| Legume-rhizobium symbiosis is a complex process that is regulated in the host plant cell through gene expression network. Many nodulin genes that are upregulated during different stages of nodulation have been identified in leguminous model plant(M. truncatula and Lotus japonicus). However, no nodulin genes in woody legume trees, such as black locust(Robinia pseudoacacia), have yet been reported, and research of the molecular mechanisms of nodule formation has not yet been carried out. Therefore, a comprehensive analysis of gene expression profiles at the transcriptome level during R.pseudoacacia-M.amorphae symbiotic nodulation, and isolation and identification of nodulin genes is the effective means to research the mechanisms of nodule formation in black locust. Using reverse genetics technology, RNA interference, which could induced gene silencing to analysis nodule-related genes function, and these work will lay the foundation for the molecular mechanism of locust nodualtion.To identify the nodulin genes involved in R. pseudoacacia-Mesorhizobium amorphae CCNWGS0123 symbiotic nodulation, a suppressive subtractive hybridization approach was applied to reveal profiling of differentially expressed genes in locust roots and two subtracted c DNA libraries were constructed to clone differentially expressed genes. The expression patterns of these upregulated genes were further analyzed by quantitative RT-PCR at different stages of nodule development. The full-length c DNAs were successfully identified using the RACE technique, and bioinformatics forecasting were used to obtain the genes information. RNA interference was used to preliminary analysis the functions of candidate genes.The main research contents and results were as follows:1. To observe the infection process of M. amorphae CCNWGS0123 clearly, enhanced green fluorescent protein(e GFP) gene was used to mark the bacteria. Plac Z–egfp plasmid p MP2444 was transformed into M. amorphae CCNWGS0123 by electroporation. Then, the marked bacterium was named G186. The infection process was observed by fluorescence microscopy. Based on experiment results, the process of G186 infection into locust roots is through infection threads and the nodulation process of black locust is longer than that of M. truncatula and L. japonicus. The infection of G186 into locust roots was artificially categorized into the following phases: root hair deformation and G186 was wrapped by curled root hairs at early stage(0 dai to 7 dai), formation of integral infection threads and nodule primordia and rhizobia in the infection threads were released into the nodule primordia at medium-term stage(7 dai to 18 dai) and nodule maturity at late stage(from 18 dai).2. To identify the nodulin genes involved in R. pseudoacacia-M. amorphae CCNWGS0123 symbiosis, total RNA was isolated from inoculated and uninoculated control roots at 1, 4, 6, 8, 11, 14, 18, and 22 d. and m RNA samples were isolated from total RNA. Suppressive subtractive hybridization was applied to reveal profiling of differentially expressed genes in locust roots and two subtracted c DNA libraries each containing 600 clones were constructed. Forward subtracted c DNA library included genes which upregulated in inoculated root, and reverse subtracted c DNA library included genes which upregulated in uninoculated root. Dot blotting was used to remove false positives in subtracted c DNA libraries. 371 forward c DNA library clones and 247 reverse clones were blotted onto nylon membranes and four identical blots were made. Clones with more than 5 signal ratios after hybridization to c DNA probes from forward probes were finally selected for sequencing. The data presented here offer the first insights into the molecular foundation underlying R. pseudoacacia–M. amorphae symbiosis.3. According to the results of dot-blot hybridization, 251 SSH clones were selected for sequencing. 212 expressed sequence tag(EST) sequences were obtained when removed those poor quality sequences. The sequences were grouped into singletons and contigs using TIGR Assembler and then 162 unigenes were obtained(114 forward/48 reverse sequences). The size of the selected clones ranged from 200 bp to 900 bp. The nucleotide sequences of ESTs obtained in this work were registered in db EST and assigned Genbank accession numbers from JK974084 to JK974197. Homology search of 162 unigenes were performed in the non-redundant(nr) protein database using the BLASTX program. The sequenced ESTs were then manually classified into 14 functional categories based on the BLASTX descriptions. Among the 14 functional categories, the ESTs with no hits or no functional annotations had a relatively high abundance(23%), which illustrates