黑莓RuMYB10基因的克隆和表达

Cloning and expression analysis of RuMYB10 in blackberry(Rubus spp.)

【目的】从黑莓中克隆RuMYB10的编码区全长序列,并分析其在黑莓果实发育过程中的表达情况与果实花青素苷积累的关系。【方法】以黑莓基因组DNA为模板,通过同源克隆和SON-PCR技术获得了RuMYB10基因全长编码序列(Accession No.:JQ359611);并以黑莓果实mRNA为模板进行验证。采用实时定量PCR技术检测该基因在果实不同发育阶段的表达水平。【结果】结果表明,该基因编码区全长共1 837 bp,编码216个氨基酸,推导蛋白分子质量为24 863 Da。具有MYB转录因子家族特有的R2R3保守序列。含有两个内含子,第2个内含子长度为750 bp,占全长40.8%;在R3重复单元中存在与bHLH因子互作的‘[DE]Lx2[RK]x3Lx6Lx3R’序列;而促进花青素苷合成的MYB转录因子3个特征氨基酸残基中的丙氨酸(A)被丝氨酸(S)取代。RuMYB10基因在黑莓果实发育后期,即果实变红到最后变黑的过程中大量表达,与花青素苷的积累相一致。【结论】从黑莓中克隆到包含完整ORF的转录因子基因RuMYB10,具有MYB因子家族结构特征和bHLH因子结合域,且在果实花青素苷积累高峰期表达量最高。

[Objective] The objective of this study is to clone and identify the RuMYBIO gene from black- berry fruits and to elucidate its expression patterns along with anthocyanin accumulation. [Method] By using gDNA template, full coding sequences of RuMYBIO （Accession No.： JQ359611） was obtained by degenerate primers and SON-PCR techniques. The sequence was subsequently verified by RT-PCR cloning. Finally, expression patterns of RuMYBIO were accessed by using real-time quantitative PCR. [Result] The full coding sequences of RuMYBIO, 1 837 bp in length, encoding 216 amino acids, had the typical R2R3 conserved MYB domain. It had 2 introns, the second of which ranked 750 bp, consisting 40.8% of the full length. The motif implicated in bHLH cofactor interacting [DE]Lx2[RK]x3Lx6Lx3R was included in the R3 region. Among the deduced amino acid residues in anthocyanin-promoting MYBs, the alanine residual was substituted by serine. The isolated RuMYBIO demonstrated an expression pattern coordinated with anthocyanin accumulation in blackberry fruit, with largely increasing at the stages of turning black process. [Conclusion] The RuMYB10 gene was isolated from blackberry fruits and possessed the bHLH co-acting motif in the R2R3 MYB domain. It showed highest expression level when anthocyanin largely accumulated in blackberry fruits.