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Expression and Functional Analysis of Correlative Enzyme Genes of Lignin Phenylpropanoid Biosynthetic Pathway in Betula Platphylla

Author: SongFuNan
Tutor: YangChuanPingï¼›LiuGuiFeng
School: Northeast Forestry University
Course: Tree Genetics and Breeding
Keywords: Birch Lignin Phenylalanine ammonia lyase ( PAL ) Coffee coenzyme A 3 - O - methyl transferase Enzyme ( CCoAOMT ) Coumarate coenzyme A ligase ( 4CL ) Function Genetic regulation
CLC: S792.153
Type: PhD thesis
Year: 2009
Downloads: 227
Quote: 0
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Abstract


Lignin is an aromatic polymer, cellulose content after the plant body of a class of polymeric organic substances, mainly in presence of plant cell walls in the secondary thickening. Provide mechanical support for the plant to protect plants from fungal invasion, terrestrial plant lignin synthesis is to adapt one of the important characteristics of the terrestrial life. Of research in recent decades has been to clarify the lignin monomer biosynthetic routes, and prove that the lignin content can be artificially regulation woody plant pulp rate and forage digestibility and lignin regulation can improve and so on. Meanwhile, the plants can also adapt to the the three lignin monomer (H / G / S) (?) Division larger proportion change. Ten kinds of enzymes involved in the lignin biosynthetic pathway, wherein the coffee coenzyme A 3-O-methyl transferase enzyme (CCoAOMT) is a key enzyme in lignin biosynthesis pathway, the major catalyst hydroxy coenzyme A ester A base reaction, coffee coenzyme A into ferulic coenzyme A; 4 - coumarate: CoA ligase (4-coumarate: CoA ligase referred 4CL) is specific to lignin biosynthetic pathway connecting the styrene-acrylic acid pathway key enzyme, the major catalyst for cinnamic acid to generate the corresponding cinnamic acid coenzyme A ester synthesis of lignin and other phenylpropanoid compounds metabolic flow of regulation point. The paper first isolated birch actin (Actin) gene full-length cDNA of the gene can be used as a real-time quantitative expression technology internal reference. Actin protein coding region of conserved sequences of primers were designed according to different plants, RT-PCR, and the full-length sequence of the actin gene cDNA was amplified using RACE technology. Birch actin cDNA of full-length 1785bp, in which the 5 'non-coding region of 157 bp, and 3' non-coding region of 495 bp coding region of 1134 bp in encoding 377 amino acids. Similarity are registered in the resulting sequence with the GenBank other plant actin nucleotide sequences at least 80%, and the similarity of the amino acid sequence of up to 96% or more, and its highly conserved and shows that the gene is closely related to the cell life activities, and birch 4CL protein structure analysis and homology modeling. In addition, using RT-PCR and RACE cloned from birch secondary xylem cDNA encoding phenylalanine ammonia lyase (PAL), 2322 bp ORF encoding 773 amino acids, and the deduced amino acid sequence contains PAL-HAL and the the PAL two functional domains and the enzyme active center sequence GTITASGDLVPLSYIA. BplPALl have different transcriptional expression of genes in various tissues, highest expression in the secondary xylem, followed by young leaves, lower expression inflorescence, different BplPAL1 gene regulation and expression in various tissues. Secondly, in the previously separated birch CCoAOMT and 4CL full-length cDNA (respectively the named BplCCoAOMT1 and Bpl4CL1), based on the use of real-time quantitative PCR and Northern the BplCCoAOMT1, and Bpl4CL1 gene expression analysis of mRNA levels. Northern analysis showed that the BplCCoAOMT1 gene transcription was higher in July and August, the level of expression. In addition, the use of real-time quantitative PCR analysis birch organs and secondary xylem in relative expression the of each period BplCCoAOMT1 Bpl4CL1 gene, results showed that the period from the end of June to early July BplCCoAOMT1 gene transcriptional level in the secondary xylem higher expression levels peaked at the end of July. Bpl4CL1 genes with similar BplCCoAOMT1 gene, but Bpl4CL1 gene expression differences in level of transcription in plant development in different periods. , Bpl4CL1 genes and BplCCoAOMT1 of gene expression in the secondary xylem highest, followed by leaf petioles least. By Agrobacterium tumefaciens-mediated antisense BplCCoAOMT1 genes were introduced into tobacco and birch, pre-built plant expression vector p121-Bp1CCoAOMT1 two tobacco varieties Longjiang 911 and SR-1 and birch gene transformation and optimization of a tissue culture system. The tobacco transgenic varieties procedure is the same, the final total of 252 kanamycin-resistant tobacco plants. Exogenous gene-specific primers for PCR testing, 252 of 115 positive. The histochemical reaction test results show that, the S lignin reduce the Longjiang 911 transgenic plants positive plants. The five transgenic tobacco lignin content than the control mean reduction of 39.0%, analysis of variance show lignin content of transgenic and non-transgenic plants was extremely significant difference. In addition, to optimize the the birch regeneration system and transgenic program, initially speculated transgenic birch plants lignin fractions change. These results show that antisense transforming the birch BplCCoAOMT1 gene to some extent affected the transgenic plants lignin biosynthesis initially speculated the birch BplCCoAOMT1 genes involved in lignin synthesis and S-type monomer synthesis.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Broad-leaved trees > Birch > Birch
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