薄壳山核桃MADS-box基因保守片段的克隆与序列分析

Cloning and sequence analysis of MADS-box gene conservative fragment from pecan (Carya illinoensis)

【目的】克隆薄壳山核桃MADS-box基因的保守片段,进行系统发育分析,为研究薄壳山核桃花发育相关MADS-box家族基因及其发育的分子机理奠定基础。【方法】以薄壳山核桃品种‘马罕’雄花花序为材料,提取总RNA反转录cDNA,采用RT-PCR克隆MADS-box基因的保守片段,并将其推导氨基酸序列与已知拟南芥的MADS-box家族基因进行系统发育分析。【结果】分离获得28条MADS-box基因的cDNA片段,片段长度均为137bp,包含基因起始密码子,核苷酸序列同源性为65.7%～98.5%,其推导氨基酸序列中有11个存在差异。系统发育树分析结果表明,这些基因片段分别归入拟南芥MADS-box基因不同亚家族中,包含ABCDE模型中的各类基因。【结论】薄壳山核桃中存在多种MADS-box家族基因,克隆的片段包含ABCDE模型中的各类花发育基因。

[Objective]The conservative fragment sequence of MADS-box genes from pecan was cloned and their phy- logeny was assayed in order to provide references for cloning pecan new MADS-box family genes related to flower development and exploring molecular mechanism of pecan flower development. [Method]The total RNA was extracted from male flower of pecan variety ＇Mahan＇ and cDNA was synthesized using inverse transcription. The conservative fragment of MADS-box genes was cloned using RT-PCR, and the phylogenetic analysis was conducted using the deduced amino acid sequences with known Arabidopsis MADS-box gene subfamily. [ResultlTwenty-eight cDNA fragments of MADS-box gene were obtained by RT-PCR, and the length of these fragments was 137 bp including the initiation codon of genes. The homology of these nu- cleotide sequences was between 65.7% and 98.5%. The 11 deduced amino acid sequences were different. According to phyloge- netic tree analysis, all these gene fragments involving different types of gene in ABCDE model were divided into the Arabidopsis MADS-box gene subfamily. [Conclusion]There were many MADS-box genes in pecan. These gene fragments included all types of floral development genes in ABCDE model.