Isolation, Expression And Functional Identification Of Monolignol Biosynthesis-related Genes From Moso Bamboo (Phyllostachys Edulis)

Phyllostachys edulis belongs to Bambusoideae subfamily in Gramineae family, and it has the largest cultivation area and highest values of usage in China. Ph. edulis is rich in fiber and suitable for making paper. However, due to the high level of lignin content in Ph. edulis, a large quantity of chemicals must be used to separate fiber from lignin, with lignin left in the waste, resulting in serious environmental pollution. Moreover, the chemicals for separation, and the re-collection of fiber using alkali have greatly increased the cost of paper making process. Hence, altering lignin content and/or monolignol composition by biotechnology and directionally cultivating new varieties have become an important research project in current biotechnological field of Ph. edulis.In this research, we constructed four full-length cDNA libraries of different organs of Ph. edulis, and on the basis of mass sequencing and data analysis, several genes encoding the key enzymes in monolignol biosynthetic pathway in Ph. edulis, namely cinnamate 3-hydroxylase (C3H), cinnamoyl CoA reductase (CCR), cinnamyl dehydrogenase (CAD), and ferulate 5-hydroxylase (F5H)-like genes, were isolated from the cDNA libraries. And we made a further research on the expression patterns of C3H and F5H-like genes, and transgene experiments were also carried out with three F5H-like genes, with the purpose of deducing their possible functions. The results are listed as follows.(1) Five CCR genes were isolated from full-length cDNA libraries of Ph. edulis. The five deduced CCR proteins have conserved Epimerase domain, and they belong to NAD dependent epimerase/dehydratase family. Four of the five deduced CCR proteins have no obvious signal peptide or trans-membrane structure, and are predicted to be localized in cytoplasm. PheCCR5, however, a signal peptide-like structure is detected on its N-terminal and might be predicted to be localized in mitochondria. Using maximum likelihood method, the phylogenetic tree was constructed. The result shows that the CCRs appeared before the differentiation of eucaryotes and that lineage-specific expansion and divergence events occurred in some angiosperms, after the divergence from lower land plants. (2) Five CAD genes were isolated from the cDNA libraries. Four of the five deduced PheCAD proteins contain two functional domains of ADH_N and ADH_zinc_N, which are the conserved domains for most CAD proteins. PheCAD4, however, has a domain of Epimerase similar to that of CCR proteins rather than ADH_N and ADH_zinc_N domains, but it has low similarity with CCR proteins. So it is a CAD protein of another group, namely CADs with Epimerase domain. CAD proteins of this group have very low similarity with CADs containing the ADH_N and ADH_zinc_N domains, so we deduce that the two kinds of CAD proteins might be another group of CAD. Using maximum likelihood method, the phylogenetic tree was constructed. It is shown that the angiosperm CADs containing ADH_N and ADH_zinc_N domains are located at two branches, and that such differentiation took place before the appearance of higher plants; after the divergence of angiosperms, lineage-specific expansion and divergence events occurred, resulting in the multiple CADs in the genome. The angiosperm CADs with Epimerase domain shared a common ancestor.(3) We got one putative C3H gene from the cDNA library and named it as PheC3H. The molecular mass of its deduced protein is 58.38 kDa with pI 8.77. PheC3H has very high similarity to TraCYP98A protein and it should belong to the subfamily A of CYP98 family. The sequence of the first 21 amino acid residues at N-terminal is predicted to be the signal peptide, and the mature protein might be located on endoplasmic reticulum. Quantitative real-time PCR was used to detect the organ-specific expression in nutritive organs of Ph. edulis seedlings. The highest expression level of PheC3H is in 3-year-old stems followed by leaf sheaths of one-year old seedlings. The relative expression level of PheC3H in one-year-old seedlings is ranged as: leaf sheaths > roots > leaves > stems.(4) Using similarity blast, we found 10 genes having relatively higher similarity to some known F5H genes. Sequence analysis shows that the ten proteins belong to cytochrome P450 superfamily, so we name them as PheCYP-1, PheCYP-2,…, PheCYP-10. The similarities between the ten proteins are not high, and the similarities between the ten proteins and know F5H proteins are not high, either, which indicates that the ten genes might have different functions. According to sequence similarity, PheCYP-2 belongs to CYP81A subfamily, PheCYP-4 belongs to CYP81 family, PheCYP-5 is a new member in CYP706 family, and PheCYP-9 might be a putative flavonoid 3',5'-hydroxylase. Each of the ten proteins contains a predicted signal peptide sequence at its N-terminal, and they may be located on endoplasmic reticulum.(5) Quantitative real-time PCR was carried out to detect the expression patterns of the ten PheCYPs in different organs of Ph. edulis seedlings. According to the relative expression level, the ten genes can be divided into three groups:①High expression genes, including PheCYP-1,PheCYP-2 and PheCYP-7. PheCYP-1 and PheCYP-2 are mainly expressed in leaves and PheCYP-7 mainly in shoots.②Intermediate expression genes, including PheCYP-3, PheCYP-4, PheCYP-6 and PheCYP-9. PheCYP-3 has relatively higher expression in leaves, stems and roots, PheCYP-4 in leaves, PheCYP-6 in shoots and PheCYP-9 in leaf sheaths and stems.③Low expression genes, including PheCYP-5, PheCYP-8 and PheCYP-10. PheCYP-5 has relative higher expression in shoots, PheCYP-8 in shoots, roots and leaf sheaths, and PheCYP-10 in leaves. According to the documents on known F5H genes and lignin biosynthesis-related genes, we can deduce that the ten genes might have some relationship on lignification process in Ph. edulis.(6) PheCYP-5, PheCYP-6 and PheCYP-10 were cloned into plant expression vector and transformed into Arabidopsis thaliana. We have got the transgenic Arabidopsis T1 generations. Xylem development of PheCYP-5 and PheCYP-10 transgenic Arabisopsis T1 generation were detected by tissue section. Transgenic Arabidopsis plants overexpressing PheCYP-5 or PheCYP-10 have significant increased width of vessel walls in the secondary xylem, indicating that PheCYP-5 and PheCYP-10 may be involved in secondary cell wall development of vessels in xylem.