Functional Characterization Of An BpCCR Gene From Betula Platyphylla Suks. And Transcriptome Analysis Of Reaction Wood

Lignin is second abundant phenolic polymers in the plant inferioring to cellulose, it fills in the cellulose skeleton to strengthen the mechanical strength of plants, but when wood is used in the paper making industry, lignin is a big barrier. Using the genetic engineering method to change lignin content and composition becomes an effective measure to solve the above questions.Therefore, excavating the essential genes in lignin biosynthesis and identifying their function becomes a key step in lignin genetic engineering research. The paper carries out our research from the following two aspects, the first is using the transgene technical to identify the function of a BpCCR gene which we have obtained, the another is using the transcriptome sequencing technology to excavate the key enzyme gene in the lignin biosynthesis.The cinamyl coenzymeA reductases gene is a key gene in the lignin biosynthesis, from the former work we obtained a CCR gene of birch(Betula platyphylla Suks.) in the cambium cDNA library, here naming it BpCCR, this gene encodes a multi-peptides with323amino acid residues, the protein molecular weight is35.4kD, and the theory isoelectric point is5.35belonging to the NADB-Rossmann superfamily, the protein sequence is highly similar with other various species. Then we carried out the following research:First we used the real-time PCR to study the expression pattern of BpCCR gene in different tissue of birch and the cambium of different growth time. The results show that the BpCCR gene prefers to express in the newborn stem, the petiole and the xylem; The expression in the opposite wood is higher than that of tension wood; The cambium expression reaches the peak on April26to June11and August16to September18. All of the above demonstrated that the BpCCR gene participate in the birch cambium activity and the xylem growth process, it is predicted to be a lignin biosynthesis-related gene.To further study the expression characteristic of BpCCR gene, we located the fusion protein BpCCR-GFP in cytoplasm using the instantaneous transformation technology; We cloned the1156bp BpCCR promoter using the genome walking which included kinds of cis-element binding to the flavanone synthesis related protein, the carbon metabolism Dof protein, the dehydration-responsing erdl protein; We established and optimized the birch instant transformation system,by this system we found out that the BpCCR gene promoter mainly expressed in the birch stem, the petiole and the the major veins.Using the Agrobacterium tumefaciens-mediat leaf disc method, we tansformed the BpCCR overexpression vector into birch; PCR examination, RT-PCR analysis and the Northern hybrid finally confirmed we have obtained three transgenic lines;Compared with the non- transgene birch, the lignin content of three transgenic lines differently increased, and the CP3line enhanced11.85%showing significant difference with the non-transgene birch.The reaction wood system is an effective tool when studying the cell wall formation and the wood forming process-related genes. In order to uncover the gene expression pattern of birch when responsing to gravity stimulation, further excavated the lignin biosynthesis related genes, three transcriptome libraries were constructed, which was from xylems of brich tension wood (TW), opposite wood (OW) and up-right wood(UW); A total of52139846,54878244and54696478raw reads were generated from the three libraries, respectively,and a total of77783,66909and74068tentative unigenes (TUGs) were generated respectively. A total of80909non-redundant unigenes were obtained with a mean size of768nt; All the80909non-redundant unigenes were searched using BLASTX against Nr database and the Swissport database for functional annotation, and43245showed significant similarity to known proteins in Nr database and29977had significant hit in Swiss-Prot database;Gene Ontology (GO) classification results showed that cellular component is the most abundant term(38088TUGs,47.07%), followed by biological process (33904TUGs,41.9%) and molecular function(21306TUGs,26.33%). Differentially expressed gene analysis demonstrated that TW and UW had5966differentially expressed genes, OW and UW had4334, OW and TW had3742; We identified a series of lignin biosynthesis related-genes, including five PAL genes,six COMT genes, four C3H genes, seven4CL genes, six CCR genes, seven CAD genes; cellulose synthesis-related gene SUSY,XTH, CseA; transcription factor family genes as18NAC genes,26MYB genes,22WRKYand so on.