Molecular Cloning And Characterization Of Transcription Factors Involved In Lignin Biosynthetic Pathway And Phenylpropanoid Pathway In Ginkgo Biloba L.

Phenylpropanoid pathway together with two primary branches of flavonoid metabolic pathway and lignin biosynthetic pathway is an important metabolic pathway in plants.The phenylpropanoid pathway,with its products can act as plant anti-toxin,pigmentation of flowers and fruits,components of cell wall and molecules of signal transduction,plays important roles in growth and development of plants. Furthermore,some active components in phenylpropanoid pathway,such as flavonoids,are also applied broadly in medicine and health of human beings. Therefore,modification of this pathway using metabolic engineering is important for application.Ginkgo biloba L.is ancient and precious in plant kingdom.It is usually called as the 'living fossil'.Because containing the active ingredients like flavonoids,G.biloba is thought to be valuable as medicine.On the other hand,G.biloba is also an expensive wood applied broadly.Phenylpropanoid pathway in G.biloba is very important because both the active ingredient flavonoids and lignin are all derived from this pathway.Analysis and modification of Phenylpropanoid pathway in G. biloba would have practical value in breeding and pharmaceutical research.The functions of an increasing number of plant transcription factors are being elucidating,and many of these factors have been found to impact flux through metabolic pathways.Because transcription factors,as opposed to most structural genes,tend to control multiple metabolic pathway steps,they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants.In this thesis,a novel R2R3-MYB transcription factor gene GbMYB1,which involved in phenylpropanoid pathway and lignin biosynthetic pathway has been cloned and analyzed from G.biloba for the first time.Using RACE method,a 1384 bp full-length cDNA,designated as GbMYB1,was cloned from G.biloba,which contained an open reading frame encoding a deduced protein of 347 amino acids with a predicted molecular mass of 38.4-KDa and a calculated pI of 5.09.Sequence alignments with Arabidopsis and other plant species showed that GbMYB1 was a member of the R2R3 MYB transcription factor family. The predicted protein GbMYB1 had the highest homology with PgMYB1 from Picea glauca and PtMYB1 from Pinus taeda.Furthermore,the conserved domain R2R3 of GbMYB1 shared almost the complete similarity with the two proteins.Phylogenetic tree analysis showed that GbMYB1 was close to AtMYB20,AtMYB43 and AtMYB99 from Arabidopsis.Amino acid sequence alignment of these proteins revealed that all five proteins shared a small conserved motif adjacent to the R2R3 conserved domain.The presence of this small conserved motif may define a new R2R3 MYB subgroup,which possibly reflects the similar functions of its members.Subcellular location of GbMYB1 demonstrated that GbMYB1 was a nuclear-localized protein.Semi-quantitative RT-PCR analysis showed that GbMYB1 expressed preferentially in the wood forming tissues,such as stems and roots, especially in xylem,implying that GbMYB1 might be involved in the lignin biosynthetic pathway.EMSA of GbMYB1 protein analysis showed that GbMYB1 could bind to ACII element involved in lignin biosynthetic pathway.Transgenic Arabidopsis plants overexpressing GbMYB1 gene just grow to only half of the size of the control plants after several weeks' growth,and the flowering time of transgenic lines overexpressing GbMYB1 was postponed greatly compared with the control plants.The life cycle of the transformed GbMYB1 transgenic lines was longer than that of the control plants for about 3-4 weeks.Moreover,transgenic lines overexpressing GbMYB1 showed the reduction to a certain extent in seed production. All the phenomena demonstrate that the transgenic Arabidopsis had dramatically lagging growth.After stained with phloroglucinol-HCl,the majority of the transformants overexpressing GbMYB1 were stained more intensely than the control plants.Compared with the control plants,the xylem cell walls of transgenic plants were thicker.These results indicate that GbMYB1 is involved in the regulation of the lignin biosynthetic pathway.In order to analyze the regulation of GbMYB1 transcription factor with genes in lignin biosynthetic pathway,a series of quantitative RT-PCR were carried out on the genes encoding lignin biosynthetic enzymes.The results showed that the expression of CADc,CADd and C3H genes in transgenic lines were up-regulated,especially the expression of CADc.Meanwhile,the gene PAL2 encoding the first primary enzyme in phenylpropanoid pathway was also up-regulated dramatically indicating that GbMYB1 transcription factor could up-regulate important genes involved in phenylpropanoid pathway and lignin biosynthetic pathway.On the other hand,Arabidopsis mutants AtMYB43,AtMYB98 showed opposite results to transgenic arabidopsis.The expressions of CADd and COMT genes encoding lignin biosynthetic enzyme were reduced obviously.All of these demonstrated directly and indirectly that GbMYB1 transcription factor is involved in phenylpropanoid pathway and lignin biosynthetic pathway.This study will be helpful for modification of phenylproanoid pathway and lignin biosynthetic pathway of G.biloba using metabolic engineering method in the near future.Another work in this study is the construction and screening of yeast one hybrid library from T.cuspidate.A yeast one hybrid library of T.cuspidate and five cis-elements involved in MeJA and SA induction were successfully constructed.And using GCC-box as bait,two putative transcription factors homeodomain TF and remorin TF were obtaind.