Establishment Of GAL4/UAS Ectopic Expression System And Functional Analysis Of Floral Patterning Gene SFL In Rice

Tempo-spacially controlled transgene expression in vivo, one of the majorconcerns for transgenic research, is a very reliable and precise way to identify genefunction. Binary GAL4/UAS transactivation system established in some modelspecies is one of such techniques to control specific tempo-spacial expression oftransgene and unveil the gene functions. This binary system consists of two types oftransgenic lines, pattern lines and target lines. Pattern lines (or driver lines) containenhancer trap vector and can display expression patterns of reporter with the sameexpression pattern of driver protein GAL4-VP16 (a transcription factor). Thus,selection of lines with specific expression pattern of reporter is required fortempo-spaciaUy controlled transgene expression. Target lines contain target vectorwith the target gene controlled by the UAS promoter that can be specificallyrecognized by GAL4-VP16. Target gene is silent since no GAL4-VPI 6 protein existsin the target lines. However, the target gene can be transactivated by GAL4-VP16 inthe hybrid of pattern line and target line and should exhibit the same tissue-specificexpression pattern as the pattern line. The objective of this study is to establish theGAL4/UAS ectopic expression system and, using this system, to characterize thefunction of some transcription factor genes in rice. The main results are as follows.1. Five tissue-specific pattern lines (P1, anther; P2, stigama; P3, anther; P4, glume;P6, leaf and stamen) and a constitutive expression line (P7, expressed in all tissuesexamined) were identified from a pattern line population in the whole-life-cycle ofrice under normal growing conditions. This pattern line population was generatedfrom an enhancer vector (pSMR-J18R)-transformed rice (japonica variety Zhonghua11) callus that showed no expression of GUS reporter at the differentiation stage inAgrobacterium-mediated transformation.2. Basic target vectors pGOFS1 and pGOC17 were constructed. The target genescan be loaded into the basic target vector pGOC17 through high efficientrecomibinational cloning approach.3. A total 23 target genes (20 transcription factors and 3 non-transcription factorgenes) were introduced into the pGOFS1 vector. Target lines for these genes were produced through Agrobacterium-mediated transformation of these vectors in ricevariety Zhonghua 11. Meanwhile, transgenic lines were generated for the vectorpGOFS1 that contains the enhanced GFP (EGFP) as reporter (ER).4. Copy number of T-DNA was checked for moer than 13 T₀ plants of each targetgene and 2-5 phenotypically normal plants with single copy of T-DNA wereidentified for each target gene for making crosses with the pattern lines.5. Hybrid seeds were generated for about 50 crosses between target lines for 14different target genes and 6 pattern lines.6. In the hybrid of ER line and pattern lines, the GFP reporter was transactivatedby GAL4-VP16 with expression pattern similar to the expression pattern of the GUSreporter in the corresponding pattern lines.7. In the F₁ plants of various combinations, various phenotypic changes wereobserved. These changes included early death of shoot, delayed growth, few tillers,narrow leaf, enlarged leaf angle, delayed flowering, male sterility and multi-carpels.Moreover, these phenotypic changes co-occurred with GFP and GUS reporter geneexpression.8. Phenotypic changes, such as drooped leaf, narrow leaf, reduced tillers, in theP7/T11 combination were further confirmed to be resulted from the activation oftarget gene (a MADS transcription factor) by the constitutive pattern line P7.9. A gain-of-function mutant SFL was characterized in detail. The SFL mutant,displaying multi-carpels (Seaflower), was identifierd in the hybrids of P3(anther-specific) pattern line and T10 target line containing a MYB-like target gene.10. Scanning electric microscope (SEM) and tissue anatomy indicated that themulti-carpels were homeoticly transformed from stamen.11. Expression analysis demonstrated that many genes controlling femaledevelopment, such as C class genes OsMADS3, OsMADS58 and DL (Drooping Leadand D class gene OsMADS13 were upregulated in the SFL mutant.12. In situ hybridization analysis showed that DL (a carpel identity gene) wasectopicly expressed in the third whorl of SFL mutant, indicating that activated SFLexpression in stamen induced DL expression. Meanwhile, another C class gene, OsMADS3, was enhanced in the third whorl of SFL mutant with certain expansion ofits expression.13. Ectopic expression of SFL in stamen induced not only multi-carpels but alsotumor-like structures. In situ hybridization results suggested that the tumor-likestructures had characteristics of ovule organ. Although ectopic expression of SFLinduced cell proliferation in flower, ectopic expression of SFL in mature organ such asleaf caused cell death. This result indicated cell activity regulated by SFL iscell-context dependent.14. Data suggested that SFL may be a non-cell-autonomous protein and can movebetween cells, which can partially explain the carpelloid structures formed sometimesin other whorls, such as palea membrane margin and lodicule and cell proliferationappeared in the fourth whorl organ.15. The lemma development was not influenced. Only the palea membrane margincan be transformed into carpel-like structures. Together with the analysis of othermutants, this result may indicate that the membrane-like margin of palea is equivalentto sepal, the first whorl organ of dicot species.16. SFL expression is mainly restricted in the floral meristem revealed by in situhybrizization analysis.17. A total of 85 SFL RNAi and antisense transgenic plants were generated andonly 4 SFL-suppressed plants were identified. Howerer, all these SFL-suppressedplants showed no obvious phenotypic change, suggesting that SFL may befunctionally redundant.Conclustion In this study, GAL4/UAS ectopic expression system had beensuccessfully established in rice. Based on this system, a transcription factor SFL wascharacterized. It was found that carpel-identity gene DL was ectopically activated in floralorgan of SFL mutant, which in turn caused homeotic transformation of stamen. However,ectopic expression of this gene in other mature organs caused cell or plant death,indicating a strict regulatory role of SFL in the development of flower organs in rice.