Study On The Translation Initiation Efficiency Affected By The Nucleotides Before Start Codon In The Sperm Cells Of Arabidopsis Thaliana

Translation initiation is crucial for protein synthesis efficiency.The results of previous studies have shown that in animal cells,translation initiation has the highest efficiency in the presence of an optimized background-Kozak sequence.However,in plant cells,the details of the nucleotide upstream of the initiation codon affecting translation initiation efficiency still require to further be studied.In this study,Arabidopsis thaliana sperm cells were used as experimental materials.By mutating the four hot nucleotides upstream of its start codon AUG,the nucleotides at different positions on the translation initiation efficiency were evaluated.In addition,this study also explored whether translation can initiate at a non-AUG start codon under an optimized or poor context.The previous results indicate that the base A appears most frequently in the upstream of the start codon AUG-1 ~-4.Under the background that the upstream of the start codon AUG is poor,four vectors with a base A at positions-1,-2,-3,or-4 were constructed in this study.Through genetic transformation mediated by Agrobacterium,the transgenic plants corresponding to the respective vectors were quantitatively observed with a fluorescence microscope.The statistical results showed that,compared with the transgenic plants with a poor upstream background,the transgenice plants with a nucleotide A at various positions exhibited the different effects on the improvement of translation initiation efficiency.Among them,the-1 position had the smallest effect on the translation initiation efficiency,but the-4 position had the strongest effect on it.Meanwhile,the nucleotide A at the positon-2 or-3 had a moderate effect on the translation initiation.In the presence of the Kozak sequence,translation in animal cells can start at a non-AUG start codon.However,in plant cells,this subject has not been investigated.In the context of the optimized upstream before the start codon AUG(-1 to-4 positions are A),this study constructed four vectors whose start codon AUG was mutated to AGG,ACG,CUG,and AUC,and the transgenic plants corresponding to the respective vectors were obtained.The observation results from the fluorescence microscope indicated that all the four kinds of transgeneic plants had the sperm cells which expressed the GFP protein.These results suggest that the translation can still start at the AGG,ACG,CUG,and AUC codons in the optimized context.Under a poor nucleotide context before the start codon AUG,this study also constructed four vectors whose the start codon AUG was mutated to AGG,ACG,CUG,and AUC,and obtained the transgenic plants corresponding to the respective vectors.Fluorescence microscope results showed that there was no EXPRESSION of GFP in sperm cells of these four transgenic lines.This indicates that translation cannot start at the non-AUG codons of AGG,ACG,CUG,and AUC in a poor context in the upstream of AUG.This study reveals that the four hot-spot nucleotides upstream of the initiation codon AUG have different effects on the efficiency of translation initiation in A.thaliana sperm cells.Furthermore,this study also shows that translation can start at the non-start codon AUG under a optimized background but can not start at the non-start codon AUG under a poor background.These findings indicate that sperm cells do not have a mechanism different from that of somatic cells in the regulation of translation initiation,thus contributing to our understanding on sperm cell development.