Functional Verification Of Wood Properties Candida Te Genes In Populus Deltoides

The research of wood trait improvement has become an important part of forest genetics and tree improving. The research of regulation mechanisms of wood properties has been a focus in research field of forestry molecular biology. Populus deltoides Marsh. have owned distinguishing features such as fast growth, high yield and fine quality. The previous established cDNA microarray based on gene expression of the different height of main stem of P. deltoides(15 years old), which was confirmed having distinct with wood properties(microfibril angle, woody density). Based on the cDNA microarray, we found that the expression of four genes,for example, small GTP binding protein gene PdRAN, remorin binding protein gene PdREM, extensin protein gene PdEXT, cytokinin binding protein gene PdCYTOB, showed high correlation with wood density trait and microfibril angle distribution. These genes related to wood properties of P. deltoides have been isolated using RT-PCR technique and their molecular functions or mechanisms have also been preliminarily identified. This information has laid a solid foundation for cultivating new varieties of poplar for fast growing, high yield and fine quality. The main results are described as follows:1. A small GTP binding protein gene denominated PdRAN related to wood properties was isolated from poplar. The length of PdRAN is 666 bp and contains an open reading frame encoding a 221 amino acid polypeptide. Analyses of expression patterns indicated that PdRAN transcripts was expressed predominantly in leaf bud, immature xylem, immature phloem, with some expression in the mature leaf, phloem, floral bud, and scarce in the mature xylem zone of the stem. A plant expression vector EGFP-PdRAN with a green fluorescent protein was constructed and transformed into onion epidermal cells and Populus davidiana×Populus bolleana by particle bombardment method and Agrobacterium tumefaciens-mediated leaf disk transformation method. Transient expression experiment showed that PdRAN was located in the cell nucleus. In order to analyze the regulation mechanism of PdRAN in wood properties in P. deltoides. Plants with over-expression vector, antisense expression vector and RNAi expression vector were constructed, and then transformed into P. davidiana×P. bolleana, Nicotiana tabacum and Arabidopsis by A. tumefaciens-mediated leaf disk transformation method or floral dipping method. PCR detection, reverse transcriptase PCR and Southern blot were used to detect PdRAN-transgenic P. davidiana×P. bolleana and Arabidopsis. The results showed that the PdRAN gene had been integrated into the genome of the poplar. Real-time PCR analysis showed that the expression levels of PdRAN has significant difference between transgenic and non-transgenic poplar. Phenotype observation found that transgenic poplar with pBI121-CaMV35S-sensePdRAN owned multiplied branches, transgenic poplar with pBI121-CaMV35S-antisensePdRAN had promoted growth and thickier stem. Compare with the wild type Arabidopsis, the transgenic plants showed sturdier stem, wider leaves, increased height and postponed reproductive stage. Anatomical studies showed that xylem and phloem zone of plants with pBI121-CaMV35S-sensePdRAN was narrowed compared to wild plants, whereas xylem and phloem zone of plants with pBI121-CaMV35S-antisensePdRAN was widen obviously. This study shed light on the molecular mechanism of PdRAN in wood growth in P. deltoides and it suggested that PdRAN might be a negative regulator involving in wood formation. Microfibrillar angle of PdRAN–sense transgenic plants were decreased in greenhouse, so it indicated that the wood properties of those transgenic poplar were improved.2. A remorin binding protein gene denominated PdREM was isolated from poplar. The length of PdREM is 612 bp and encodes a protein of 203 amino acids. Tissue differential expression analysis indicated that PdREM transcripts was expressed predominantly in leaf bud, immature xylem, phloem, with some expression in the mature leaf, immature phloem, mature xylem zone of the stem, and lowest detection in the floral bud. A plant expression vector EGFP-PdREM with a green fluorescent protein was constructed and transformed into onion epidermal cells and P. davidiana×P. bolleana by gene gun method and Agrobacterium tumefaciens-mediated leaf disk transformation technique. Transient expression experiment showed that PdREM was located in the plasma membrane. In order to analyze the regular mechanism of PdREM in wood properties in P. deltoides. Plants with over-expression vector, and antisense expression vector have been constructed, and then transformed into P. davidiana×P. bolleana, N. tabacum and Arabidopsis by A. tumefaciens-mediated leaf disk transformation method or floral dipping method. PCR detection was used to detect PdREM transgenic P. davidiana×P. bolleana and Arabidopsis. The reverse transcriptase PCR technology showed that the expression levels of PdREM had significant difference between transgenic and non-transgenic poplar. Visible phenotype properties found that transgenic poplar with pBI121-CaMV35S-antisensePdREM had thicker and stronger branches, increscent caliper and larger number of leaves related to non transgenic poplar. Transgenic poplar with pBI121-CaMV35S-sensePdREM had smaller shoot and diminished caliper. Compared with the wild type Arabidopsis, transgenic plants showed sturdier branch, wider leaves, increased height and postponed anthesis. Anatomical observation indicated that xylem zone of sense PdREM transgenic poplar was narrowed, whereas xylem zone of antisense PdREM transgenic poplar was widen evidently. These transgenic results suggested that PdREM might be a negative regulator involving in poplar wood formation. Microfibrillar angle of PdREM transformed plants were decreased, and it suggested that PdREM gene may play a important role in microfibrillar angle improving.3. A extensin protein gene denominated PdEXT was isolated from P. deltoides. PdEXT is 423 bp long and is capable of encoding a protein of 140 amino acid polypeptide. Based on vascular tissue-specific promoter vector proNAC068, over-expression vector and antisense expression vector have been constructed, and then transformed into Populus alba×Populus glandulosa and Arabidopsis by Agrobacterium tumefaciens-mediated leaf disk transformation method or floral dipping method. PCR detection was used to detect PdEXT-transgenic P. alba×P. glandulosa and Arabidopsis. The results initially showed that several transgenic poplars were identified upon PCR amplification. Compared with the wild type Arabidopsis, the leaves of transgenic lines showed characteristic of green and viridity, sturdier shoot, postponed florescence and ideal plant architecture.4. Functional identification of wood properties candidate gene PdCYTOB in P. deltoides. Tissue expression patterns of PdCYTOB gene, which related to wood properties of P. deltoides, were investigated using quantitative real-time PCR(RT-PCR). The results indicated that PdCYTOB was expressed predominantly in immature xylem, immature phloem and phloem zone of the stem of P. deltoides. Southern blot and RT-PCR were used to detect antisense Pd CYT- OB-transgenic P. davidiana ×P. bolleana plants. The results showed that the PdCYTOB gene had been integrated into the genome of the poplar and expressed. Phenotype, anatomical studies and microfibrillar angle analysis showed that plant height of transgenic poplar were higher than untransformed lines, and xylem and phloem zone of plants with pBI121-CaMV35S-antisense PdCYTOB were wider than untransformed trees. In addition, microfibrillar angle of transgenic plants were decreased in test field evidently, it suggested that PdCYTOB might be resulting in improved pulping and papermaking properties in PdCYTOB antisense transformed lines. This study shed light on the molecular mechanism of PdCYTOB in wood formation in P. deltoides.