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4 - coumaric acid coenzyme A ligase (4-coumarate: CoA ligase, 4CL) is a key enzyme in the phenylpropanoid pathway , plants provide a large number of secondary metabolites , such as lignin , flavonoids . Lignin synthesis process , 4CL contact lignin precursors and reaction branching link , respectively catalytic caffeic acid , p - coumaric acid, erucic acid and ferulic acid to generate the corresponding CoA ester . In this study, RT-PCR and RACE methods , and successfully cloned the birch Bpl4CL2 Bpl4CL3 gene , cDNA full-length 2069bp and 1974bp , the ORF respectively 1629bp and 1656bp , encoding 543 and 552 amino acids . Bioinformatics amino acid sequence encoded by the the Bpl4CLs gene structure and function prediction results confirm a discrepancy , Bpl4CL3 protein in Bpl4CL1, Bpl4CL2 and other species are conserved motif position may represent the control of lignin synthesis other than biological functions. Cloned full-length DNA three Bpl4CLs gene : Bpl4CL1 the DNA total length of 1755bp insert a 126 bp intron between 1194 and 1195 of the cDNA sequence ; the DNA total length of Bpl4CL2 3185bp cDNA sequence 1553 between 1554 and 1658 and between 1659 inserted 318bp and 798bp intron ; the DNA Bpl4CL3 a total length of 3280bp , between 1333 and 1334 of the cDNA sequence , respectively , between 1480 and 1481 , between 1546 and 1547 insert the 758bp, 173bp, 283bp intron . Determine birch 4CLs gene control gene expression analysis experiments , the use of semi - quantitative RT-PCR analysis of birch housekeeping gene 18S rRNA , actin gene Actin the ubiquitin gene Ubiquitin and tubulin gene Tubulin expression in birch . The results showed that the 18S rRNA , actin and Ubiquitin no significant differences in gene expression between samples , indicating that these three genes can be used as the reference gene of the the correction the Birch target gene expression amount . Select Actin as a reference gene , using real-time PCR technology were analyzed Bpl4CLs Birch different times and in different parts of the expression , to prove Bpl4CL3 higher expression levels in the mature parts of , and expression Bpl4CL1 young parts higher than mature parts , Bpl4CL2 the male flowers the highest expression level .
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