The Effect Of A Non-coding RNA Gene Zm401 On Microspore Development In Maize

Male reproductive processes in flowering plants take place in the stamen. The male gametophyte (pollen grain) of flowering plants is the producer and the carrier of gametes. In a single unit each grain contains all the genetic information required to unite with the female gamete at fertilization and to form a new sporophyte. The developmental events leading to anther formation and pollen release are exquisitely timed and choreographed. Transcriptional activation of specific gene sets is required for the reestablishment and maintenance of differentiated cell types and functions during anther development. The begaining and mature of male gametophyte of flowering plants are controlled by a series of genes in the male stamen. Genes regular meiosis of pollen mother cells, microspore forming stage and the forming process of the male gametophyte. Because of crucial function in the plant's reproductive cycle, pollen is the object of biochemical and molecular biological research. Identifying genes that are responsible for pollen development is very important.In our previous study, a cDNA library to poly (A) RNA isolated from mature pollen of zea mays was constructed. Differential screening and Cold-plaque screening obtained one cDNA fragment; designated zm401 (Zea mays), full length of zm401 cDNA was generated by 5' and 3' RACE method. zm401 cDNA was 1149 bp in length. zm401 was specifically or preferentially expressed in mature pollen. The analysis results by biologic software suggest that zm401 was one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as RNA. We performed in vitro translation to further study if zm401 could be translated in vitro. The result that zm401 cDNA could not be translated confirmed the conclusion that zm401 could be a member of a new category of non-coding RNA. But we could not know if zm401 can be translated in vivo, or execute its function by RNA form. The exact functions of zm401 remain to be elucidated, and further analysis of zm401 may provide a way to elucidate the mechanism of anther development.To gain more information about the function of zm401 gene, plant expression vectors with zm401 RNAi construct and sense zm401gene promoted by ZM13 promoter were constructed. Maize embryogenic callus were transformed by microprojectile bombardment with these plant expression vectors. After selecting for hygromycin resistance, 10 plants with zm401 RNAi construct and 6 plants with sense zm401 respectively were obtained by genomic PCR, southern blotting analysis. The aberrant phenotypes were observed in the anther of the transgenic plants with zm401 RNAi construct and sense zm401 gene. The tassels of transgenic plants with zm401 RNAi construct could not open when the tassels of control plants did, and the top tassels of transgenic plants with sense zm401gene were dysplasia, shriveled and withered. Histological differences were observable for transgenic plants. Comparing the non-transformant with transgenic plant, microspores in the non-transforming were rounded normally; the shape of most of the pollen grains of transgenic plants became distorted. The degradation of the tapetum was delayed for transgenic plants. Most of the pollen grains in the transgenic plants were distored and were not stained with the solution of I2-KI, indicating the absence ofaccumulated starch. In contrast, most of the pollen grains in the non-transformant were rounded and were deeply stained by solution of I2-KI. To further study the function of zm401, we transformed it into tobacco. All transgenic tobacco plants expressing zm401 showed various levels of sterility, ranging from abortive flower development to male sterility. Further analyses on anther development of transgenic plants indicated multiple abnormalities of anther development at late stages. These abnormalities include lagged degradation of the tapetum and connective tissue, failed deposition of fibrous bands in endothecium cells, and aborted pollen grain development. All these results strongly suggest that zm401 has...