The Mechanism Of Boron Inhibiting The Sporangiogenesis Of Phytophthora Nicotianae And The Transcription Characteristic Of Phytophthora Nicotianae Csn4 Gene

Phytophthora nicotianae Breda de Haan is a common and destructive pathogen belonging to oomycetes.As one of the most important soil-borne plant pathogens,P.nicotianae is widely found in temperate,subtropical and tropical,and has a host range of more than 250 genera of plants in 90 families such as tobacco,tomato and citrus.The disease caused by P.nicotianae gravely affects the growth and physiology of crops,which causes economic losses to agricultural production.However,there is no effective method to control this disease.Many researchers have reported that the main method of P.nicotianae dispersal is by the production of zoospores.In most cases,the motile zoospores are responsible for primary infection and disease dissemination.Therefore,the research on methods to control the generation of sporangium is becoming increasingly important to the disease management.In the early experiments,we found that 0.5 m M boron significantly inhibited the sporangiogenesis of P.nicotianae.In order to explore the mechanism of B inhibition on sporangiogenesis,suppression subtractive hybridization(SSH)was employed.A high-quality gene fragment(COP9 signalosome subunit 4,csn4)was found in the SSH library.This suggests that csn4 gene could relate to sporangium generation of P.nicotianae.However,further research is needed to show that whether the csn4 gene is associated with sporangiogenesis of P.nicotianae.Therefore,on the one hand,the effects of boron on the antioxidant system and the csn4 gene transcription of P.nicotianae were studied in this paper.On the other hand,the transcription of csn4 gene in different stages of P.nicotianae was studied.The results showed that,at the stage of sporangiogenesis,the antioxidant system of P.nicotianae was significantly changed,and the transcription level of csn4 gene was significantly inhibited by treated with boron.The results showed that the CSN4 subunit of COP9 signalosome is related to the sporangiogenesis of P.nicotianae,and provides the theoretical basis to control the sporangiogenesis of P.nicotianae.The main results are as follows:1)The effects of boron on antioxidant system of P.nicotianae at the stage of sporangiogenesis was studied.The results showed that the SOD and CAT activities significantly decreased for all treatments compared to the control.The MDA content treated with boron displayed a significant increase(p < 0.05)contrasted with the control.The increased MDA levels indicated the increased level of membrane lipid peroxidation and a condition of oxidative stress.Integrated analysis imply that the activity of antioxidant enzymes was decreased,which makes the cells to be in oxidative stress and prevents normally develop of pathogen.2)In this study,a quantitative method for csn4 gene was established.The beta-tubulin gene was used as the internal reference gene.Two pairs of primers were designed,and finally a pair of primer was obtained by screening for each gene.In addition,the recombinant plasmids and standard curves of csn4 and beta-tubulin genes were established.The standard curve equations were y =-4.007 x + 26.889,y =-4.109 x + 28.453 and correlation coefficients were 0.9976,0.9936,respectively.Using the value of Ecsn4/Etub(Ecsn4 and Etub respectively represent the transcription of csn4 and beta-tubulin gene)to evaluate the transcription level of csn4 gene.This method can efficiently eliminate the influence of differences of sample amount on the experimental results,and this study proves the feasibility of this quantitative method.3)The transcriptional analysis of csn4 gene of P.nicotianae at different concentrations of boron treatment showed that the transcription level of csn4 gene was significantly lower in the boron-treated group than in the boron-free treatment.The low concentration of boron(0.5 m M)can inhibit the transcription of csn4 gene significantly,and the inhibition rate was about 40%.The transcription level of csn4 gene was not significantly changed with the increase of boron concentration.4)The results of quantitative analysis of csn4 gene transcription in different stages of P.nicotianae showed that the transcription level of csn4 gene was significantly increased from the mycelial growth to the sporangiogenesis.And the transcription level of csn4 gene at developmental stage was significantly higher than growth period.The order of transcription level of P.nicotianae csn4 gene in different stages is: sporangiogenesis > zoosporogenesis > mycelial growth> zoospore phase.In the stage of sporangiogenesis,the transcription level of csn4 gene increased with the extending of induction time,and reached the highest level at 12 h for inducing sporangiogenesis.The results showed that the csn4 was involved in the regulation of the whole life cycle of P.nicotianae,but it was mainly involved in the developmental stage of P.nicotianae.The large-scale synthesis of sporangium material may be when induced 12 h.