【目的】β－胡萝卜素羟化酶( BCH)是催化β－胡萝卜素经中间产物β－隐黄素合成玉米黄质的关键酶，玉米黄质在植物光保护过程中发挥着重要作用。通过研究毛竹β－胡萝卜素羟化酶基因( PeBCH)的结构特点、表达特征和功能，为揭示强光胁迫条件下PeBCH在竹子光保护中的作用提供证据，为培育抵抗强光胁迫的植物新品种提供新的基因资源。【方法】以毛竹为对象，通过同源克隆的方法分离 PeBCH，利用生物信息学软件分析其结构特点，采用实时荧光定量 PCR技术分析基因的组织表达特性，构建 PeBCH 基因的过量表达载体，并在拟南芥中异位表达，通过对转基因拟南芥植株的表型和生理变化来鉴定 PeBCH 基因的功能。【结果】从毛竹中获得了1个BCH同源基因序列，命名为 PeBCH；该基因的全长1385 bp，编码区为927 bp，编码区对应的基因组序列为1566 bp，包含5个内含子、6个外显子，内含子完全符合 GT-AG剪接原则。PeBCH编码1个308个氨基酸的蛋白， PeBCH蛋白具有BCH家族的特征结构域PD095142和PD011050，存在4个跨膜结构，二级结构含有无规则卷曲、延伸链和α螺旋3种，其中以无规则卷曲覆盖的氨基酸残基最多。组织表达特异性分析表明，PeBCH 基因在毛竹的根、幼茎、叶片、叶鞘、节中均检测到表达，但表达丰度存在着显著差异，其中在叶片中的相对表达丰度最高。不同光照处理影响 PeBCH 基因的表达，随着光强的增大该基因的表达丰度呈现先上升后下降的趋势，其中光强1000μmol·m －2 s －1处理后基因的表达丰度最高，约为对照的1.5倍，但光强1500μmol·m －2 s －1处理后，PeBCH 基因的表达丰度显著下降，仅为对照的5%，明显受到强光的抑制。利用 PeBCH 基因转化拟南芥后，RT-PCR 分析表明PeBCH基因在转基因植株中得到表达；与野生型拟南芥相比，转PeBCH基因拟南芥植株生长健壮，叶绿素、类胡萝卜素、胡萝卜素和叶黄素含量均有所增加；在实验室环境光强(145μmol·m －2s －1)和强光(530μmol·m －2s －1)条件下，转 PeBCH基因植株的 NPQ明显高于野生型植株，二者 NPQ 的稳定值间差异达到极显著水平( P ＜0.01)。【结论】PeBCH在毛竹中为组成型表达，光照处理(＜1000μmol·m －2 s －1)诱导其在叶片中的表达。该基因的过量表达有助于提高转基因植株的热耗散能力，抵抗强光胁迫。该基因将是今后植物抗逆分子育种的重要基因资源之一。%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 -2 s -1 ,and reached the highest level ( about 1. 5 times of the control) after 2 h treatment with 1 000 μmol·m -2 s -1 . However,it was inhibited significantly after 2 h treatment with 1 500 μmol·m -2 s -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 530μmol·m -2 s -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 -2 s -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.
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