Cloning And Functional Analysis Of Wood Properties Candidate Genes In Populus Deltoides

Exploration of how wood develops and forms has been a focus in research field of woody plant molecular biology in recent years, but the study of what genes and how they control wood properties is far from attaching importance to scientists. Micro-array technique, with its advantages of high throughput, sensitivity and reliability over other tools developed for investigating genes expression pattern, is competent for assaying thousands of genes in a short time at functional genome era. The cDNA micro-array, prepared from two cDNA libraries of developing xylem tissues of poplar, was used to assay gene expression pattern in immature xylem tissues at different height of main stem of Populus deltoides (15 years old), which was confirmed having distinct wood properties (micro-fibril angle, woody density) along the main stem, and the dots with various expression profile between chips were screened out and the single-clones related to these dots were subjected to 5'sequencing. After 5'sequencing of these single-clones, the bio-informatics tools were employed to identify the candidate genes which may influent the wood properties of poplar. A versatile RNAi vector, adapted to poplar functional genome research, has been constructed with the intron of the candidate gene, which obtained from PCR amplification of poplar genomic DNA according to sequences comparison results between the full length cDNA sequence and the genome sequence of ZF-RNG gene cloned from poplar specimens. Two constructs of RNAi and anti-sense of candidate gene were formed with partial CDS of CytoB gene, and were transformed into the poplar plantlet. The transformed plants were screened out by PCR technique and the target gene expression level assayed by RT-PCR. The results show as follow:(1) The difference of wood density and micro-fibril angle exist along main stem of Populus deltoids stock tree. From down to up of stem, wood density decreases, and micro-fibril angle decreases at first and then rises. The relation between wood density and micro-fibril angle along stem is low.(2) Two cDNA libraries, which sampled from developing xylem tissues of Populus deltoids and F2 progeny of Populus deltoids×Populus cathayana, were constructed. After that, cDNA expression micro-arrays were manufactured with 11181 unitary probes of cDNA fragment which amplified from single clones of the two libraries with 10000 and 5000 clones respectively. A total of 125 singlets were obtained after analysis of 274 Clones sequences, which were confirmed having different expression patterns along the main stem of poplar by cDNA expression micro-arrays. 62 of single-pass sequences, occupying 49.6% of total singlets, show similarity to previously described sequences in Genbank. About 11% of the recognized genes participate in pathway of resisting environment stress. 4% of genes are involved in controlling other genes expression. Genes, that synthesize cell components and belong to transcript factor, hold 6% of recognized genes respectively. Genes which felled into the group of metabolism or secondary metabolism and the pathway of signal transduction occupy 8% respectively. The 63 remaining sequences were subjected to homological gene search in poplar ESTs database and annotated with matched gene function. After that, 3 sequences could not be found similar ESTs in poplar database and were presumed specific expression genes of Populus deltoids.(3) Genes involved in constructing rRNA components expressed more in wood tissues showing high density than that of low density. However, H2B1 gene expresses in high level with wood tissues of low density. PAL and 4CL are related to wood density trait with the correlation coefficient of 0.69 and 0.95 respectively. The plant gene, Remorin, shows high correlation between its expression profiles and micro-fibril angle distribution along main stem. Among the transcript factor genes, zinc finger RING protein (including CCCH, C3HC4 and PIF1), elongation factor, CCR4-associated factor, DNA/RNA biding proteins, ethylene responsive element binding factor 4, ethylene responsive element binding factor 5 and ethylene-responsive transciptional coactivator-like protein (ERT) share high correlation coefficient with wood density distribution and are presumed their expression pattern influence formation of wood density. But, the ethylene responsive element binding factor 3 has low correlation coefficient with wood density distribution. The genes, including cell cycle-related protein (EXO), vascular tissue pattern formation (Q058H06 [POPLAR.104]) and cell growth / morphogenesis (P058D03), may affect the wood density. According to the results of micro-array experiment, genes that control wood properties may be regulatory genes rather than functional genes which participate in process of cell components synthesis directly. (4) Full length cDNA fragments of ZF-RNG, CytoB and EXO were cloned by RACE technique. The Visual Genome Library Walking experiment was employed to clone full length genes of ZF-RNG and CytoB on chromosome, and the Spidey software was used to assay the splicing pattern of ZF-RNG. After that, the genome locations of ZF-RNG and CytoB were searched by blastn algorithm with poplar genomic database. The result shows that ZF-RNG gene, located on LG_VI ranged from 16611332 to 16616623, consists of 9 introns and 10 exons, and has partial homological sequences on LG_XVIII. However, there are two copies of CytoB gene on poplar genome, located on LG_IX ranged from 8914871 to 8915817 and on LG_I ranged from 16611332 to 16616623.(5) The expression profiles of candidate genes were detected in developing xylem, leaf, alabastrum and leaf bud tissues by RT-PCR technique. The high expression level in floral bud, media expression level in developing xylem, and low expression level in leaf and leaf bud tissues of ZF-RNG gene suggest that this gene relate to plant development. EXO gene transcript is more abundant in leaf, and has similar levels in other parts. The transcript of CytoB gene is more abundant in alabastrum and leaf bud tissues, and is rare in leaf tissues.(6) A RNAi vector, named pRNAi, had been constructed with pUC19 plasmid,"35S-GUS-Nos"fragment of pBI121 and the intron9 of ZF-RNG gene which was cloned from poplar genomic DNA . Two constructs of CytoB, which were anti-sense construct pAntiCytoB and RNA interference construct pRNAi-CytoB2EX, were constructed with pRNAi vector, vector fragment of pBI121 digested with Hind III and EcoRI, and partial CDS of CytoB. The constructs were transformed into plantlets of Populus davidiana via agrobacterium tumefaciens-mediated transformation and 34 plants transformed with pRNAi-CytoB2EX and 7 plants transformed with pAntiCytoB were screen out by PCR. Four transformed plants, two of each from transformants of pRNAi-CytoB2EX and pAntiCytoB respectively, were selected randomly for gene expression level assaying of CytoB by RT-PCR. The expression level of one pRNAi-CytoB2EX transformant, labeled with A24, decreased to one fifth that of controls.