Mining And Expression Analysis Of Regulatory Genes And Lignin Biosynthesis During Pear Fruit Rust Formation In "Nijisseiki"

Pear(Pyrus)is one of most widely cultivated throughout the world.The history of pear cultivation was more than 3,000 years in China.Fruit rust is one important factors affecting the appearance quality of pear fruit,which result from formation of cuticle and cork of epidermal cells during the fruit development.Fruit spots are formed due to corky epidermal cells.The higher the cork,the more rust spots.The cork is related to level of lignin and phenolic compounds.Lignin synthesis is a complex process involving multiple pathways.We pay attention to the content of lignin and the activity of related enzymes in the formation of pear rust in "nijisseiki".High-throughput sequencing technology was used to analyze genes and pathways associated with fruit rust formation.To study the expression of related genes in bagged and non-bagged pericarp,and to identify key genes that regulate fruit rust formation.This paper expounds the molecular mechanism of pear rust formation,and its main research results are as follows:1.It is certified that phenylalanine ammonia-lyase and peroxidase are the key enzymes in the lignin synthesis of pear pericarp.The results show that the content of the lignin is highest 45 d after anthesis,the lignin content tended to decrease with the development of fruit;and the lignin content after 75 d anthesis was gentle.The key enzyme in phenylpropanoid metabolism-the changes of phenylalanine ammonia-lyase(PAL)activity and lignin content were highly consistent.The activity of PAL in bagging and no-bagging was highest 45d after anthesis,and the PAL activity of bagging pericarp was lower than that no-bagging in each period.The key enzyme that controls the synthesis of lignin monomers-Peroxidase(POD)also had the highest activity 45d after anthesis,and the POD activity in bagging pericarp is much higher than that no-bagging.Polyphenol oxidase(PPO)is a key enzyme in the synthesis of phenolic compounds,it had the highest activity 30d after anthesis,and the PPO activity in bagging pericarp is significantly lower than that of no-bagging.2.It is determined that fruit rind with fruit rust has different morphological structures.Using an optical microscope was found,there was no difference in the structure of the bagged and no-bagged pericarps 30 days after anthesis.pericarp cuticle smooth,epidermal cells have 1-2 layers,and the epidermal cell is rectangular.At this stage,the cells are in division period.At 90d after anthesis,the cuticle is not smooth and wavy in the no-bagged pericarp,and the epidermis layer cell gap is big.In the bagged pericarp,the cuticle is relatively flat,and the epidermal cells are arranged closely.Using scanning electron microscopy was found,there was no significant difference in the cuticle of the bagged and no-bagged pericarps 30 days after anthesis,and the cuticle was smooth.At 90d after anthesis,in the no-bagged pericarp,the horny membrane is rough,uneven,and the surface of the pericarp have waxy.There are no stoma on the surface of the fruit,the corneous membrane is extensively damaged,the intercellular connection is completely destroyed,and the cells have voids.The fruit spot on the surface of the bagging pericarp is small,the cuticular membrane is relatively flat,and there is no gap between the cells.3.Dig out key genes related to the formation of pear rust.Transcriptome sequencing was performed on the pericarp 30 d,45 d and 90 d after anthesis by the Illumina HiSeq 2500.and the clean reads were 65,568,13,60,125,170 and 113,392,425,gc%in 46,Q20 is around 96%.After differential gene screening,2177 differential genes between 30 days and 45 days after anthesis were used,and 2543 differential genes between 30 days and 90 days after anthesis were used.Using GO enrichment,it was found that the main enrichment of secondary cell wall metabolism and lignin metabolism in biological process,Cell and cell part of cell,molecular function of binding and catalytic activity class.Using KEGG analysis,a total of 218 metabolic pathways related to pericarp-were obtained,23 differential genes were screened for phenylalanine metabolism,and 94 differential genes were associated with phenylpropanoid biosynthesis.4.It is proved that the key genes for lignin synthesis during fruit rust formation.According to high-throughput sequencing results,differential genes were screened by FRKM>2 and Q value<0.05.A total of 42 genes with significant differences in lignin synthesis and expression in different periods were obtained.Real-time PCR was used to analyze the expression differences of these genes between bagged and no-bagged fruits.The results showed that among the 21 POD coding genes,the expression of five genes pbr013214.1,pbr000691.1,pbr022326.1,pbr035815.1 and pbr030045.1 was significantly higher than that of bagged fruit in the no-bagged pericarp.The highest expression levels of pbr035815.1 and pbr022326.1 in non-bagged pericarp at 45 days after anthesis.The expression of these genes is positively correlated with the lignin content.for the 4 4CL-encoded genes,pbr012851.1 was not expressed in no-bagged pericarp,this genes is negatively related to the lignin content.Pbr013510 is a gene coding for COMT.pbr022402.1,phr022405.1 and pbr022403.1 are genes encoding CCR.Expression of these four genes in no-bagged pericarp was higher than that in bagged,and is positively correlated with the lignin content.