| Baphicacanthus cusia(Nee)Breek,is a plant of Acanthaceae family.The Indigo Naturalis(Qingdai)is made from its leaf and stem,which quality is best in Fujian province and is known as "Jian Qingdai".And its root is used as a drug named "Nan-Ban-Lan-Gen",which is a represtantive drug of anti-bacterial and inflammatory,heat-clearing and detoxicating,it’s also a famous-region drug in Fujian.In recent years,as the the scale of the pandemic such as swine flu,avian flu and SARS increases,and the decline of high-quality trueborn herbs and shortage of ecological resources,which has becoming more and more imperative to strengthen the protection for its germplasm resources and quality improvement.It has brought a new opportunity for molecular biology with the development of "Omics era",also provided a new technical tool for the research of species genetic information.The popularity of the next generation sequencing(NGS)provides a fast,high-throughput approach for the medicinal plants in which short of genetic information,which is used for getting batch sequences.It will provide a new view for the molecular reasearch on B.cusia,of which the main active substances are indigo and Indirubin,and it will provide a support for protecting germplasm resources of B.cusia by sorting and revealing function genes.The research contents and results are as follows:1.We have finished the transcriptome sequencing and annotation of B.cusia(wild type and MeJA treatment type)by using the Illumina Hiseq sequencing platform and established a transcriptome database which contains 795663624 unigenes.After blasting,5-Enoltypeshikimic acid-3phosphate synthetase and chorismate synthase involved to the pathway of indole alkaloids were obtained,and the reasarches on B.cusia will be performed with the B.cusia transcriptome database subsequently.2.The gene sequences were isolated based on transcriptome sequencing data of B.cusia,and the characteristics of cDNA sequences were predicted through bioinformatic softwares,and it will reveal the role of both genes played in synthesis of indole alkaloids by the function study in vivo and vitro.(1)The full-length cDNA sequences of BcEPSPS and BcCS genes were obtained by RT-PCR,which ORF are 1554 and 1326 bp,also the characteristics of ORF sequences were predicted through a variety of bioinformatic softwares.(2)Tissue-specific expression of BcEPSPS and BcCS was performed and the results showed that both genes expressed differently in root,stem and leaf,which expressed highest in stem,but similarly in root and leaf.The expression of BcEPSPS and BcCS was examined at different time points(0,2,4,6,8,12,24,36 h)after MeJA,SA and ABA treatment by real-time quantitative PCR analysis,which indicated that both genes were also induced up-regulated by MeJA,SA and ABA,and differently in the induced-up time and expression level.The results of subcellular localization showed that both genes located in the chloroplast,BcEPSPS gene also expresses in the cytoplasm.(3)The recombinated protein were purified through prokaryotic expression and BcEPSPS’s enzyme activity was determined in vitro and the results showed BcEPSPS is of catalytic activity in vitro.(4)The gene overexpression vector was built for regeneration plants and hairy root,which were tranfered respectively by agrobacterium impregnation of EHA105 and C58C1.The RT-qPCR results showed the highest expression of BcEPSPS and BcCS were 37.8 and 52.3 times than control in trangentic hairy roots.Meanwhile,the content of target compounds though HPLC method were testfied and both genes accelerated the downstream products,which confirmed that both genes play an important role in the indole pathway.The T0 generation of transgenic arabidopsis was obtained by agrobacterium of EHA105,which finds that its phenotype is different from the control and the further experiments will be performed subsequently. |
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