CAJ | 학술논문

采用基因芯片技术,从大豆中鉴定了一个荚优势表达基因.利用生物信息学的方法,拼接了该基因的全长序列,并通过RT-PCR克隆了该基因.Blast检索分析表明,该基因编码一个具有AP2结构域的转录因子,且与拟南芥DREB类蛋白TINY的氨基酸相似度最高,将该基因命名为GmTINY1.GmTINY1包含一个735 bp的开放阅读框,编码244个氨基酸残基.GmTINY1与拟南芥TINY和TINY2蛋白的相似度分别为59%与62%.系统发生分析表明,GmTINY1、TINY和TINY2位于一个分支,且同属于DREB亚家族.实时定量RT-PCR检测表明,GmTINY1基因在荚中高丰度表达,在花中的表达量也较高,在根中的表达量较低,而在叶片中未检测到表达.基因芯片信息分析结果表明,GmTINY1在种子发育的子叶期的种脐部分高丰度表达.由此推论,GmTINY1基因在大豆生殖器官发育中可能发挥调控作用,可能与种子发育过程中种脐的形成有关.
채용기인심편기술,종대두중감정료일개협우세표체기인.이용생물신식학적방법,병접료해기인적전장서렬,병통과RT-PCR극륭료해기인.Blast검색분석표명,해기인편마일개구유AP2결구역적전록인자,차여의남개DREB류단백TINY적안기산상사도최고,장해기인명명위GmTINY1.GmTINY1포함일개735 bp적개방열독광,편마244개안기산잔기.GmTINY1여의남개TINY화TINY2단백적상사도분별위59%여62%.계통발생분석표명,GmTINY1、TINY화TINY2위우일개분지,차동속우DREB아가족.실시정량RT-PCR검측표명,GmTINY1기인재협중고봉도표체,재화중적표체량야교고,재근중적표체량교저,이재협편중미검측도표체.기인심편신식분석결과표명,GmTINY1재충자발육적자협기적충제부분고봉도표체.유차추론,GmTINY1기인재대두생식기관발육중가능발휘조공작용,가능여충자발육과정중충제적형성유관.
Plant reproductive development involves the coordination of a lot of genes encoding transcription factors. The AP2 domain transcription factors have been proved with critical roles in plant reproductive development. By microarray analysis, we identified a gene which showed higher expression in pod as 500 folds as that in leaf of soybean. The Blast searches indicated this gene encodes a dehydration responsive element binding protein (DREB)-like protein showing highest similarity to Arabidopsis TINY, therefore named as GmTINY1. By searching soybean genome and EST databases, the putative full-length cDNA sequence for GmTINY1 was in silico assembled. The GmTINY1 gene was cloned from soybean seeds at 15 DAF (days after flowering) by RT-PCR. GmTINY1 contained a complete open reading frame (ORF) of 755 bp which encoded a peptide of 244 amino acids. The predicted molecular mass and isoelctric point of GmTINY1 are 26.76 kD and 5.12, respectively. An AP2 domain and a Ser-rich domain were identified in GmTINY1 amino acid sequence by Motif Scan server. The GmTINY1 encoding product showed 59% and 62% sequence similarities with Arabidopsis TINY and TINY2, respectively. Multiple sequences alignment revealed that a highly conserved AP2 domain was present in each AP2 domain transcription factor. In this AP2 domain, it was found that a Ser~(66) was specifically present in GmTINY1, DREB1B, TINY and TINY2 proteins but a Cys~(66) in DREB1A and DREB1C proteins, indicating, like Arabidopsis TINY, TINY2 and DREB1B, GmTINY1 might be able to bind both the dehydration responsive ele-ment(DRE) and ethylene responsive element (ERE) motifs while DREB1A and DREB1C only bind DRE motif. Besides, it was found that a Ser-rich domain probably involving translational modification on GmTINYl protein was located close to AP2 domain. The neighbor-joining phylogenetic tree showed that the AP2 domain transcription factors were grouped into four subfamilies: DREB, ERF, AP2, and RAV; and GmTINY1, TINY, and TINY2 were grouped into a branch which attributed to the DREB subfamily. With an attempt to understand the biological role for GmTINY1 and to verify the microarray result, we used the Real-time quantitative PCR approach to analyze the expression pattern of GmTINY1 in various soybean organs. We found that expression of GmTINY1 was the highest in pod, relatively lower in flower and root, but undetectable in leaf, indicating that GmTINY1 may play some roles in soybean reproductive organs and root, but not in leaf. The Blast search results against soybean EST database also supported the specific expression of GmTINY1 in soybean pod and root. We analyzed GmTINY1 expression during the course of seed development based on publicly available microarray data and found that GmTINY1 was expressed with a low level at the globular stage and heart stage but highly expressed in hilum at the cotyledon stage embryos. Taken together, it is suggested that GmTINY1 may play some regulatory role in soybean reproductive development, such as the formation of hilum in soybean.