| Jingu34(Jinyigu85-2) is a new varietiy of millet with a high quality, high yield, high resistance to disease and high tolartance to drought and other abiotics which was breeded by Department of Crop Genetics, Shanxi Academy of Agricultural Sciences in1996and examined as well as approved the National Crop Variety Approval Committee and Shanxi crop variety Approval Committee in2002. This variety has excellent drought resistance, but the current regulation of its molecular mechanism in response to drought stress profound lack of awareness bacause of the less adequate reference transcriptome information which led the less useful genes being discoveried. To solve this problem, this study we use Illuminate Hiseq2000sequencing platform for Jingu34sequencing and assembling by De nove for transcript profiling. The main finding are as follows:(1) Illuminate Hiseq2000platform was used for transcriptome sequencing and obtained4,047,015,420bp raw reads, included44,966,838of Clean reads, which the Q20value was97.92and GC content was55.77%. By use of Trinity(release-20130225),110073contigs with average length of398and61226non-redundant genes(unigenes) with average length of712were obtained.(2)50067of61226unigenes get the annotation information by using of BLAST software, which46615was from Nr database,47429was from Nt database,30845was from Swiss-port database.19181unigenes was from COG database and of which were classified into25entries.29487unigenes were obtained annotations which were assigned to the128metablic pathways.36238unigene get enriched GO annotation features, these GO entries are grouped into three main categories and59sub-categories entries entries.(3)61,226of unigene were analysised for variable-shear detection of single nucleotide polymorphism using SOAP package and5,192of unigenes were found with variable shear phenomena involved in the six kinds of variable shear type. Single nucleotide polymorphisms detection was showed that61,226unigene contains21,775detection sites, including14,075sites occurring Transition variability and7,700loci occurring Transversion variation.(4) Set transcriptome as the reference sequence, the digital expression profiles were constructed, which obtain3463228and3483434raw Tags in control group and treatment group, separately.3,343,583and3,359,588clean Tags were obtained after fitration, more than86%of which can be compared to the Gene Tag and72percent of which can be unique compared to Gene Tag.(5) The digital expression profiling showed666differentially expressed genes,368genes of which appeared upregulated and298of which appeared downregulated. GO functional analysis showed that403genes of which involved in cellular components,350of which involved in molecular functions,338genes of which involved in biological processes. Functional pathway enrichment analysis showed that among449genes differentially expressed with KEGG annotation information which participated in the95reference metabolic pathways.(6) An F-box genes with full-length of513bp, CG content of48.9%was cloned from the ubiquitin-mediated protein degradation pathway metabolic pathway. F-box protein encoded170amino acid residues and its protein molecular mass was18.552kD. Expression pattern analysis showed F-box are able to produce differential expression in the PEG, ABA and without water stress.(7) The pTF101-35s-F-box expression vector was constructed and transferred into mode tobacco crop using Agrobacterium-mediated method. Finally, the seeds from TO were obtained.(8) The use of transgenic T1generation plants for gene function analysis, the results showed that physiologies and enzyme in both the transgenic plants and control were not affected. When subject to five days without watering for drought stress treatment, SOD activity in transgenic plants showed increased, the content of MDA showed reduced and proline content showed increased. By15days of drought stress and water recovery, the transgenic plants showed higher survival ratio(76.5%,77.2%,80.9%) than wild type(63.3%). |
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