| Gardenia jasminoides Ellis is a traditional Chinese medicine(TCM)commonly used in China,and it is also an important resource for both medicine and food.In food production,the natural pigment extracted from G.jasminoides Ellis is widely used in food dyeing.In recent years,with the vigorous development of TCM industry,the standardized planting scale of G.jasminoides Ellis in China is also expanding.However,G.jasminoides Ellis is weak in disease resistance and easy to be infected with diseases and pests.Brown spot is one of the common diseases of G.jasminoides Ellis.It not only affects the normal growth of G.jasminoides Ellis,but also causes the whole plant to wither.At present,the commonly used control measures of brown spot are spraying Carbendazim and other antibacterial chemicals.Pesticide residues caused by long-term use not only seriously affect the quality of G.jasminoides Ellis,but also cause immeasurable damage to the ecology and environment.Under the stress of disease,the signal transduction system senses the invasion of disease,the plant body produces stress response,regulates the defense system to resist disease,and regulates the expression of related functional proteins to play the corresponding defense function.In this paper,the physiological indexes of G.jasminoides Ellis leaves were measured by different time pathogen infection,and the physiological changes of G.jasminoides Ellis responding to brown spot disease were studied.Scanning electron microscope(SEM)and transmission electron microscope(TEM)were used to observe the ultrastructure of G.jasminoides Ellis leaves in the process of interaction with pathogenic bacteria.At the same time,transcriptome sequencing of leaves in different susceptible periods was carried out to screen the key genes of G.jasminoides Ellis in response to brown spot infection.In order to explore the infection mode and pathway of the pathogen of brown spot,and the response mechanism of G.jasminoides Ellis to the pathogen,and provide the basic theory and basis for the prevention and control of brown spot.Objective:Explore the infection methods and pathways of brown spot pathogens,the response of defense enzyme system to pathogens,and the key genes of gardenias in response to brown spot pathogen infections,providing a theoretical basis for subsequent brown spot control and disease-resistant varieties cultivation.(1)The response of defense enzyme system of G.jasminoides Ellis to pathogens was revealed by detection of defense enzyme.(2)The infection of G.jasminoides Ellis leaves was observed by SEM and TEM,and the infection pathway and the effect on mesophyll cells were studied.(3)Transcriptome sequencing was used to analyze the gene expression changes of G.jasminoides Ellis infected by pathogenic bacteria,and then to identify the resistance related genes of G.jasminoides Ellis.Method:(1)The leaves of one-year-old G.jasminoides Ellis seedlings were inoculated with the pathogen staphylomyces grapevini by the bacterial cake method,and the activities of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),as well as the contents of malondialdehyde(MDA),proline(Pro)and soluble sugar were determined after 0,4,12,24,48,72 and 96 h.(2)The leaves of G.jasminoides Ellis inoculated with pathogenic bacteria for 0,2,4 and 6 days were sectioned and observed by scanning electron microscope(SEM)and transmission electron microscope(TEM).(3)The transcriptome was sequenced by high-throughput sequencing technology,the data quality was controlled by seqprep and sickl,the clean reads were de novo assembled by Trinity database,the differentially expressed genes were analyzed by deseq,the differentially expressed genes were analyzed by go and KEGG databases,and the protein interaction network of the three pathways was analyzed by string database,The network visualization analysis is carried out by using Cytoscape.Result:(1)The activities of SOD,POD and CAT increased first and then decreased,the content of MDA decreased first and then slightly increased,and the content of PRO and soluble sugar decreased generally.(2)The results of SEM showed that the changes of physiological indexes were accompanied by the corresponding changes of ultrastructure;At the middle stage of infection,the mycelium grew and spread inside the leaves and broke through the epidermis;At the later stage of infection,hyphae crisscross into a network covering the surface of leaves.The results of TEM showed that the shape of chloroplast changed from spindle shape to oval shape in the early stage of infection,and the number of starch grains increased significantly.In the middle stage of infection,a few leaf cells had partial dissolution of cell wall,and the shape of chloroplast became round,and osmiophilic granules increased significantly.At the later stage of infection,the plasma wall of the cell membrane was separated from the cell wall,the cell membrane was deformed,part of it was broken,the chloroplast bilayer membrane was dissolved and destroyed,the boundary became blurred or even integrated,and the thylakoid lamellae and stromal thylakoid lamellae were arranged irregularly and dissolved.(3)The transcriptome was sequenced by high-throughput sequencing technology.The leaves of G.jasminoides Ellis were used as experimental groups at 12,24,48 and 72 h after infection with pathogens,and 0 h as control.A total of 680463948 clean reads were obtained.The sequencing results of each experimental group and the control group(0 h)were analyzed.When compared with the control group(0 h),there were 3696 differentially expressed genes,2117 down regulated genes and 1579 up-regulated genes in the four groups after 12 h infection.One day after infection,the number of differentially expressed genes in all groups was at least 884,of which 407 were upregulated and 477 were down-regulated.There were 3208 differentially expressed genes,1329 of which were up-regulated and 1879 down-regulated.Three days after infection,there were 2864 differentially expressed genes,1411 of which were upregulated and 1453 down regulated.Venn analysis showed that there were only 339 common DEG at all time points,accounting for a small part.There were 6421 differentially expressed genes.6421 differentially expressed genes were analyzed by go and KEGG.After the leaves of G.jasminoides Ellis were infected by the pathogen,the differential genes were significantly enriched in phenylpropane biosynthesis,plant hormone signal transduction,starch and sucrose metabolism pathway.The protein interaction networks of the three pathways were analyzed by using String database.According to the connectivity of each node in the network,the key genes of the interaction network are obtained.COMT1 is the key gene of phenylpropane biosynthesis pathway,AUX1 is the key gene of plant signal transduction pathway,and SUS3 is the key gene of starch and sucrose metabolism pathway.Conclusion:(1)After being infected by pathogens,it can reduce and resist the damage of pathogens by increasing the activity of antioxidant enzymes.(2)The results of SEM showed that the pathogen mainly penetrated into the leaves by dissolving the epidermis of G.jasminoides Ellis Leaves.The results of TEM showed that the pathogen infection had certain effects on cell wall,cell membrane and organelle of mesophyll cells,especially on chloroplast.(3)It is mainly through regulating the expression of COMT1,AUX1 and SUS3 genes,improving the growth and development of plants and strengthening the cell wall to achieve the effect of disease resistance. |
