| Nicotine whose content could affect the quality of tobacco leaf and cigarette product is an important substance in tobacco. In order to discover the key genes associated with nicotine synthesis and applied on the molecular control about of nicotine content, so that the nicotine content is in an appropriate range, this study selected the different material to construct c DNA librarians, through the determination of nicotine content in different period. By high-throughput Illumina sequencing, we got the original sequence. The original sequence was filtered to obtain a clean sequence, and the clean sequences of different materials were compared to find out the deferentially expressed genes between the different material. Referring to the tomato genome,these deferentially expressed genes was classified by GO functional enrichment, to find the pathway about nicotine synthesis.then according the KEGG Pathway analysis, we could identify key up-and-down regulated genes which associated with nicotine biosynthesis. Finally, by NCBI,searching and grasping the information about these key genes in detail. The main results are as follows:1 ? Hybrid combinations have different performance in 2014. The performance of G70×GDH88?Va116×Basma and NC82×TN90 show over high parent's heterosis. The behavior of G70×Maryland?Va116×Meitandamanyan and K326×meitandamanyan display over low parent's heterosis. The manifestation of G70×Qinggen ? G70×TN90 ? G70×Basma ? G70×GDH02 ?K326×Maryland ? K326×Yongsheng ?K326×GDH02 ?K326×Nanjiang 3 ?K326×GDH88 ?Va116×Qinggen?Va116×Nanjiang 3?Va116×GDH88?NC82×Yongsheng and NC82×GDH88show weak advantages. There are different performance of hybrids in 2015. The behavior of Va116×Basma and Va116×Yongsheng manifest positive heterosis. Va116×TN90?Va116×GDH88and K326×TN90 show weak advantages. Experimental results indicate that screening of the strong advantages of hybrids as Va116× Basma and weak heterosis as Va116×GDH88, these two crosses as the experimental materials constructed c DNA librarians.2?Applying the technology of Illumina Hi SeqTM, in accordance with the transcriptome sequencing process and data analysis method of reference genome sequence. In this experiment, to four materials, the number of those clean sequence pieces are accounted for more than 95% of the number of pieces in original sequence. Error rate were 0.02%. Q20 was more than 96%,Q30 was more than 91%. In the context of allowing two bases mismatched, in four clean sequence pieces that sequence of reference genome was more than 80%, and the reference sequence have a unique position more than 78%, many position sequencings and sequences was less than 2.5%, The sequence was more balanced that in the genome Positive-strand and Minus strand. The results show that it can satisfy the following the sequence analysis with reference genome selection with a reasonable standard, there is no pollution in the experiment and the high quality of library.Besides, illumina high throughput sequencing is a effectively method for obtaining tobacco transcription data.3?Based on four experimental materials of transcriptome sequence alignment to the genome,Sequence of the exons have a maximum number that more than 85%, and 2% or so the clean sequence is located in the intron. The rest sequence alignment to gene interval. The results showed that the four transcripts were more complete in the reference genome, and higher accuracy of Library, it's good for the subsequently searching for differentially expressed genes.4 ? Based on FPKM method, between Va116×Basma and Va116 material, there are 4119 differentially expressed genes were found, they are 2224 up regulation genes and 1895 down regulated genes. Va116×Basma compared to their male paren, there are 1109 differentially expressed genes were found, they are 537 up regulation genes and 572 down regulated genes.Va116×Basma compared to Va116×GDH88, here are 2119 differentially expressed genes were found, they are 1338 up regulation genes and 781 down regulated genes. The results showed that the hybrid combinations of different advantages and there are a large number of differentially expressed genes between strong heterosis combinations and their parents.5 ? Through the analysis of the GO enrichment, the results showed that most of the differentially expressed genes were related to biological processes. In biological process, most of the differentially expressed genes are enriched in the metabolic and biochemical processes.The proportion of the metabolic processes associated with nicotine biosynthesis is less, but theenrichment degree is significant. In the GO classification of molecular function, most of the genes are related to the catalytic activity. In term GO with significant difference in gene expression, due to the different classification, the distribution of up regulated and down regulated genes have different expression. In the three biological processes involved in the synthesis of nicotine, the number of up-regulated genes are more than that of down regulated genes. The results showed that there were a large number of differentially expressed genes which were differentially expressed in the biological process, molecular function and cell composition. These differences were likely to be the cause of Heterosis in some traits. |
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