Study On The Cloning And Expression Characterization Of XTH Genes In Three Compositae Plants

Xyloglucan endotransglycosylase/hydrolase(XTH), an important cell wallmodification enzyme, plays a key role in the expansion of cell and the life activities ofplant. In order to study the function, expression pattern and action mechanism of XTH,XTH cDNAs were cloned from petals of Chrysanthemum×morifolium, Tagetes patulaand Dahlia hybrida, and on the basis of previous work, the structure and expressionpattern of XTH gene were further researched with dahlia petals as the experimentmaterials in this study. The results provided important basis for clarification of themolecular mechanism of development and its regulation of petals as well as enrichmentof the developmental biology theory of plants.1. Two XTH cDNAs were cloned from the petals of Chrysanthemum×morifoliumand Tagetes patula by RT-PCR, and were named as CmXTH (Genbank:HM752243) andTpXTH (Genbank:JN164663) respectively. Their opening read frames were both882bp,and encoded293amino acid residues which contained the XTH active site ofDELDFEFLG. Blast and Cluster analysis revealed that CmXTH and TpXTH had highhomology with XTHs of other compositae such as dahlia and gerbera. The encodedpolypeptides were clustrued into the same group with At-XTH7which belong to thegroup1of Arabidopsis XTH, suggesting that CmXTH and TpXTH might havexyloglucan endotransglucosylase activity as well as xyoglucan hydrolase activity.2. The3’ terminal sequence of DpXTH1(Genbank: HM053613.1) was gained withthe use of3′RACE, and its length was542bp, including190bp of untranslated regions.A full-length XTH genomic DNA sequence was gained by subsection PCR methodfrom dahlia petals, and its full length was1293bp, including4exons which wereseparated by3introns. The sequence similarity between exon parts of XTH genomicDNA and the DpXTH1mRNA was93.52%, and the sequence similarity between theencoded amino acid sequences was93.49%, so it was speculated that the gained XTHgenomic DNA(tentatively named as DpXTH2) might be one homologous gene of DpXTH1. According to the analysis of comparing with the At-XTHs in Arabidopsisthaliana, the DpXTH1showed higher homology to At-XTH7, while the exon parts of thegained XTH genomic DNA showed higher homology to At-XTH6, and the At-XTH6andAt-XTH7both belong to the third group of At-XTHs, so DpXTH1and DpXTH2mighthave both xyloglucan endotransglucosylase activity and xyoglucan hydrolase activity.The upstream sequence of start codon of DpXTH2was cloned using hi TAIL-PCRwith the genomic DNA as template, and its length was125bp. Based on the aboveresults, the structure characterizations of DpXTH genes in Dahlia petals were exploredpreliminarily.3. The expression of DpXTH1during the growth and development of dahlia petalswas detected by semi-quantitative RT-PCR withβ-actin, GAPDH and18S rRNA asreference genes. The results withβ-actin and GAPDH as reference genes showed thatthe expression level of DpXTH1continually increased during the growth anddevelopment of petals, suggesting that DpXTH1was not only involved in growth ofpetals, but also involved in the senescence of petals. On the other hand, the result with18S rRNA as reference gene was different from the results withβ-actin and GAPDH asreference genes, indicating that18S rRNA might be not a inappropriate reference genefor detecting gene expression in the process of the growth and development of dahliapetals.