Rice functional genomics study with T-DNA insertion mutants --Characterizetion and gene expression analysis of a growth and development mutant M0019081

Abstract

In this study, the T-DNA insertion mutant pool created by Dr Yu's group of Academia Sinica was used for screening distinct phenotype in grain development such as sterile, low fertility or low grain yield. Twenty-two mutants were selected initially for flanking sequence identification through plasmid rescue and/or thermal asymmetric interlaced PCR analyses. Ten mutants of their flanking sequences were resolved. Among them, a dwarf and low fertility mutant M0019081 showing a single copy of T-DNA insertion in the chromosome 7 within the BAC clone OJ1316_A04 was selected for further study. The genomic DNA hybridization pattern of rice endogenous copia-like retrotransposon, Tos17, of mutant M0019081 was the same as that of TNG67. The T-DNA insertion locus was confirmed by PCR and the genotype of T-DNA insertion in the progenies was co-segregated with the dwarfism and low fertility phenotype. Three hypothetical protein genes, designated as A04.104, A04.106 and A04.107, were revealed flanking by the T-DNA insertion locus in the BAC clone using RiceGAAS assay. While the RNA expression level of A04.104, and A04.106 showed no difference between TNG67 and M0019081, the expression level of A04.107 was about 2-fold increase in the 15-days-old seedlings in the mutant. The expression of A04.107 was observed ubiquitously in various stages of leaves and panicles in the TNG67. Sequence analysis revealed that the A04.107 was most similar to the mammalian Mo25 protein, a protein involved in regulating cell proliferation and polarity through LKB1. A putative Mo25-like gene in Arabidopsis, At5g18940 was identified to be the highest homology (82%) to A04.107 from the GenBank database. Although the possible function of Mo25-like protein in plant remains unclear, the enhanced expression of this novel gene A04.107 could cause dwarf and low fertility in rice plant will be an interesting subject for further investigation.

本研究共篩選約6,000株T-DNA插入突變株系，根據生長發育及稔實異常的明顯外表性狀，選取22個突變株系，其中可藉由TAIL-PCR及質體救援之方式得到10個T-DNA插入鄰近序列。M0019081為矮株且稔實率低之突變株，其T-DNA為單一插入於水稻第七對染色體上BAC clone OJ1316_A04，而且其內生性跳躍因子Tos17的拷貝數目與台農67號同樣均為3個，並未因轉殖過程而增加。T-DNA插入點經由聚合酶連鎖反應確認而且後代植株之基因型也與株高及稔實率的外表性狀有相關性，推測其外表性狀的特異是由T-DNA插入所造成的。逆轉錄聚合酶連鎖反應發現T-DNA插入點下游3.4 kb之A04.107基因表現量有增加的情形，可能是由於T-DNA上之35S加強子所影響。A04.107為穩定表現於葉片及穗之基因而且在90-100天之葉片有較大之表現量，分析15天幼苗的基因表現情形後發現，T-DNA插入之異質與同質配子結合體的基因表現量均為台農67號的兩倍之多。透過RiceGAAS之胺基酸序列比對，發現A04.107相似於哺乳動物之Mo25蛋白，分析A04.107與其他11個物種之Mo25胺基酸相似度，發現具有40-60%的一致性及60-80%之相似性，而且與阿拉伯芥擬Mo25蛋白At5g18940最為相似。雖然可以推測A04.107為一擬Mo25蛋白，但其真正功能目前尚不明確，經由突變株外表性狀特異，推測A04.107為水稻上一個新發現基因且其扮演之功能可能與生長及種子發育有關。