Study On The Physiology And Biochemistry And Mechanism Of Different Organs Of Sarcodactylis Infected HLB

Sarcodactylis is the dry and ripe fruit of Citrus medica L.var.sarcodactylis Swingle,which has the functions of soothing the liver,relieving pain in the stomach and eliminating phlegm due to dampness.It is mainly distributed in tropical and subtropical areas and cultivated in Zhejiang,Fujian,Guangdong,Guangxi and Sichuan,and so on.In contrast,Sarcodactylis produced in Guangdong and Guangxi has these characteristics of fast growth,large fruit,good quality,high yield.In recent years,its yield and quality have seriously reduced due to HLB.This study established a method for the identification of Sarcodactylis infected with HLB,discussed the effects of HLB on the physiology and biochemistry of its different organs,and explored its mechanism.This study provided the theoretical basis for the identification of Sarcodactylis infected with HLB and the utilization of disease fruit,and provided also certain reference significance for prevention of the disease.Specific research contents and results are as follows:1.Leaves infected by HLB showed mottled yellow,and fruit were relatively small,even deformed,without “red nose fruit”.According to the PCR test results,there was specific band of 1160 bp in infected group,which could be cut into 520 bp and 640 bp by Xba I enzyme.Therefore,it can be concluded that the pathogen carried by samples in this study is Candidatus Liberibacter asiaticus.From the point of qPCR detection results,Sarcodactylis infected with HLB had amplification curve,dissolved curve and peak,and a certain Ct value.Normal group had not amplification.In a word,character analysis can roughly distinguish HLB in the field,and PCR or qPCR can identify Sarcodactylis infected with HLB in a timely and accurate manner,and qPCR detection is more sensitive and quantitative.2.By measuring contents of starch,sucrose,fructose and glucose in different organs of Sarcodactylis,it was found that the infection of HLB had different effects on carbohydrate metabolism in its different organs.Starch content of leaves and stems and soluble sugar(sucrose,fructose and glucose)content of leaves in infected group significantly increased,starch and fructose content of fruit and soluble sugar content of stems were significantly decreased,the ratio of soluble sugar to starch in infected leaves and stems decreased significantly.The results showed that the infection of HLB not only affected carbohydrate metabolism in plants,but also affected their normal life activities and reduced the ability to resist the adverse environment.3.Through the determination of chlorophyll content,MDA content and activities of various defense enzymes in different organs,it was found that the infection of HLB significantly reduced contents of chlorophyll a,chlorophyll b and carotenoids(P<0.01),thus affecting the photosynthesis of the host and reducing the resistance of the host.The increase of MDA content indicated that HLB had destructive effect on host cells,and the increase of related defense enzyme activity indicated that the infection of HLB stimulated the resistance reaction in host.4.By measuring contents of naringin,hesperidin,5,7-dimethoxycoumarin and total flavones,it was found that contents of naringin and hesperidin from infected group were significantly increased(P < 0.01),while contents of 5,7-dimethoxycoumarin were not significantly changed.The samples in normal group had a high similarity,while those in infected group had a low similarity.The content of total flavones in infected group was significantly higher than that of normal group(P < 0.05),which may be related to the disorder of hormone metabolism.5.Transcriptome sequencing found that DEGs were found in the leaves,and most of them were down-regulated.Most of DEGs in stemsand fruit were up-regulated,and the number in stems was higher than that in fruit.The amount was consistent with the degree of physiological and biochemical changes in plants after infection by HLB.The database analysis showed that there was significant enrichment of DEGs related to defense response,growth and development,and the results were consistent with the increase of defense enzyme activity in infected group.The proportion of DEGs related to carbohydrate(starch and sucrose,etc.),inorganic ion transport and metabolism was higher,which was consistent with the disorder of carbohydrate metabolism in infected group.6.Based on transcriptional data,four sucrose invertase genes were successfully cloned and expressed in vitro.It was speculated that CmNIs proteins were all unstable hydrophilic proteins with molecular weight of62.21 ? 72.07 kDa,and the secondary and tertiary structures of proteins were predicted.The influence of HLB infection on the relative expression levels of different organs and different CmNIs was also analyzed.It was found that the relative expression levels of CmNI2 and CmNI3 were significantly up-regulated in infected leaves and stems,while the relative expression levels of CmNI4 were significantly decreased in infected stems,which was only 27% of that of normal group.