Rice functional genomics study with T-DNA insertion mutant- Characterization and gene expression analysis of a heading development mutant M0034845

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-three mutants were selected initially for flanking sequence identification through plasmid rescue and/or thermal asymmetric interlaced PCR analyses. Among them, eleven mutants of their flanking sequences could be resolved and 5 of them revealed only single T-DNA insertion with the same Tos17 hybridization pattern as that of TNG67 after Southern blotting assay. The T-DNA insertion loci of 3 mutants, M0034845, M0019179 and M0023235, were confirmed through PCR analysis. The mutant M0034845 showed late flowing phenotype was selected for further study. The late flowing phenotype in the T1 and T2 progenies was observed to co-segregate with the existence of T-DNA insertion. This mutant has T-DNA inserted in the chromosome 11 and located within the BAC clone OSJNBa57E15. Two hypothetical protein genes, designated as JA-01 and JA-02, were revealed flanking by the T-DNA insertion locus in the BAC clone using RiceGAAS assay. While the RNA expression level of JA-01 showed no difference between TNG67 and M0034845, the expression level of JA-02 was about 3-fold increase in the seedling and leaves of 90-day-old plant in the mutant. The expression of JA-02 was observed in roots, various stages of panicles (from 2 to 13 cm) and in the leaves of all developmental stages in the TNG67. Dark treatments of rice seedling decrease the RNA expression of JA-02 in the TNG67. On the contrary, dark treatment did not reduce the expression of JA-02 in the mutant. This different response may explain our observations that the late flowering phenotype was more significant for the plants grown in the area with low light intensity (about half of the normal one) than those grown in the area with higher light intensity. Although JA-02 expression was detected in all materials tested, Blast analysis showed no annotation for JA-02 gene. So, how does JA-02 involved in the regulation of late flowering and what role it may plays during development requires further investigations.

本研究藉由中研院余淑美博士等團隊所建立之水稻T-DNA插入突變體庫來探討與穀粒發育之相關基因，由其中約6,000株突變株選取23株穀粒發育或稔實異常之突變株，藉由質體搶救 ( plasmid rescue ) 及thermal asymmetric interlaced PCR ( TAIL PCR ) 方法，共解讀出11株突變株中被T-DNA插入的染色體組位置鄰近的核酸序列 ( flanking sequences )。以南方墨點法 ( Southern blot ) 分析，發現所選取的5個突變株其T-DNA均為單一個插入點的插入模式，而突變株之Tos17拷貝數與TNG67的拷貝數皆同為三個。進一步藉由PCR技術確認T-DNA插入點，M0034845、M0019179及M0023235等三個突變株T-DNA插入點可以確認。選取M0034845突變株為主要探討材料，經RiceGAAS比對發現M0034845突變株之T-DNA插入於水稻第十一條染色體之BAC OSJNBa0057E15，插入點附近有JA-01及JA-02基因。以RT-PCR分析， M0034845突變株七天幼苗及九十天葉片之JA-01基因表現量與TNG67沒有差異，而JA-02基因表現量約為TNG67之3倍。利用RT-PCR分析發現JA-02基因在TNG67生長各時期中皆有表現，且根部及穗部也會表現，因此可能屬於一個持續表現之基因。以正常光照及無光照處理TNG67及M0034845突變株，發現突變株經由無光照處理後，其根部比TNG67約長10公分，進一步分析其JA-02基因表現情形，TNG67經無光照處理後JA-02基因在葉片及根部之表現量均下降，而M0034845突變株表現量在有光照及無光照環境下，JA-02基因表現並無太大差異。另外，M0034845突變株在光照較弱環境下有晚開花現象，但在正常光照環境下則無晚開花現象。將JA-02基因經由資料庫比對，無法與其它物種比對得到更多資訊，針對此基因在水稻中所扮演的角色，需要更多實驗證明。