果梅PmKNAT2基因全长cDNA克隆及表达分析

Isolation and Expression Analysis of PmKNAT2 Gene from Japanese Apricot

【目的】从‘大嵌蒂’果梅中克隆PmKNAT2,并对其结构特征及表达模式进行分析,为研究果梅雌蕊败育的分子机理和分子育种奠定基础。【方法】在NCBI中查找与果梅相似性很高的桃KNOPE2(KNAT2的同源基因)序列(EF093491),根据桃的KNOPE2序列设计特异引物;采用改良CTAB法提取‘大嵌蒂’果梅花芽总RNA;通过RT-PCR和RACE技术获得基因cDNA序列全长;使用DNAMAN软件进行序列拼接;利用NCBI数据库中的BLASTn和BLASTp程序进行相似性分析;通过生物信息学方法分析结构特征;用DNAMAN软件分析基因的ORF和氨基酸序列;MEGA4.0软件进行系统进化树的构建;利用Bioxm2.6推测分析蛋白分子量和等电点;根据NCBI中Conserved Domains程序进行蛋白质保守域结构预测;用ExPaSy提供的在线SOPMA程序进行蛋白质二级结构预测;构建PJIT166-PmKNAT2-GFP的融合亚细胞定位表达载体,采用基因枪法转化洋葱表皮细胞,经暗培养后在激光共聚焦显微镜下观察;利用实时荧光定量RT-PCR研究其表达模式,分析其在‘大嵌蒂’花芽发育不同时期、不同器官中的表达特性。【结果】在‘大嵌蒂’果梅中获得一个KNAT2的同源基因,命名为PmKNAT2,其cDNA全长为1 402 bp,5'UTR47 bp,3'UTR 293 bp,编码区全长为1 062 bp,编码353个氨基酸,预测蛋白质分子量为40.41 kD,理论等电点为4.85。蛋白结构分析表明该基因编码的氨基酸具有2个结构域,MEINOX结构域(KNOXⅠ和KNOXⅡ2个亚结构域)和HD(homomeodomain)结构域,属于KNOX蛋白;相似性分析发现该基因与GenBank中其它来源的KNOX蛋白序列的相似性为50%—100%;系统进化树分析显示果梅PmKNAT2与桃KNOX聚为一类,表明与其亲缘关系最近。二级结构预测结果表明PmKNAT2所编码蛋白由47.14%的α-螺旋(Alpha helix)、3.43%的β-转角(Beta turn)、3.14的β-折叠(Extended strand)和46.29%的随机卷曲(Random coil)组成。亚细胞定位结果显示该基因编码的蛋白定位于细胞核和细胞膜中。实时荧光定量RT-PCR分析表明,在‘大嵌蒂’果梅不同时期花芽中PmKNAT2的表达量存在一定差异,PmKNAT2在11月份的表达量最高,9月、10月和11月的表达量差异不明显,但与12月和1月花芽中的表达量差异明显;生长素在1月份含量最高,9、10、11月份差异不明显,其含量变化趋势与PmKNAT2表达趋势相反。对该基因表达量最高的11月份的花芽进行实时荧光定量分析发现:PmKNAT2在各种花芽中均有表达,在不完全花(雌蕊褐色、雌蕊畸形和无雌蕊)中的表达量高于完全花(雌蕊正常)。进一步分析PmKNAT2在完全花与不完全花的不同花器官中的表达量,结果表明PmKNAT2在完全花与不完全花的各部位的表达量趋势相似均为:萼片>雄蕊>花瓣,在完全花雌蕊中的表达量最低。【结论】推测PmKNAT2在雌蕊发育阶段的异常表达可能与‘大嵌蒂’果梅雌蕊败育有关。

【Objective】 This paper aims to isolate the PmKNAT2 gene from Japanese apricot（Prunus mume Sieb. et Zucc.） cv ‘Daqiandi＇, and analyze the structure and expression pattern of this gene, for further studying the molecular mechanism of Japanese apricot pistil abortion and molecular breeding. 【Method】 Specific primers were designed based on the peach sequence（EF093491） in NCBI, which is the highest homologue with peach gene KNOPE2. The improved CTAB method was used to isolate total RNA and the full length of PmKNAT2 cDNA was obtained by using RT-PCR and RACE. The sequencing data were assembled by DNAMAN software; BLASTn and BLASTp in NCBI were used to do the similarity analysis. PmKNAT2 gene structural characteristics were analyzed by the following software： DNAMAN was used to analyze the ORF and amino acid sequences and MEGA4.0 was used to create the phylogenetic tree; the protein molecular weight and isoelectric point were speculated by using Bioxm2.6; the conserved domain structure of protein was predicted by Conserved Domains program in NCBI; the protein secondary structure was predicted by using SOPMA program. The fusion expression vector PJIT166-PmKNAT2-GFP was constructed and then introduced into onion epidermal cells by the particle bombardment method; green fluorescence was monitored under a laser scanning confocal microscope. Quantitative real-time PCR（qRT-PCR） was performed to determine the expression pattern of PmKNAT2 in different developmental stages of flower buds and different flower organs. 【Result】 The full length of PmKNAT2 cDNA was 1 402 bp and contained 47 bp 5′UTR, 293 bp 3′UTR and a 1 062 bp ORF which encoded a 353 amino acids polypeptide with a calculated molecular weight of 40.14 kD and an isoelectric point of 4.85. Protein structure analysis showed that PmKNAT2 contained two kinds of domain namely MEINOX area（KNOXⅠand KNOXⅡ） and HD area, which indicated that it belongs to the KNOX protein. Similarity analysis showed that the predictive amino acid sequence of PmKNAT2 compared with other sequences of KNOX in GenBank shared 50%-100% in homology. The phylogenetic tree analysis showed that PmKNAT2 was clustered together with peach KNOX protein, which was consistent with the morphological classification. In addition, the predictive secondary structure showed that PmKNAT2 protein was made up of 47.14% alpha-helix, 3.43% beta-turn, 3.14% extended strand and 46.29% random coil. Subcellular localization results showed that the PmKNAT2 protein localized in cell nucleus and cell membrane. Real-time PCR analysis showed that the expression level of PmKNAT2 was various in different stages of flower buds of ‘Daqiandi＇, the highest level in November. There were no significant difference in September, October and November. However, there was a significant difference between December and January. As for the determination of auxin content, the results showed that the highest level in January, and there was no significant difference in September, October and November; in contrast with the trend of gene expression. The expression analysis of flower buds in November showed that PmKNAT2 expressed in all the tissues, the expression level of imperfect flower（pistil brown, pistil deformity and no pistil） were higher than perfect flower. There was no tissue specific expression of PmKNAT2 gene between perfect flower and imperfect flower. The expression level in the sepal was higher than that in the stamen and the expression level in the stamen was higher than that in the petal. The lowest expression level of pistil was in perfect flower. 【Conclusion】 The abnormal expression of this gene might be related to pistil abortion in ‘Daqiandi＇.