猕猴桃花青素合成途径基因AcCHS 和AcLDOX的克隆与表达分析

 根据物种水平上蛋白保守序列设计特异引物，扩增获得‘红阳’猕猴桃花青素合成途径中查尔酮合成酶（chalcone synthase，CHS）和无色花青素双加氧酶（leucoanthocyanidin dioxygenase，LDOX）基因的特异片段，用RACE（rapid-amplification of cDNA ends）技术克隆出这两个基因的cDNA全长，长度分别为1501 bp（AcCHS）和1381 bp（AcLDOX），分别编码389个和355个氨基酸。通过比对发现AcCHS与棉花（Gossypium hirsutum）、山茶（Camellia japonica）和黄蜀魁（Abelmoschus manihot）的CHS序列相似性较高，达到95%，与葡萄（Vitis vinifera）和苹果（Malus × domestica）的相似性分别为94%和93%；AcLDOX与山葡萄（Vitis amurensis）和葡葡的相似性分别高达94%和93%。用实时荧光定量PCR分析AcCHS和AcLDOX在‘红阳’（红肉）、‘金魁’（绿肉）和‘金农’（黄肉）3种不同果肉颜色的猕猴桃内果皮中的表达，发现AcCHS的表达量在‘红阳’果实转色期（花后65 d）较高，而在‘金农’开花后表达量呈持续下降趋势；AcLDOX在‘红阳’果实发育早期呈上升趋势，花后65 d后迅速下降，在‘金魁’果实发育后期呈明显上升趋势，在‘金农’开花后呈持续下降趋势。

Cloning and Expression of Anthocyanin Pathway Genes,AcCHS and AcLDOX,in Actinidia chinensis

Based on the highly conserved sequences of other plants，counterpart sequences of CHS（chalcone synthase，CHS）and LDOX（leucoanthocyanidin dioxygenase，LDOX）of Actinida chinesis，‘Hongyang’were obtained using the special primers. Full-length cDNAs encoding CHS and LDOX were cloned respectively from fruit of‘Hongyang’by RACE（rapid amplification of cDNA ends）. The AcCHS was 1501 bp in length，encoding 389 amino acids. AcLDOX was 1381 bp in length and encoded 355 amino acids. Amino acids sequence of AcCHS exhibited over 95% homology with CHS of Abelmoschus manihot，Camellia japonica and Gossypium hirsutum. It also shared 94% and 93% homology with Vitis vinifera and Malus × domestica respectively. AcLDOX showed 94% and 93% identity with Vitis amurensis  and Vitis vinifera respectively at amino acids level. The expression of AcCHS and AcLDOX in the inner pericarp of three kiwifruit cultivars with red，green and yellow color was analyzed by Real-time PCR. AcCHS expressed highly in flesh of‘Hongyang’at 65 DAF（days after flower）. Expression of the gene in yellow-fleshed‘Jinnong’decreased permanently after flowering. Expression of AcLDOX increased during the early developmental stage in‘Hongyang’，but dropped quickly after 65 DAF. Interestingly，expression of AcLDOX raised markedly in the late developmental stages of green-fleshed‘Jinkui’，which was the highest among three cultivars. AcLDOX in‘Jinnong’also deregulated after flowering.