Cloning Of Plant Embryogenesis Gene LEC1 And Genetic Transformation Of Rice

Plant embryogenesis represents the critical transition from the single-celled fertilized egg to the mature and multicellular embryo by undergoing a series of controlled cell divisions and cell differentiation events. It is a process of gene expression and regulation with order and alternation. The genes expressing or regulating during embryogenesis and development stages are being discovered in succession.Arabidopsis embryogenesis gene LEC1, whose protein is homology of HAP3~a kind of eukaryotes transcriptional activator, can lead the transformation from somatic cells to embryonic cells alone, expressing only in embryonic cell and endosperm in the normal situation.In the article, we cloned the embryogenesis gene LECl by PCR using special primers P1, P2 and cDNA of Arabidopsis legument in globular embryoid - heart shape embryoid stage as templet, and also cloned the homeotic gene from rice DNA by using special primers UP, LP. The interested fragments were linked into pMD18-T, then sequenced using M13+/-, and aligned in GenBank. The results of sequence analysis showed that the sequence obtained were the same as LEC1 in GenBank.Then plant expression vectors were constructed. The target genes were linked into the expression vector downstream to Camv35S promoter and upstream to terminator in pWM101. PCR and restriction endonuclease digestion were followed to detect the target。 The recombined plasmids which were extracted then digested by Nhe I in order to be linear were transferred into rice restorer line 0099 and rice sterile line Pei'ai 64S by pollen-tube pathway.We harvested seeds which were named first transgenic generation (T1). The seedlings were planted in field after being treated by Hygromycin. Positive clones of restorer line were obtained by molecular detection - PCR using special primers P35S, P2 and leaf genome DNA as templet, and the transgenic rice will be used in further research. The efficiency of transformation is 9.3%.Transgenic sterile line rice developed seeds without integrity endosperm. The seeds without integrity endosperm were cultured in 1/2 MS culture medium, one germinates and another grows root. DNA of the bud was extracted, then detected by PCR using the primers-P35S and P2, it is positive.We harvested seeds possessing the possibility of germination from sterile rice without pollination. It proved that the LECl gene was integrated into the genome of rice. The results of experiments suggest that gene LECl can accelerate embryogenesis in rice, a kind of monocotyledon crop. These experiments suggest that apomixis of rice will be realized through genic engineering-artificial controling expression of LECl in rice. And then the dream of heterosis fixation will come true.