Subcellular Localization Of Pf40 Gene Product And Function Analysis

The central philosophy in the study of the regulation of plant shoot architecture is the concept of apical dominance, whereby the growing apical meristem supresses the growth of axillary meristems. The molecular mechanism of apical dominance is studied further using transgenic plants and mutants. Pf40 is obtained from millet seeds cDNA library. It has high similarity with mental transporter family. by blasting in GenBank. Software analysis predicted that PF40 has 8 transmembrane domains.The similarity of the structure and the transmembrane distribution between PF40 and HKE4(a member of ZIP zinc family) suggested that PF40 may be included in ZIP family.PSORT software analysis predicted that PF40 may localize in membrance system. But which membrane system it localized in is unclear. A full length of PF40 coding region fused at the 5' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco by Agrobacterium—mediated transformation. PCR and Southern blot revealed that fusion gene have integrated in tobacco genom. The subcellular localization was analyzed by fluorescence microscope and confocal. The fluorescence of PF40-GFP fusion protein was detected mainly in the ER, demonstrating that PF40 was mainly localized in the ER. Golden labell confirms the location.PF40 has no conventional location signal. To determine which transmembrane domain could locate PF40 in the ER, 4 truncated PF40 fragments were amplified by PCR and fused with gfp.The truncated fusion genes were introduced and stably expressed in tobacco. The fluorescence detection revealed that F56-GFP and F36-GFP appear in the cytoplasm and nucleus while F12-GFP (including TM1-3) and F14-GFP (including TM1-5) appear in the ER surrounding the nucleus and the cortical ER. This means loss of C-terminal region has no effects on location while truncation of N-terminal region results in mislocation. N-terminal 93 amino acids can locate the protein in the ER.Phenotype of p/40-gfp transgenic tobaccos in which fluorescence could be detected changed with a branch appearing..The other pf40-gfp transgenic tobaccos in which fluorescence could not be detected have same phenotype with nontransgenic tobaccos. Southern blotting indicates that the pf40-gfp fusion gene integrate into the genome of tobacco transgenic plants from different transformed cases. gfp transgenic tobacco has only 1 stem. The phenotype of only gfp transgenic tobacco is same with non-transformed one. This indicates the branching phenotype was caused by pf40 gene expression.Plant expression vector PBI40S (35S promotersence pf40 gene) and PBI40AN (35S promoter: antisence pf40 gene) were transformed into Arabidopsis mediated by Agrobacterium GV3101.The stable transgenic plants were analyzed by PCR and Southern blot to verify the integration of pf40 gene into Arabidopsis genom.Branches of PBI40S transgenic Arabidopsis appear more and earlier than those of non transgenic ones. The differences of phonotype between PBI40AN transgenic Arabidopsis and non transgenic ones are not significant. PBI40S transgenic Arabidopsis have lower levels of plant hormone IAA than non transgenic ones while PBI40AN transgenic Arabidopsis have normal IAA levels. The roots ofboth transgenic Arabidopsis have normal sensitivity to IAA.Two genes coding key enzymes in IAA biosynthesis pathway were clonged with RT-PCR method. They were asb(coding anthranilate synthase beta chain,) and tsb(coding tryptophan synthase beta chain).AS enzyme activity in PBI-PF40S transgenic Arabidopsis decreased significantly while TS β enzyme activity was not altered. In PF40AN transgenic Arabidopsis, both AS and TS P enzyme activity were not changed. Genes(asb and tsb) expressions were not changed at RNA level detected by semi-quantitative RT-PCR and Norther blot. It was predicted that pf40s gene expression resulted in lower AS P enzyme activity, so the levels of IAA decreased. At last pf40s transgenic Arabidopsis shoot architecture altered.