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以‘油青49’和‘油青甜菜薹80’菜薹茎尖为材料,采用RT-PCR和RACE技术克隆获得组蛋白甲基转移酶基因Brcu PRMT5的全长cDNA和gDNA序列。BrcuPRMT5 cDNA序列全长为2 117 bp,其中完整开放阅读框为1 929 bp,编码642个氨基酸,相对分子量为71.55 kD,理论等电点(p I)为5.87;多序列比对结果表明,Brcu PRMT5编码的氨基酸序列含有高等植物PRMT5基因1个高度保守的结构域;系统发育分析结果显示与大白菜、油菜及甘蓝的亲缘关系最近;亚细胞定位软件分析得知,Brcu PRMT5蛋白无跨膜区域,可能定位于线粒体中;对应的gDNA全长为4 151 bp,含有23个外显子和22个内含子,最长的外显子长度为140 bp,内含子的长度范围为50~150 bp。利用半定量RT-PCR和实时荧光定量PCR技术分析基因的表达,BrcuPRMT5在菜薹不同组织中均有表达,其中在花中表达量最高,叶次之,根中最低;BrcuPRMT5从苗期至完全抽薹开花期的表达量呈现上升趋势。BrcuPRMT5在菜薹花发育过程中可能起一定的调控作用。
The full-length cDNA and gDNA sequences of the histone methyltransferase gene Brcu PRMT5 were cloned by RT-PCR and RACE techniques using the stem tips of ’Youqing 49’ and ’Youzai Beitai 80’. The full-length cDNA of BrcuPRMT5 was 2 117 bp with a complete open reading frame of 1 929 bp encoding 642 amino acids with a relative molecular mass of 71.55 kD and a theoretical isoelectric point (pI) of 5.87. The results of multiple sequence alignment showed that Brcu The amino acid sequence of PRMT5 encoded a highly conserved domain of PRMT5 in higher plants. Phylogenetic analysis showed that the genetic relationship with Chinese cabbage, rape and cabbage was the closest. The subcellular localization software showed that there was no transmembrane domain in Brcu PRMT5 , Which may be located in mitochondria. The corresponding full-length gDNA is 4 151 bp in length with 23 exons and 22 introns, the longest exon length is 140 bp and the length of intron is in the range of 50 ~ 150 bp. The expression of BrcuPRMT5 was detected in semi-quantitative RT-PCR and real-time fluorescence quantitative PCR. The expression of BrcuPRMT5 was the highest in flowers, followed by the leaves and the lowest in roots. The expression level at bolting and flowering stage showed an upward trend. BrcuPRMT5 may play a regulatory role in the development of Campanulaceae flower.