Genome-wide Identification Of CCR Family In Moso Bamboo And Functional Analysis Of PeCCR1

Bamboo is one of the most valuable forest resources in China,which plays an important role in safeguarding ecological safety and wood safety.China has the most abundant bamboo resource in the world,and moso bamboo(Phyllostachys edulis)is the most dominant species with excellent wood properties,which plays an important role in alleviating the shortage of wood supply.Lignin is a major component of bamboo,and its content plays a decisive role in the material properties of bamboo.Therefore,it is of great value to study the lignin biosynthesis of bamboo.Cinnamoyl-Co A reductase(CCR)is one of key enzymes involved in lignin biosynthesis,which plays a vital role in the regulation of carbon flow in lignin biosynthetic pathway.In this study,moso bamboo was used as a material to carry out genome-wide analysis of the gene members of CCR family,and an in-depth study of PeCCR1 was performed.The main results are as follows:1.Genome-wide analysis of CCR family members in moso bamboo.Bioinformatics analysis methods was used to identify the CCR family gene members in Bamboo GDB,and fourteen candidate sequences of CCR homologous genes were obtained,among which ten had the complete conserved domain of CCR,and were named as PeCCR1?PeCCR10 successively.There were significant differences in the number,length and position of introns in PeCCRs.For examples,PeCCR2 has 5 introns,while PeCCR5 has no introns;the longest intron is 4090 bp,and the shortest one is 89 bp.The length of amino acid sequences encoded by PeCCRs ranged from 136 to 391 aa,with the predicted molecular weight of 4.971?43.05 k Da and the isoelectric point of 5.60?8.31.Sequence analysis showed that the sequences of PeCCRs were significantly different at the N-terminus and C-terminus,which were further consistent with their secondary and tertiary structures.However,the central sequences were highly consistent,containing the conserved domain and catalytic sites specific to the cinnamoyl-Co A reductase family,which indicated CCRs were more conservative in evolution.2.Expression analysis of PeCCRs based on transcriptome data.The tissue specific expression analysis showed that the expression patterns of PeCCRs in different tissues were significantly different.For examples,PeCCR3 was not detected in roots and leaves.Rhizome and shoot,PeCCR3 was the lowest one in root and PeCCR9 was the highest one of all the PeCCRs in flowering inflorescence.These results suggest that PeCCRs maybe have different function.Two genes(PCCR1 and PCCR5)were further validated by using real-time quantitative PCR(q RT-PCR).The result showed that the relative expression levels of PCCR1 and PCCR5 in different tissues had a certain differences.The expression level of PeCCR5 was significantly higher than that of PeCCR1,and the highest level of PeCCR1 was in roots,while that of PeCCR5 was in shoots.The expression differences indicated PCCR1 and PCCR5 maybe function differently in different tissues.Besides,the expression of both PCCR1 and PCCR5 were increased with the increasing shoot height and up to the highest level in 6.7 m.Moreover,the increase of PeCCR5 was significantly higher than that of PeCCR1.These result indicated that PCCR1 and PCCR5 might play similar roles in the process of bamboo shoot of lignification.3.Functional analysis of PeCCR1.Specific primers were designed according to PeCCR1 sequence,and the complete open reading frame(1026 bp)was obtained by homologous cloning method.The overexpression vector of PeCCR1 was constructed and transformed into Arabidopsis thaliana(wild type,Col-0)using the dipping flower method.Two lines of resistant plants were obtained by hygromycin screening,and the expression of PeCCR1 in the transgenic plants were validanted by RT-PCR.Phenotype analysis demonstrated that there were some differences between transgenic and Col-0 plants.The rosette leaves were bigger and the flowering time was earlier in the transgenic plants than those in Col-0 plants.Moreover,the lignin content was determined by section histochemical staining and bromoacetyl method,which showed an increased lignin content in the stem of transgenic plants compared to that of Col-0 plants.These results indicated that overexpression of PeCCR1 promoted the growth and development of transgenic plants with increased lignin.This study laid a foundation for the understanding of CCR genes in moso bamboo,which was of great significance to reveal how the CCR genes affect the bamboo properties by participating in lignin biosynthesis,and it was helpful to further utilize genetic engineering technology to regulate the lignin content of plants.