关键词: 毛竹, β-胡萝卜素羟化酶基因, 拟南芥, 色素含量, 叶绿素荧光参数

Abstract: [Objective] Zeaxanthin plays an important role in light protection for plants under light stress. β- carotene hydroxylase (BCH) is a key enzyme in catalyzing β-carotene to form zeaxanthin via β-cryptoxanthin. To reveal the role of BCH in light protection for bamboo under stress conditions of high light intensity and provide new genetic resource for the breeding of new varieties, the study of bamboo β-carotene hydroxylase gene (PeBCH) structural features, expression and functional characteristics will be carried out. [Method] Moso bamboo (Phyllostachys edulis) was used for the isolation of PeBCH by homologous cloning method, the structural characteristics of PeBCH was analyzed using bioinformatics software, the analysis of gene expression in different tissues was performed with real-time quantitative PCR, gene function was identified through the phenotypic and physiological analyses of transgenic Arabidopsis thaliana with overexpressed PeBCH.[Result] A homologous gene of BCH was obtained from P. edulis and named as PeBCH. The full length cDNA of PeBCH was 1 385 bp including an open reading frame (ORF) of 927 bp, which encoded a 308 aa protein with the characteristic domains, PD095142 and PD011050 of BCH family. The genomic sequence corresponding to the ORF was 1 563 bp containing five introns and six exons, which was in full compliance with the intron splicing principles of GT-AG. There were four transmembrane domains in PeBCH, which comprised three kinds of second structures such as random coil, extended strand and α helix, of which the random coil covered the largest number of amino acid residues. Tissue specific expression showed that PeBCH was differently expressed in root, young stem, leaf, sheath and node, with the highest level in leaf. The expression of PeBCH was affected by light treatment in a trend of upward followed by downward with increasing light intensity. The expression was in an upward trend with the light intensity no more than 1 000 μmol·m^-2s^-1, and reached the highest level (about 1.5 times of the control) after 2 h treatment with 1 000 μmol·m^-2s^-1. However, it was inhibited significantly after 2 h treatment with 1 500 μmol·m^-2s^-1, which was only 5% of the control. RT-PCR analysis demonstrated that PeBCH was expressed in the transgenic plants of A. thaliana. Compared with wild type, transgenic plants were vigorous, the content of pigments including chlorophyll, carotenoid, β-carotene and lutein were all increased compared with those of wild-type plants. Meanwhile, the values of non-photochemical quenching (NPQ) of transgenic plants were all increased under the laboratory light intensity (145 μmol·m^-2s^-1) and the intensity of 530 μmol·m^-2s^-1, and the difference between the stable values of NPQ reached a significant level (P <0.01). [Conclusion] PeBCH was constitutively expressed in moso bamboo, and it was induced in leaf by high light intensity (<1 000 μmol·m^-2s^-1). The overexpression of PeBCH was helpful to improve heat dissipation capability of transgenic plant to resist high light stress, indicating that PeBCH will be an important genetic resource for molecular breeding of plant resistence in future.

Key words: Phyllostachys edulis, β-carotene hydroxylase gene, Arabidopsis thaliana, pigment content, chlorophyll fluorescence parameter