the current lack of genomic knowledge on locust. In order to screen nodulin genes involved in nodulation, our studies focused on forward library. The 114 unigenes were again classified into 13 functional categories based on the BLASTX descriptions and GO functional classification. BLASTX annotations of the assembled unigenes demonstrated that 80(70%) sequences had significant similarities with the sequences in the NCBI nr database, whereas the rest of the unigenes had no homologues at the amino acid level. Among the annotated genes, those categories related to Transcription(10.5%), Protein metabolism(14.0%), Defense/stress response(10.5%) and Signaling(7.9%) were overrepresented. So, it is suggested that different levels of regulation, including transcriptional, post-transcriptional, translational and post-translational, may occur during the development of the locust nitrogen-fixing symbiosis.Regulatory genes are expected to perform crucial functions during nodulation. With a particular interest in these genes, 28 unigenes were preferentially chosen based on the putative annotations linked to transcription, protein metabolism, defense/stress response and signaling. Through RT-PCR analysis, our results revealed that 21 genes were found to be upregulated in infected roots. Among the 21 upregulated genes, nine encoded putative TFs and RNA-binding proteins, four encoded membrane proteins for signaling, and eight encoded post-translational regulators. The expression patterns of these genes were accurately quantified by q RT–PCR. The full-length c DNAs were obtained using the RACE technique. Multiple alignment of some protein sequences were displayed using Clustal W.4. Five late nodule-specific genes were screened from forward SSH library by q RT-PCR, which are: 2zheng43, 2zheng44, 2zheng45, 2zheng190 and 2zheng287. Gene expression profiles revealed that these genes were not expressed in the early infected roots(0, 1, 4, 7, 10 dai), 30-d-old control roots, leaves and stems(30 dai;except 2zheng43) but were expressed in mature infected roots(after 18 dai) as well as nodules(30 dai). So, these genes maybe involve in nodule development and maintain nodules function. The full-length c DNAs were obtained using the RACE technique, 2zheng43(1455 bp), 2zheng44(608 bp), 2zheng45(656 bp), 2zheng190(580 bp) and 2zheng287(689 bp). The deduced numbers of amino acid sequences of the five genes were 412, 152, 106, 86 and 113, respectively, encoding mannan endo-1,4-beta-mannosidase(2zheng43), leghemoglobin(2zheng44), mini zinc finger(2zheng190), conserved membrane protein(2zheng45) and root nodule-specific m RNA(2zheng287, BLASTN). Signal peptide sequence have been predicted in 2zheng45 and 2zheng43 amino acid sequences using Inter Pro Scan, and 2zheng45 amino acid sequences has a transmembrane domain.5. RNA silencing was applied to investigate the four genes(2zheng43, 2zheng45, 2zheng190 and 2zheng287) funtion in nodule development.At the same time, Agrobacterium rhizogenesis-mediated transformation systems of R. pseudoacacia were established. The nodules transformed with the empty vector were used as the control and the efficiency of silencing were detected by q RT-PCR. The result indicated that 2zheng43 and 2zheng45 were silenced effectively in nodules harvested at 20, 30 and 40 dai. RNA silenced nodules should have some differences with the control nodules in nodulation and internal structure.For the nodulation, the difference in average nodule numbers between the two samples was significant at the harvest time of 10, 20, 30 and 40 dai, that nodule numbers reduced but white nodules increased on RNA silenced roots. Expression levels of leghemoglobin gene in nodules at various harvest times(20, 30 and 40 dai) were estimated by q RT-PCR.The results show that leghemoglobin gene in silenced nodules clearly declined compared with it in control nodules.For the nodule internal structure, microscopic analysis of nodule paraffin-embedded slides showed that mycetocyte in control nodules were normal with regulated arrangement and regular shape at the time of 20 and 40 dai. In contrast, mycetocyte in the silenced nodules were not normal, which the numbers of mycetocyte were decrease with no regulated arrangement.For example, it was found that in 2zheng45 and 2zheng43 gene silenced nodules, the numbers of mycetocyte were very few, and in 2zheng43 gene silenced nodules the number of mycetocyte very few and mycetocyte nucleoids was observed.The results above indicated that silencing of these genes delayed nodule development, suggesting the importance of this gene and its product in the regulation of nodule formation and nitrogen fixation. |
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