Functional Analysis Of ZmEG1 Gene In Maize Spikelet Development And Male Sterility

Maize (Zea mays L.) is one of the most important crops in the world, whose seeds are developed from spikelets. As a well-known model plant for floral development research, the spikelets of maize are composed of paleas, lemmas, lodicules, stamens, and pistils. Apart from the above floral organs, which also presented in rice, maize has its unique glume developmental feature. Thus, the research on the molecular mechanism underlying the maize floral development is undoubtedly important, theoretically and agriculturally.EG1 encodes a lipase and plays a vital role in rice floral development as a floral meristem gene and an organ identity gene for extra glume, while it still remains unclear whether its homologous gene in maize (ZmEG1) is functionally conserved. Our previous work has proved that ectopic expression of ZmEGl caused multiple floral defects in rice, indicating that ZmEG1 might act as a meristem gene and get involved in development of pollen grains. To evaluate the function of ZmEG1 more precisely, ZmEG1 was overexpressed in maize via Agrobacterium tumefaciens-method. By tissue culturing and the following molecular methods, including anti-hygromycin test, GUS staining, Tail-PCR, two transgenic maize plantlets were verified that Ubil:ZmEGl were successfully inserted into chromosome 6 and 5, independently. Furthermore, phenotypic comparison of spikelets was conducted between transgenic and non-transgenic maize, by using stereomicroscope, paraffin section, SEM, TEM, and QPCR. The results showed that ZmEG1 acts as a key meristem gene and impact the maturity of pollen grain. From the phenotype of the Ubi1:ZmEGl transgenic maize, it can also draw a conclusion that the glume of maize is homologous organ to the extra glume of rice. The main results are as follows:1. Bio informatics analysis showed that ZmEG1 is located on chromosome 3 in maize, having highly phylogenetic relationship with OsEGl; ZmEG1 protein has the same lipase conserved sequence GXHXG-motif with OsEGl, AtDGL and AtDADl; the promoter of ZmEG1 gene contains regulatory element related to meristem expression and MeJA-response element, which indicate that ZmEG1 might be a meristem genes associated with JA,and encode a protein that belongs to the family of phospholipase A1.2. The pCAMBIA1300-ZmEG1 was transformed in maize Hi-II callus by Agrobacterium tumefaciens.2 of 9 were confirmed that Ubil:ZmEG1 was successfully inserted into chromosomes 6 and 5 respectively in maize by GUS staining, anti-hygromycin gene detection and Tail-PCR. QPCR analysis demonstrated that the expression of ZmEG1 in transgenic maize was increased more than 3 times.3. Phenotypic comparison was conducted between transgenic maize and NT by using multiple method, including microscopic observation, crossing and pollen grain staining. The results showed that the transgenic maize displayed obvious tassel floral defects in numbers and patterns. The anthers of transgenic maize failed to dehisce and the pollen grains of transgenic maize could not be I2-stained. Extra ubisch body, extra starch in anther locule and undegraded endocecium cell impacted the maturity of pollen grains and resulted in male sterility. Besides, some extra glume-like organs occurred in transgenic tassel spikelets and the glumes in both tassel and ear elonged, implicating that the glume of maize is homologous organ to the extra glume of rice.4. In addition, together with the increased expression of ZmEG1 in transgenic maize, the expression of some floral mersitem genes and class C gene was significantly increased, suggesting that ZmEG1 may positively regulate these genes to involve in the tassel development of maize.