关键词: 鹅掌楸, 基因克隆, 组织表达特征, 异源表达, 功能分析

Abstract: [Objective] Based on the previous results of association analysis between growth traits and EST-SSR markers in Liriodendron, a gene corresponding to the marker locus associated with growth traits was cloned from Liriodendron chinense, the function of this gene was studied by analyzing its sequence characteristics, structural features, genetic relationship with other species homologous genes and expression characteristics. Finally, the gene function was validated through over-expression in Arabidopsis thaliana, the aim of this study was to understand the molecular mechanism of its growth and development and contribute to the further utilization of functional genes.[Method] Based on the transcriptome database of L. chinense leaf, RT-PCR and RACE were used to clone and separate the EST related gene corresponding to 642 locus that associated with growth traits of hybrid Liriodendron for analyzing bioinformatics of the sequences. Quantitative real-time PCR was used for the analysis of gene expression in different tissues such as floral bud, flowering leaf buds, leafs, petals, stamens and pistil of L. chinense. Using the Gateway Recombination Cloning Technology, the over-expression vector of LcPAT8 was constructed, and transformed into A. thaliana with the floral dipping method of Agrobacterium-mediated transformation, the T2 transgenic plants were obtained and then the phenotype was analyzed.[Result] The full-length cDNA of 1 968 bp was cloned(GenBank accession number:KU883608), containing 1 269 bp ORF and encoding 422 aa protein. Bioinformatics analysis showed that the protein encoded by this gene had a typical DHHC-CRD domain, and it belonged to the DHHC-type zinc finger protein family. BLAST analysis indicated its high similarity to predicted protein S-acyltransferase(PAT) of other plants, and according to the alignmentresult with AtPAT genes in NCBI, we named it LcPAT8. Gene expression suggested that the LcPAT8 was expressed in various tissues, it had the highest expression in pistil but the least in stamen, and the expression in petal and leaf were higher than that in flower bud and leaf bud. The over-expression vector of the LcPAT8 was successfully constructed by the Gateway technology, the T2 transgenic A. thaliana were obtained. The rosette leaf number and bolting time of transgenic plants over-expressed LcPAT8 had no significant difference from the wild type, but when most pods of the wild type ripen, and leaves turned yellow and fell, the transgenic plants were still vigorous in growth, their leaves were fresh and green,and a large number of flower wads were present; when the wild types withered and died, the transgenic plants still had a large number of branches growing and flowering.[Conclusion] The experimental data confirmed that the LcPAT8, a DHHC-type zinc finger protein gene from L. chinense, was highly-expressing in the pistil and petal, it might have been involved in petal expansion and the development of carpel and embryo. Functional analysis showed that the over-expression of LcPAT8 in A. thaliana led to obvious extension of growth cycle in the transgenic plants. This result indicated that the LcPAT8 played a vital role on the regulation of plant growth and development.

Key words: Liriodendron chinense, gene clone, tissue expression characteristics, heterologous expression, functional analysis