月季突变体抑制差减杂交cDNA文库构建及分析

Construction and Analyses of SSH cDNA Libraries of Rose Floral Color and Scent Mutant

为了分析月季花色花香突变机理和揭示花色花香代谢的相关基因,利用抑制性消减杂交技术分离了月季红花无香突变体‘往日情怀’(以下简称突变体)与其野生型金黄色浓香品系‘金银岛’(以下简称野生型)之间表达差异cDNA片段。分别以突变体作为驱赶子,野生型为检测子,以及以野生型作为驱赶子,突变体为检测子建立了两个差异表达cDNA文库WSSH和JSSH,分别代表在突变体和野生型中特异表达的cDNA;再经文库高密度点阵膜的杂交差示筛选分析,在WSSH库中获得特异表达的27个阳性克隆,在JSSH文库中得到25个阳性克隆。差异表达克隆测序后经BLAST比对分析发现WSSH文库中含有与红花突变体的花青素积累直接相关的CHS、DFR、细胞色素P450加单氧酶、乙二醛酶Ⅰ、己糖转移因子、MYB1转录因子、S-腺苷蛋氨酸转移酶、ADR等花色相关EST;JSSH文库中含有与野生型花香形成相关的月季甲基间苯二酚O-甲基转移酶、转醛醇酶、Acyl-CoA结合蛋白、钙调素结合蛋白、MYB92转录因子的EST以及导致芽变的Ty1-copia-like逆转座子。

In order to analyse molecular mechanism of rose flower and scent metabolism and to explore the genes involved in rose floral color and fragrance metabolism, the suppressive subtraction hybridization （SSH） technique was used. Two differential expressing cDNA libraries were constructed with the cDNAs of the rose color and scent mutant Wangriqinghuai as driver and the cDNAs of its wild type Jinyindao （ Rosa × hybrida） as tester, vice versa. The cDNA libraries of WSSH indicated the differential expressing cDNA fragments in Wangriqinghuai and JSSH indicated the differential expressing cDNA fragments in Jinyindao. The difference of PCR bands for substracted sample and non-substracted sample was obvious. The relevant cDNA fragments expressing differentially between Wangriqinghuai and Jinyindao were revealed and isolated through micoarray analysis of the clones in SSH. Twenty-seven positive clones of WSSH and 25 positive clones of JSSH were obtained. Mter sequencing the clones expressing differentially in two cuhivars and blasting their sequences, some ESTs regulating the color of pink mutant, such as CHS, from WSSH. Some ESTs regulating the floral color and scent of yellow DFR, MYB1, ADR were obtained progenitor, such as OOMT, MYB92 were also obtained from JSSH. Tyl-copia-like retrotransposon most likely causing bud mutation was also got in this study. This is the first report on rose mutant SSH which would be useful for analysis and isolation of the genes related to rose floral color or scent.