玉米籽粒发育相关基因ZmMADS-RIN的克隆及表达分析

玉米籽粒发育及产量是玉米重要的经济性状。本研究以玉米骨干自交系郑58为试材，采用同源克隆法得到一个与玉米籽粒发育相关的基因ZmMADS-RIN。该基因的cDNA全长859 bp，开放阅读框为768 bp，编码255个氨基酸，与玉米B73中所对应的cDNA的编码区序列相比，共有6个SNPs位点的差异，3个氨基酸残基发生了变化。生物信息学分析表明，ZmMADS-RIN蛋白的相对分子量为29.23 kD，理论等电点(pI)为8.84，是一种亲水性蛋白，不含信号肽序列，也不含跨膜结构；具有5个磷酸化位点；二级结构中含有50.20%的α-螺旋(alpha helix)、27.45%的无规则卷曲(random coil)、14.51%的延伸链(extended strand)和7.84%的β-转角(beta turn)；该蛋白位于细胞核内；其氨基酸序列中含有高度保守的MADS结构域、相对保守的K结构域、低保守的I结构域和最不稳定的C结构域，是典型的MIKC型MADS-box蛋白；系统进化树分析表明，ZmMADS-RIN蛋白属于AGL6分枝，与水稻基因OsMADS6的相似性最高，为89%。ZmMADS-RIN基因在玉米自交系郑58的籽粒中特异表达，在根及不同时期的叶片中没有检测到表达信号。荧光定量PCR结果显示，ZmMADS-RIN基因的表达量在玉米籽粒发育的不同阶段呈一定规律的变化，在授粉后0~20 d，呈上调表达趋势，授粉后25 d表达量迅速下降至较低水平，而在授粉后30~40 d则检测不到其表达。推测该基因可能与玉米籽粒的发育有关。

Cloning and Expression Analysis of ZmMADS-RIN Gene for Regulating the Kernel Development of Maize

Kernel development is the most important character of maize. In this study, a maize gene named ZmMADS-RIN encoding MADS-box protein relating to kernel development was isolated. The full length cDNA of ZmMADS-RIN is 859 bp, and it contains an open reading frame (ORF) with 768 nucleotides and encodes 255 amino acid residues. The variable analysis of ZmMADS-RIN in coding region showed that six nucleotides and three amino acid residues were different between Zheng 58 and B73. Bioinformatics analyses showed that ZmMADS-RIN was a hydrophilic protein with a molecular weight of 29.23 kD, a theoretical isoelectric point of 8.84, and five phosphorylation sites, but signal peptide sequence and transmembrane domain were not found. The secondary structure of ZmMADS-RIN protein contained 50.20% of alpha helix, 27.45% of random coil, 14.51% of extended strand and 7.84% of beta turn. ZmMADS-RIN protein was tested to be a typical MIKC-type protein localized in nucleus with highly conserved MADS domain, relatively conserved K domain, less weakly conserved I domain and most variable C domain. Phylogenetic analysis indicated that ZmMADS-RIN protein belongs to the same branch of AGL6, and shows high similarity of 89% to OsMADS6 protein from Oryza sativa. RT-PCR results showed that ZmMADS-RIN expressed in kernel specially, and not in root or leaf during different phases. In addition, qRT-PCR results indicated that the expression of ZmMADS-RIN increased continually from zero day to 20 days after pollination, and reached the highest level at the 20th day, then decreased significantly. And it could not be detected during 30th?40th days. These results demonstrated that ZmMADS-RIN might be associated with the regulation of developing kernel in maize.