Transcriptomics Analysis Of Cassia Obtusifolia Seed, Cloning And Function Study Of Trypsin Inhibitor Gene

Cassia obtusifolia is a Leguminosae annual plant which contains many active metabolites, such as anthraquinone, flavone, phenylpropionic, carotenoid, lipoid, terpenoid etc. C. obtusifolia possesses biological activity including removing heat to brighten vision, dropping blood pressure, dropping blood fat, hepatoprotection, bacteriostasis, etc. It also contains bioactive macromolecules, such as trypsin inhibitors, which posses the ability of anti-insects, anti-tumor and antiviral activity. So far, the chemosynthesis of many components is difficult. Therefore, the study of genes related to active metabolites is important and significant for biosynthesis of active metabolites. Considering the lacking of genome data of C. obtusifolia, the transcriptome sequencing is a reasonable choice to mine those genes. The main work of this thesis is using transcriptome sequencing to mine the genes related to biosynthesis of active metabolites and constructed the metabolic pathway. The related genes was then cloned and subjected to expression analysis. The results of this thesis established a massy groundwork for anaphase study of active metabolites biosynthesis and transgenes plant. The main results was shown as below.1) The seed of C. obtusifolia was firstly subjected to transcriptome sequencing.40102 Unigenes was obtained and 85% of them were homology to the genes in public dabase. The Unigenes were subjected to functional classification and annotation by GO, COG, and KEGG. Many genes related to bioactive metabolites were identified based on the annotations and the relevant metabolic pathway was constructed. These results will be useful for the study of biosynthesis pathway and regulation mechanism of bioactive metabolites and significant for improving the medicine value of C. obtusifolia.2) The suitable reference genes of C. obtusifolia in different tissues and under different stresses were firstly selected and evaluated. Based on the screen and evaluation of 15 candidate reference genes, the EF1a2, CYP1, ACT2 were considered as the most stable genes and EF1a2/CYP1 was considered as the the most stable gene combination in all tissues and under different stresses. The results provided a reliable uniform standard for the gene expression analysis of C. obtusifolia.3) A new trypsin inhibitor gene CoTI1 was cloned and subjected to structure modeling, pronucleus expression and function identification. CoTI1 belonged to Kunitz trypsin inhibitor, showed the prominent inhibition to bovine trypsin and Helicoverpa armigera, Beet armyworm, Spodoptera litura. CoTIl consisted of 11 β-sheets and 12 loops, a disulfide bond which might be related to the stability of structure. Molecular docking showed that Arg86 of CoTIl inserted into the trypsin active pocket and bind with key residue Asp 189 of trypsin to inhibit the trypsin activity specifically. The residues Leu84 and Thr88 also might be important for binding trypsin. In order to confirm the result, mutants of CoTI1 were constructed and the results showed that the mutants lose most of the inhibition, which indicated the important role of three residues. CoTI1 was up-regulated under salt/drought stress and up-regulated then down-regulated under ABA treat, which indicated that CoTI1 might play important role under abiotic stress. The results were useful for the molecular mechanism and anti-insects study of CoTIl.4) A new shikimate kinase gene CoSK was cloned and subjected to sequence analysis, structure modeling and expression pattern analysis. CoSK was highly homology to shikimate kinase and contained the shikimate kinase active sites. It might be located to deferent plastids. CoSK adopted to a-β-a fold and contained 5β-sheets surrounded by 12 a-helices. According to molecular modeling, the residues Lysll8/Arg223 binding to ATP and the residue Asp 137 binding to shikimate might be the key residues for catalysis. CoSK couldn’t form dimmer and might be unstable under heat shock. CoSK showed high expression in root/leaf/callus but low expression in stem/pod/seed. The low expression in seed might be related to the tissue transport of bioactive metabolites or intermediate. The high expression in root suggested it might play important role in root physiological processes.5) A new methylcrotonyl-CoA carboxylase gene MCCase from C. obtusifolia was cloned and subjected to sequence analysis and expression pattern analysis. MCCase might be located to mitochondria/cytoplasm. There was a conserved domain in MCCase which showed similarity to acetyl CoA carboxylase and there were 4 highly conserved motifs, which might play role in catalysis. MCCase showed different expressions in different tissues. The low expression in seed might be related to the low content of Leu and the role of MCCase in catabolism of Leu. The high expression in root/hypocotyl might be related to the inhibition of light/sugar to the expression of MCCase. The results will be useful to understand the amino acid metabolism and prove the nutritive value of C. obtusifolia.