| Agrobacterium tumefaciens is a soilbom phytopathogen.It causes crown gall tu-mor in many dicotyledonous and several monocotyledonous plants by generating and delivering a single stranded T-DNA,namely T-strand,from Ti plasmid to plant cell where it integrates into host genome.Expression of oncogenes on T-DNA result in tu-morigensis.In the process of T-DNA transfer,several virulence proteins are translocat-ed from Agrobacterium to plant cell through T4SS and play important roles in the trans-fer and integration of T-strand.In order to essentially understand the impact of Agro-bacterium infection on gene transcription in plant cell,uncover how Arabidopsis genes respond to the T-DNA-encoded oncogenes or to bacterial effector proteins codelivered into the plant cells by Agrobacteria,and study association of development of plant tu-mor with gene transcription,we used a distance-variance way to reanalyze Deeken et al's(2006)and Lee et al's(2009)microarray data and compared differentially tran-scribed(DT)genes functioning in photosynthesis(PS),respiration,nucleotide,RNA,DNA,protein,lipid metabolisms,defense,hormone metabolism and signaling pathway in reference and GV3101 stalk tissues and in young and mature tumors.1.DT genes found in Arabidopsis thaliana inflorescence stalks between 3h and 6 days after wounded and innocunated with Agrobacterium GV3101 are distinct from those found in reference stalks.It was showed 4651 DT genes were found in Arabidopsis thaliana inflorescence stalks between 3h and 6 days after wounded and innocunated with Agrobacterium GV3101,among which 270 genes were up-regulated and 4381 were down-regulated.While 3885 genes were found differentially transcribed during the same interval in ref-erence stalks.There were 1045 DT genes shared in both condition.1.1 Far less DT genes are found in photosynthesis and respiration metabolism in stalks treated with GV3101 than those in reference stalks.In control,there were 33 DT genes distributed into the pathway PS,among which 31 were negatively regulated of which 20 linked to light reaction.And in stalks treated with GV3101,7 DT genes were defined in pathway PS,of which 6 were down-regulated and associatd with light reaction.As far as respiration pathway was concerned,the number of DT genes belonged to glycolysis,OPP,TCA and mitochondrial electron transport metabolisom in control were 17,12,31 and 45,repectively,and all genes were down-regulate,with the excep-tion of 2 in OPP.In stalks treated with GV3101,it was 7,1,17 and 10 DT genes as-sighned to glycolysis,OPP,TCA and mitochondrial electron transport metabolisom,repectively,and all of which were negatively transcribed.1.2 DT genes coding for NB-LRR disease resistance proteins increase six times in stalks innocunating with GV3101 more than those in references.In the functional categary of biotic stress,there were 119 and 50 genes were found differentially transcribed in stalks treated with GV3101 and not treated,respectively,among which 105 and 39 were down-regulated.10 DT genes were common in both conditions.Further subcategorization showed that 67 PR proteins-encoding genes were regulated differentially after innocunating with GV3101,among which 37 genes coding for NB-LRR disease resistance proteins were negatively transcribed.While in control,there were only 18 DT genes encoding PR proteins among which 6 DT genes for NB-LRR disease resistance proteins were down-regulated.1.3 DT genes assigned to hormone metabolism in GV3101-treated stalks differ from those in referencesIn the pathway of hormone metabolism were 19 genes up-regulated and 60 genes down-regulated found in reference stalks,and were 13 genes with positive transcription and 60 with negative transcription defined in stalks treated with GV3101.12 DT genes were shared.2.Statistic variation in gene transcription in young tumours differ from that in mature tumorsFunction analysis showed that 1223 genes were defined as transcribed differential-ly in young crown gall tumors of which about two thirds(824)genes were up-regulated.And 3551 DT genes were found in mature tumors,among which nearly equal halves were positively and negatively transcribed,respectively.2.1 Inhibition of gene transcription in pathway PS in mature tumors and en-hancement in youngA total of 106 DT genes were detected in the PS pathway in mature crown gall,among which nearly all were down-regulated(103).And in yount crown gall,only six genes were found transcribed differentially,in which four were positively regulated.2.2 Transcription of the DT genes related to respiration pathway in young tumors is enhanced and decayed in mature tumorsIn processes TCA and mitochondrial electron transport,which are two main path-ways of respiration metabolisom,5 and 10 genes with positive transcription were found in young crown galls,respectively.Only 1 gene negatively transcribed was detected in the pathway of mitochondrial electron transport.While in mature tumors were 7 and 15 genes positively regulated in pathways TCA