Study The Function Of OsCEN2 In Rice

The transition from stage of vegetative to reproductive is one of critical process in the life span of rice(Oryza sativa).Starting from the transformation of reproduction,rice spikelets begin to take place,followed heading,flowering,seeding.These development processes are complicated.In Arabidopsis thaliana,the genes that control flowering are called FT/TFL1 gene families.Although some research about flowering regulatory genes have been reported,the regulation mechanism of flowering network is remained to further elucidated in rice.In this study,the function of OsCEN2 is illuminated through methods of molecular biology and genetics.The main results are as followings:1.To study OsCEN2 gene expression characteristics,different tissues and period material were used to analysis the relative expression level using real-time fluorescent quantitative PCR.The results demonstrate that OsCEN2 expression level is relatively high in in two-week leave;While in the spikelet,it is relatively high at the stage of S11(mitotic);it is also highly expressed in the grains after 18 days flowering.2.In order to elucidate the function of OsCEN2,two vectors of overexpression and RNA interference of OsCEN2 were constructed,and transformed into rice calli through agrobacterium-mediated methods,and many lines of transgenic plants were obtained respectively.The expression levels significantly increase more than 1000 in the overexpression lines,while significantly decrease in RNAi lines with detection methods of real-time fluorescent quantitative PCR.3.The main changes among the transgenic and wild type(WT)plants are two aspects,flowering time and seed size.The heading time in OsCEN2 overexpression plants(CEN2)significantly delayed,while advanced in RNAi plants(CEN2i)in the early season in 2017.For more detailed heading date in the later season in 2017,more precise data demonstrated that the heading date in two CEN2 plants delayed 33 and 31 days respectively compared with WT,while the heading date in the CEN2 i lines advanced 7 and 6 days respectively compared with WT.At the same time,the seed size significantly decreased in CEN2 lines compared with WT.While there is no significantly difference between RNAi plants and WT.4.To explore OsCEN2 regulation mechanism on heading date,14 important genes about heading and flowering date were selected from literatures.Real-time fluorescent quantitative PCR results indicted,relative expression level of MADS14,MADS15,and MADS34 changed greatly compared with WT in both CEN2 and CEN2 i lines when the phenotypes were considered.For further detected the interaction relationship between the OsCEN2 and MADS14,MADS15,MADS34 respectively,Yeast Two Hybrid(Y2H)experiments indicated that co-transformation yeast of BD-OsCEN2 and AD-MADS34 can grow on the 4 defect culture medium board,and the bacterial colonies were blue on the medium coated with x-alpha gal.Bimolecular Fluorescence Complementation(BiFC)experiments revealed there is obvious fluorescence when YC-OsCEN2 and YN-MADS34 co-transformed into the rice protoplast.These results of Y2 H and BiFC demonstrated that OsCEN2 can interact with OsMADS34 in regulating heading date.5.In order to further study how OsCEN2 control seed size,firstly,the seed glumes were scanned with electron microscope.The results illustrated that the cells both in lemma and palea become significantly smaller than WT.This is maybe one of the reasons of the small seed size.Secondly,the seed filling rate also measured,and the results displayed the seed filling rate were significantly decreased in CEN2 plants than WT plants.Thirdly,some seed development related genes’ expression levels were detected.Some genes expression level,such as D1,BRD1,BZR1,SRS5,ETR2,are obviously changed.