利用c DNA-AFLP 技术研究苹果柱型与非柱型c DNA的差异表达

 以苹果(Malus×domestica Borkh. ) 柱型品种舞姿、非柱型品种富士及其杂交F₁群体中柱型、非柱型单株为研究试材, 应用cDNA-AFLP ( cDNA-amp lified fragment length polymorphism) 技术, 研究柱型与非柱型苹果cDNA的差异表达, 探讨了苹果柱型基因Co表达的分子机制。通过64对引物组合的cDNA-AFLP分析获得了1 630条在柱型与非柱型苹果中差异表达的片段。经过BSA ( bulked segregant analysis) 分析和反向Northern杂交鉴定, 获得了11个差异表达的cDNA片段, 并进行了克隆、测序和序列同源性检索分析。有5个片段在GenBank数据库中发现同源序列, 其余的6个功能未知。核酸和氨基酸比对分析中同源性最高的是片段A7和A16。A7的核苷酸序列同源于红叶石楠Photinia fraseri 26S核糖体RNA (97% ) 、蛋白同源于玉米的细胞色素P450单加氧酶(91% ) , A16的核苷酸(85% ) 及蛋白(93% ) 同源于直果草属Triphysaria versicolor的α-expansin 2基因。对A7、A16两个候选基因片段再进行正向Northern杂交验证,在富士中表达最强, 在非柱型后代、柱型后代及舞姿中表达依次减弱。由此推断, 由细胞色素P450单加氧酶基因所调控的赤霉素GAS变化影响了苹果柱型性状的表达。

Differential Expression Analysis of cDNA Between Columnar and Non-columnar Apples Using cDNA-AFLP

In order to illustrate the molecularmechanism of Co gene, which controls columnar tree structure in apple (Malus×domestica Borkh. ) , differential exp ression p rofile of cDNA among the columnar apple cultivar Waltz, the normal cultivar Fuji, and F₁ seedlings from the cross of Fuji ×Waltzwas analyzed through cDNA-AFLP. Sixty-four pairs of primers were used for the cDNA-AFLP amp lification. Totally 1 630 differentially expressed fragments between columnar and non-columnar app les were detected. Eleven differentially exp ressed fragmentswere confirmed by bulked segregant analysis (BSA) and reverse Northern blotting, further cloned and sequenced. Based on Database search, five cDNA fragments had high homologies to known genes,while the functions of other six were unknown. According to the alignment of Blastn and Blastx, one differentially exp ressed fragment, A7, was 97% identical in nucleotides to the 26S ribosomal RNA gene of Photinia fraseri and 91% in amino acids to cytochrome P450 monooxygenase in maize, respectively. Another differentially exp ressed fragment, A16, was 85% in nucleotides and 93% in amino acids identical to alpha-expansin 2 in Triphysaria versicolor. A16 and A7 as the candidate cDNA differential fragments, which may be related to tree architectures, were further analyzed by Northern Blotting. The express levels of them were the highest in Fuji and reduced gradually in the order of the no-columnar gene pool, columnar gene pool and Waltz. The results imp lied that the change of GA_S modulated by the cytochrome P450 monooxygenase genesmight influence the columnar growth habit.