| Sophora Tonkinensiss Gapnep belongs to the culture of bean of Dicotyledoneae, which is the dry roots and stems of Yuenanhuai. It’s major distributed in Guangxi, where is a prat of the south of China. The main part of S. Tonkinensis Gapnep which is utilized for herb is matrine and oxymatrine which is a kind of alkaloids. Besides, it contains polysaccharide, flavonoids, triterpenoids, phenolic compounds, coumarins, sterols, anthraquinones and caffeic acid, etc, which can be used for treating tumor, inflammation, and Antioxidant as well. This study is aiming at studying the transcription of S. Tonkinensis Gapnep genes by utilizing the technique of Solex High-throughput sequencing. We use the plantlet, transplanted for 6M and 12M of S. Tonkinensis Gapnep as materials and establish 3 transcriptomes libraries, then analyze the unigenes we get by aligning the genesequences in the Genebank, for studying the expressing patterns of the key enzymes of S. Tonkinensis Gapnep. This study provides the basic data for the further step of cloning the full-length gene sequences and exploring the gene functions of S. Tonkinensis Gapnep. The study also lays the foundation of the biosynthesis of alkaloids in the S. Tonkinensis Gapnep. Meanwhile, it has the theoretical and practical significance of bettering the condition of lack of resources of S. Tonkinensis Gapnep, which is endanger and provides a novel method to solve similar problems. Main result is as follow:1. In the phase of plantlet, a little amount of martrine and oxymartrine has been accumulated; the content is increasing with growing, and reaches the peak in the sixth month; indicating that is the critical time of alkaloids synthesis; The martrine and total alkaloid content of the late S. Tonkinensis Gapnep resemble to the 6th months’, but decreased after 6 month; analyze the content of matrine, oxymatrine and total alkaloid of StOM,St6M and Stl2M, the result turns out to be that they are extremely different. We can find the gene related to the alkaloid biosynthesis through this three period.2. Modified Trizol method is suitable for the RNA extraction of the root of S. Tonkinensis Gapnep. The result has a clear ridge without degradation; The luminosity rate between 28s rRNA and 18S rRNA is nearly 2:1, and the OD260/280 is 2.09,3.05 and 2.08 respectively; OD260/230 is 2.12,1.95 and 2.2, indicating that the total RNA has a relative good quality and can be used for next experiments.3. Obtaining 60 million raw sequence, which is 55 million when omitted the low quality part; Totally, we gain 107575 unigenes, whose average length is 839nt, and N50 is 1379nt.4. Using all 107575 unigenes for alignment and annotation, it turns out to be that in NR and NT 66844 and 63599 unigenes have been annotated, which has the total number of 373790, which is 68.6%.5. Founding alkaloids, terpenoids, sterols, flavones and plenty of other secondary related genes whose amount is 7971, which has the amount of known genes related to alkaloid is 151, including CYP450s,Lysine decarboxylase,diaminopimelate decarboxylase, diamine oxidase, hyoscyamine 6-dioxygenase、(RS)-norcoclaurine 6-O-methyltransferase, etc.6. Obtaining differential expression patterns of 3 transcriptomes:in StOM/St6M, 6,484 genes has been up regulated,9,784 has been down regulated; in StOM/St12M, 9,133 genes has been up regulated,9,363 has been down regulated; in St6M/St12M, 4,821 genes has been up regulated,2,412 genes has been down regulated;7. Obtaining 13,506 SSRs, which occupies 12.55% of total unigenes.1496 SSRs each 100kb; There are various SSRs. The Nucleotide repeats repeat from 1 to 6; 3 repeat is the most which has total number of 4,629,2 repeat has 4471, the least is 4 repeat, which is 236;8. There are 68301,72525 and 77560 SNPs in the StOM,St6M and St12M respectively; The rate of transversion and conversion are almost the same. The number of SNP site is increasing with the growing process of S. Tonkinensi Gapnep. |
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