Subcellular Localization And Expression Pattern Analysis Of Tillering Inhibitor Gene In Rice

Rice is the most historic food crop and feed the majority of world’s population. Over2.5billion people live on rice. Rice production was performed in more than120countries and regions all over the world. China is the largest producer of rice and provided one-third rice production. Rice plays a significant role in China’s food safety. Rice tillering is one of the most important agronomic traits that directly determine the number of the tillers of rice and further affect yields. Therefore, research on rice tillering will greatly benefit theoretical significance and applications. With the fast development of Rice functional genomics research and molecular cloning technique, resent years, several genes involved in tillers formation and endogenous hormones control were cloned and further worked on for those genes’functions, supplying a major breakthrough for tiller study.A previous work was done on identification of an excessive tillering mutant MIB-ext. A recessive nuclear gene ext-MIB located on chromosome6of rice was assessed by map based clone and functional verification, which controls rice tillering. Sequence analysis indicated that ext-MIB was highly homologous to MAX2which is branching inhibitor in Arabidopsis and was named as OsMAX2on this basis. This gene coded a F-box protein containing LRR repeat domain based on protein prediction and maybe recognized target protein during the process of ubiquitin degradation as a subunit of E3-ubiquitin ligase. For further characterization of the gene function, preliminary work was done to investigate expression pattern of OsMAX2using subcellular localization, histological specific expresses analyses and real-time PCR analyses.For subcellular localization of OsMAX2, we construct35S:OsMAX2:YFP fused gene vector, transferred it into rice and obtain positive plant after detection. At the same time we construct35S:OsMAX2:YFP transient expression vector and transferred it into Onion skin cells mediated by PEG, fluorescent signal was detected by fluorescent microscope and fluorescent confocal microscope. No fluorescent signal was detected among To line, further detection will be done in Ti line. The result of subcellular localization of OsMAX2indicated that OsMAX2is localized on the nucleus.For histological analysis of OsMAX2gene, we cloned upstream-2370bp sequence of OsMAX2coding region as the predicted promoter, insert it into pOsMAX2:GUS expression vector and transferred the vector into rice mediated by Agrobacterium. Some positive plants were obtained. GUS staining was performed on different tissues of obtained transgenic lines, The results showed that OsMAX2gene expression was detected in root, stem, nodes, spire, leafbud, leaf sheath, spike, internode and leaf.Tissues-specific distribution of OsMAX2gene was further examined by reverse transcription polymerase chain reaction (RT-PCR) analysis. Consistent with the results obtained from GUS analysis,OsMAX2mRNA were detected in all tissues examined. Expression in leaf, leaf sheath and stem was slightly higher than others.In conclusion, the analysis of expression pattern of OsMAX2gene showed that OsMAX2protein localized only in the nucleus, and expressed in all tissues of rice plant. All results of the study laid a basis for further research on the function of OsMAX2gene in strigolactone-related shoot branching pathway.