大豆種子成熟後期大量表現基因之研究：分析決定20及24KD蛋白質之基因族

Abstract

在許多高等植物中，有一類蛋白質僅在種子成熟後期大量表現，故稱此類蛋白質?late embryogenesis abundant protein，簡稱lea protein。由大豆品種十石所製備的cDNA基因庫（cDNA library）篩選而得之一lea選殖株（clone），稱pGmPM1，經核?酸序列分析結果，該選殖株共有836bp，其中僅有一個open reading frame (ORF)，可轉譯出173個胺基酸。其在試管內轉譯之產物經SDS-PAGE電泳分析，知其分子量約?24KD，且其組成胺基酸並具有親水性及對熱穩定之性質。以pGmPM1對大豆品種William's 82之染色體基因庫（genomic library）進行篩選，得到一選殖株，稱?gGmPM9，所攜帶的染色體DNA約有10kb，其中之2281bp已定序完成，經與pGmPM1之序列比較分析，發現在gGmPM9的兩個exon間有一481 bp之intron，並且在轉譯區中比pGmPM1少了69 bp，亦即有23個胺基酸的缺失，在這短少的23個胺基酸中，有22個為連續出現的胺基酸。若此染色體基因可在同一時期之大豆種子表現，則其相對應的cDNA選殖株就可能存在於cDNA基因庫中，乃設計一組primer，以聚合?鏈反應之技術篩選以pGmPM1為代表之cDNA族（cDNA family），結果証實有一組cDNA選殖株的確比pGmPM1少了69 bp，而這組cDNA選殖株經篩選及定序後，其中之一cDNA選殖株被認為是gGmPM9所對應之基因，故命名?pGmPM9。pGmPM9全長724bp，其ORF可轉譯出152胺基酸，且其胺基酸序列與pGmPM1所決定之胺基酸序列高達96％的相似度，pGmPM9經試管內轉譯而得之蛋白質，以SDS-PAGE分析後，其分子量約為20 KD，且與pGmPM1之基因產物同樣具有親水性及對熱穩定之特性。另外在不同的大豆品種，藉由聚合?鏈反應偵測種子成熟後期萃取之mRNA，發現這兩組基因皆在同一時期表現。此外，由primer extension得知染色體基因選殖株gGmPM9的轉錄起始點位於ATG上游-48 bp處。我們已對gGmPM9之5'端cis regulatory sequences進行剪接及選殖工作，將進一步研究promoter區域之調控機制。

Lea genes express to high abundance to both mRNA and proteins during late embryogenesis in many higher plant seeds. A lea cDNA clone was screened by differential hybridization from a cDNA library of pod-dried soybean seeds. The cDNA insert has a length of 836 bp, which includes 62 bp 5'untranslated region, 521 bp open reading frame, and 251 bp 3'untranslated region. The deduced polypeptide has 173 amino acids and possesses hydrophilic and heat stable proteins. The apparent molecular weight of pGmPM1 translation product is 24 KD as determined by SDS-PAGE. A genomic clone (gGmPM9) that hybridized to pGmPM1 was screened and subcloned, a fragment of 2281 bp has been sequenced. Sequences comparison shows that the genomic DNA has two exons interrupted by one intron of 481 bp and the cDNA sequence contains an additional 69 bp (66 bp encoding 22 amino acids) within the coding sequence. In order to look for the cDNA clones that were derived from gGmPM9, we designed two primers to distinguish pGmPM1 from the corresponding cDNA clones of gGmPM9 by polymerase chain reaction (PCR). The sequence of a selected cDNA clone was found to match with the exon region of gGmPM9 perfectly and named the clone pGmPM9. pGmPM9 contains 724 bp, the ORF encodes a protein of 152 amino acids and the molecular weight is 20 KD by analysis of SDS-PAGE. The pGmPM9 deduced protein sequence has 96% identity as compared with that of pGmPM1. These two genes have been demonstrated to be expressed (by PCR) at the late seed developmental stage in several soybean cultivars. This indicates that pGmPM1 and pGmPM9 may be belong to members of a multigene family. The transcriptional start site of gGmPM9 located as determined by primer extension at 48 bp upstream of the ATG codon. The 5'upstream region of 1kb sequence has been subcloned for the in vitro assay of the nuclear protein bindings by gel retardation and foot printing experiments.