甜菜无融合生殖系花期差异表达基因cDNA文库的构建

Construction of the subtractive cDNA library of buds of the apomictic monosomic addition line in sugar beet

为了克隆M14（M14是栽培甜菜与白花甜菜杂交产生的单体附加系,由于附加的白花甜菜第9号染色体上存在无融合生殖基因,其传递率可高达97.5%）品系中无融合生殖相关基因,应用抑制消减杂交方法,在甜菜开花的关键时期减数分裂期取样,建立了甜菜无融合生殖系M14与能够进行正常有性生殖的二倍体甜菜A2Y之间差异表达基因的cDNA文库,并对295个克隆进行了测序,共测得179个表达序列标签（Expressed Sequence Tags,ESTs）,通过对生物信息学数据库资料的查询,共得到了89个与已知基因同源的ESTs,90个新的ESTs,选择其中2个EST进行了RT-PCR鉴定,证明在M14中特异表达.以上结果为以后克隆与无融合生殖相关的全长基因打下了基础.

M14 is a monosomic addition line, the progeny from the hybridization of B. vulgaris L. and B. corolliflora Zoss in sugar beet. It is constituted of the normal 18 B. vulgaris chromosomes plus the No.9 chromosome of B. corolliflora and has a chromosome transmission frequency of 97.5%. The reason that the transmission frequency in Ml4 is so high is interpreted as a possible occurrence of apomixis in the No. 9 chromosome of B. corolliflora in M14. In order to clone the the apomixis gene（s）, the subtractive cDNA library of buds of Ml4 was constructed using suppression subtractive hybridization （SSH）.295 clones were sequenced, acquiring 179 expressed sequence taqs （ESTs）. 179 ESTs were blasted in GenBank, 89 of which were known genes and 90 of which were novel genes. 2 ESTs were differentially expressed in M14 using RT-PCR. From the 179 ESTs, the ESTs related to the apomixis gene（s） can be selected, which can futher be used to clone the whole length of the apomixis gene（s）.