Research Platform Development Of Lignin Synthesis And Function Exploration Of Arogenate Dehydratase

Pear is one of the most popular Rosaceae fruits in the world.The genetic composition of Asian pears is complex and shows high genetic polymorphism.Different pear varieties have different fruit taste,which directly affects the edible quality of the fruit.The size and density of stone cells are the key factors affecting fruit quality.The synthesis and accumulation of lignin is the basis for the formation of stone cells in pear fruits.The difference in lignin accumulation between different pear varieties results in differences in the content of stone cells.This topic uses the difference in stone cell content to cluster pear varieties and use transcriptome sequencing analyze the differentially expressed genes and methylation difference sites that form stone cell differences between cultivars with wholegenome methylation sequencing,and look for the potential genes for the formation of stone cell differences in pear varieties.The synthesis of lignin is a key hub from aquatic plants to terrestrial plants in the evolutionary history of plants.The differences in lignin content and composition among different plants depend on the differences in the composition of the phenylpropanoid metabolic pathway and the shikimic acid metabolic pathway.This study identified All family members in the metabolic pathways of phenylpropanine and shikimic acid in 41 representative species in plant history were collected,and the degree of amino acid similarity between families was counted to further explore the key genes that determine the initiation of lignin synthesis in the species.In this project,the above research results will be displayed on the website platform by building an LNMP environment on a cloud server.Build the LSGD online database,which provides the blsat function of the lignin synthesis gene and various online tools.Based on the differences in the content of lignin and stone cells between different pear varieties,using the content of lignin and stone cells as feature vectors,the KNN algorithm was used to establish a variety prediction model and put the functions online,providing a platform for future lignin research.As a key enzyme for the synthesis of phenylalanine,ADT(Arogenate dehydratase)is the key hub for carbon substrates to enter lignin synthesis.In this project,it was found that ADT is an important protein that is conservative in the process from aquatic algae to terrestrial plants.And it has been found in the study of differences in methylation and transcriptome between pear varieties.We analyzed ADT in the Rosaceae family for wholegenome family analysis,and quantitatively verified the expression differences of ADT family during pear young fruit development.Furthermore,the effects of the stone cells and lignin content were verified by the transient transformation of pear fruits and the stable transformation of pear seedlings.The main test results are as follows:1.The 11 common pear varieties are clustered according to the stone cell content,and divided into R type(high stone cell content)and L type(low stone cell content),and the fruits of 15 D,30D and 45 D after flowering are sampled respectively.The location of the pulp is divided into three areas: area A(adjacent to the peel area),area B(central pulp area),and area C(adjacent to the core area),a total of 18 groups of samples,each group of samples three biological replicates,transcriptome Sequencing,after the RNA-seq overall quality assessment data is credible,using DESeq for differential expression analysis,the differentially expressed genes of different varieties in the same period and the same region are taken to intersect,and 1923 differentially expressed genes between varieties are obtained,of which 31 A phenylpropanoid pathway gene and monomer polymerase directly involved in the synthesis of lignin,the results of fluorescence verification are basically the same.2.Download the complete genome files of 41 representative species in the evolutionary history of plants from the JGI database(https://img.jgi.doe.gov/)and GDR database(https://www.rosaceae.org/),and anchor There are 25 gene families in the shikimate pathway,phenylpropanoid pathway and monomer polymerization process.By using Arabidopsis genes as a benchmark for comparison,BLAST obtained 25 family members from different species,screened by HMMSCAN,and calculated the degree of similarity between each member family of each species and the Arabidopsis protein through HMMSCAN.This similar degree of cluster side confirms the evolution of plants from algae to seed plants via mosses,lycopodium,and ferns.The shikimic acid pathway is the upstream secondary metabolic pathway of the phenylpropanoid pathway,and its gene families have formed conserved domains in algae plants.However,only a few genomic groups in the phenylpropanoid pathway have been identified in algae and tend to be complete after mosses.This discovery confirms that the evolution of plants from aquatic to terrestrial is accompanied by the formation of a large number of phenylpropanoid pathway genes.The systematic synthesis of lignin monomers is a key step for aquatic plants to move to land.3.The LSGD database(blast.pyruslignin.cn)was constructed by installing the Linux system on the cloud server,configuring the Nginx network protocol,using PHP as the backend language,and creating a library of synthetic amino acids related to plant lignin identified in this topic.With the function of online BLAST of lignin-related genes,some online biological information analysis and processing tools on the website are further constructed through the call of PYTHON and R scripts from the PHP backend.Measure the stone cell and lignin content data of 30 common pear varieties,establish a KNN pear variety prediction model through machine learning,select the optimal K,and further put the prediction model on the LSGD database.4.Using ‘Dangshan Su’ pear as the test material.,in order to further study the key ADT gene function of pear,the open reading frame(ORF)of Pb ADT1 with restriction site was amplified by PCR,and the fusion of Pb ADT1 and green fluorescent protein(GFP)was constructed.The plant expression vector p35S-ADT1-GFP.The distribution of plant expression vectors in tobacco leaf epidermal cells was observed by fluorescence microscope to determine the location and expression of genes in the cells.The research results show that the recombinant expression vector of Pb ADT1 gene is located on the nucleus.According to the fluorescence PCR results during the development of pear fruit,it can be known that Pb ADT1,Pb ADT3,Pb ADT6,Pb ADT7 are consistent with the transcriptome expression pattern of pear fruit development,showing a trend of first rising and then falling,and concentrated at high levels of expression at 35 d and 45 d after flowering,Which coincides with the formation period of a large number of stone cell clusters in pear fruit.The transient expression of Pb ADT1 can cause the increase of pear fruit stone cells and lignin content,and increase the expression level of key lignin synthase.The lignin content of pear-positive seedlings infected by Pb ADT1 increased significantly.