利用T-DNA插入突變體探討水稻基因之功能-抗病能力異常突變體M0023454及穀粒發育異常突變體M0039314之特性及基因表現分析

Abstract

In this study, 12 T-DNA insertion mutant lines were selected by their distinct phenotypes in grain development such as sterile, low fertility, abnormal panicles and grains. Only six of these mutants could get flanking sequence by TAIL PCR and plasmid rescue. Among them, only mutants M0023454 and M0039314 showed single insertion event without changes of Tos17 number in their genomes. They were selected for further study. In mutant M0023454, the T-DNA was inserted in the BAC clone of OSJNBa0039C07 in the rice chromosome number 4. This mutant showed more susceptible to the pathogen, and this phenotype was co-segregated with the genotype. The expression of gene 454-10, 987 bp upstream of T-DNA insertion site, was activated in mutant while no expression was detected in TNG67. Gene 454-10, encode an AP2 domain, is belong to the AP2/ERF family and much more similar to ERFBP group, which involved in pathogen resistant pathway. This study suggests that the susceptible phenotype to pathogen in mutant M0023454 may be due to the overexpression of gene 454-10. In mutant M0039314, the T-DNA was inserted in the PAC clone of P0702E04 in the rice chromosome number 8. Mutant M0039314 showed abnormal phenotypes, such as dark green leaves, irradiative tillers, low fertility, incomplete seed development, weaker anthers and reduced length of panicle and peduncle. Almost all of these abnormal phenotypes were co-segregated with the genotypes. The expression of gene 314-5 and 314-6, flanked at both side of T-DNA inserted site, were activated in mutant M0039314 while no expression was detected in TNG67. Gene 314-5 encodes a putative transcription factor, SBP, and expressed weakly in all vegetative tissues but strongly in the reproductive tissues in TNG67. Gene 314-6, an OsMADS45 gene, has been published and showed mainly expressed in the reproductive tissues in TNG67. The results of this study suggest that the abnormal phenotypes in mutant M0039314 may be due to the overexpression of gene 314-5 and 314-6. In order to confirm the abnormal phenotypes of M0023454 and M0039314 are really caused by the overexpression of 454-10, 314-5 and 314-6 and to find out the exact function of these genes, the transgenic lines with overexpression of these genes will be created.

本研究自田間約6,000株的T-DNA插入突變株中，篩選穀粒完全不稔實、稔實率較低、穗發育特異或榖粒外型特異等與水稻榖粒發育相關的突變株，共12株系。利用質體救援或Thermal asymmetric interlaced PCR的方法，共六突變株獲得T-DNA所插入鄰近序列，由南方墨點法結果顯示其中M0023454及M0039314兩株突變株為單一T-DNA插入所致。進一步分析其後代的基因型與農藝性狀間的遺傳模式，發現有共分離的現象。M0023454突變株具有易遭病菌感染的特性，其中T-DNA插入於水稻第4對染色體OSJNBa0039C07 BAC上之113711 bp位置，經由逆轉錄酶連鎖反應分析，T-DNA的插入，可活化插入點上游987 bp處編號454-10基因的表現量。經分析發現，454-10在葉及種子發育過程中有微量的表現，而在5天的根部組織及誘導15天的癒傷組織中有大量的表現。經胺基酸比對分析，454-10蛋白內含有AP2活性區域，歸類在AP2/ERF家族基因中的ERF類，主要參與調控抵抗病菌入侵的生理反應，與阿拉伯芥中的RAP2.6最相似。因此推測454-10的大量表現可能造成植株抵抗病菌能力產生變異。M0039314突變株具有：稻桿外擴、穗型直立、花梗及穗長較短、花藥發育異常、稔實率低及種子發育不完全等特異的農藝性狀；其中T-DNA插入於水稻第8對染色體編號P0702E04 PAC的48951 bp的位置，經由逆轉錄酶連鎖反應分析，T-DNA的插入，可活化上游314-5及下游314-6兩個基因的表現量。經分析發現，314-5在營養生長組織有持續微弱的表現，在生殖生長組織中則有大量的表現，而314-6在生殖生長組織中偵測到大量表現。進一步將314-5及314-6的胺基酸序列進行比對分析，314-5蛋白含有一個植物特有且具轉錄因子特性的SBP活性區域；314-6則為水稻中已發表的OsMADS45。由M0039314突變株的農藝性狀，推測314-5及314-6的大量表現可能會影響穗、花器及種子的型態。M0023454及M0039314突變株所觀察到之特異農藝性狀是否確實因為454-10或314-5、314-6基因活化大量表現所導致，仍須進一步針對回復株作探討。