and mitochondrial electron transport,while were 3 and 8 genes negatively transcribed.2.3 Variation of gene transcription in nucleotide synthesis pathway in young tu-mours differs from that in mature tumoursIn young tumours,5 DT genes were found related to pyrimidine metabolisom,among which one genes was up-regulated and functioned in the salvage pathway of uracil,and 9 DT genes were detected in purine metabolisom pathway,in which 2 genes with positive transcription were involved in de novo synthesis of IMP and 4 up-regulated genes took part in the de novo synthesis of GTP.There were no gene tran-scribed differentially in both de novo and salvage synthesis pathways of AMP.In mature tumours was one gene with negative transcription involved in synthesis of IMP,and 14 genes differentially transcribed functioned in pyrimidine metabolisom and 21 DT genes found taken part in purine metabolisom pathway.2 up-regulated genes were detected in the de novo synthesis pathway of UMP.In both salvage pathways of uracil and AMP were one up-regulated gene defined,respectively.Other DT genes were associated with transformation of different nucleotide.2.4 RNA transcription regulation became more complex during the development of crown gall tumoursIt was shown that 110 genes were transcribed differentially in young tumors,among which 72 genes were up-regulated and 38 genes were down-regulated.Among these genes,transcription level of 15 genes involved in RNA processing and 4 genes functioned in RNA binding were up-regulated and 4 of 5 DT genes detected in RNA synthesis were positively transcribed.In transcription regulation 86 genes were tran-scribed differentially,of which 49 were up-regulated and 37 down-regulated.2.5 Gene transcription in protein synthesis in mature tumours is inhibited In young tumors were 267 genes differentially transcribed,in which 35 genes weredown-regulated.While in mature tumors there were 565 genes with different transcrip-tion of which nearly half(274)were transcribed negatively and many functioned in chloroplast.As far as the protein synthesis was concerned,there were 16 and 6 DT genes found in young tumors distributed in the process of initiation and elongation of peptide,respectively,but no genes were differentially regulated in the peptide release process.However,in mature tumors,the number of DT genes defined in initiation,elongation and released processes was 15,9 and 5,respectively,among which 10,5 and 4 genes were down-transcribed.2.6 Gene transcription change in lipid synthesis in mature tumours as comparing to young tumorsThere were 29 genes found differentially transcribed in lipid synthesis pathway in young tumours,of which 18 were negatively regulated.However,in mature tumors,transcription level of 65 gene changed in the pathway of lipid synthesis including 42 genes with lower level.2.7 Distinctive transcription change in functional categary stress in mature tu-mours happen when compariing with that in young tumorsIn young tumors were 20 and 30 DT genes fallen into the functional categaries of biotic and abiotic stress,while in mature tumours there were 54 and 52 DT genes de-fined in those both categaries,respectively.In mature tumors,a majority of DT genes related to biotic stress were PR protein-encoding genes,among which transcription of 13 genes decreased and 9 elevated.But only 3 down-regulated genes detected in biotic stress class in young tumors.As abiotic stress functional class was concerned,in young tumors,2 and 15 DT genes were related with drought/salt and heat stress,respectively,but in mature tumors,there were 23 and 16 DT genes belonging to these two stress categaries.No gene was transcribed differentially in light stress in young tumors,and 3 genes with down-transcription detected in mature tumours.There were no DT genes shared in young and mature tumours.2.8 Genes in signaling pathways were less activated in mature tumorsIn young crown galls,59 genes were transcribed differentially including 40 genes with elevated level and 19 fallen in the signaling pathways.However,in mature tumour were 185 DT genes found related to signaling,in which 101 genes were with lifted tran-scription level and 84 with decreased level.Distinction appeared in transcription of G protein-encoding genes in young and mature tumours.In 14 G protein-encoding DT genes were 13 shown negatively transcribed in young tumours.In mature tumours were 30 G protein-encoding genes transcribed differentially containing 14 genes with down-regulation.3.Expression of VirD2 and VirE2 proteinsIn this research,vird2 gene was cloned from the genome DNA of A.tumefaciens strain C58 and vire2 gene was cloned from LBA4404 using PCR method.The DNA segments containing vird2 or vire2 gene were subcloned into pET32(a)and expressed as fusion protein in E.coli BL21.VirD2 or VirE2 fusion protein was purified using Ni-NTA column.Function assay of cleavage of ssT-DNA and plasmid T-DNA by VirD2 fusion was performed. |
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