT.This indicates that changes in theexpression level of CAD1 may also regulate the expression of some genes in the entire lignin synthesis pathway.Fungal infection can induce differential expression of genes of key enzymes in the biosynthesis pathway of small sphagium moss lignin such as C4H1,C4H2,CCoAOMT2,CCR2,CCR3,CCR5,COMT1,CAD3,CAD4,and may also lead to polygenes of phenylpropane-type metabolic pathways and multipathway disease resistance response.