| Safflower(Carthamus tinctorius L.)has a long history of cultivation in China,it is one kind of Catananche annual herbs.Safflower can be used as medicine?oil?fodder?dye and so on.It has various pharmacological effects,such as promoting blood circulation for removing blood stasis,improving microcirculation,antibiotic analgesic,oxidation resistance,etc.Safflower seed contains a variety of essential amino acids,the nutritive value of Safflower in recent years has been the attention of scholars,but the content of essential amino acids of safflower seeds in the lower reach of animal feed standards,how to improve the content of essential amino acids of safflower seeds in need on the agenda.The metabolic pathway of aspartic acid is the main pathway for the synthesis of lysine and other essential amino acids in plants,DHDPS(dihydrodipicolinate synthase)is one of the most important enzymes in lysine biosynthesis in this pathway.It is urgent to improve the expression of lysine in plants through the study of DHDPS gene.In our study,through cloning the full-length Ct DHDPS gene,we built the plant expression vector of Ct DHDPS gene.After that,we transferred Ct DHDPS gene into the Arabidopsis thaliana and analyzed the content of the total free amino acid in this transgenic Arabidopsis plants,it laid a theoretical and experimental foundation for the study of the mechanism of Ct DHDPS gene in aspartate metabolism pathway.The Ct DHDPS gene and its mutants were induced by prokaryotic expression,which provided a protein for screening Ct DHDPS with high enzyme activity,it provides a new idea for the cultivation of New Safflower varieties with high essential amino acid content.The main results in our research are as follows:(1)According to the annotation information of safflower transcriptome library,we got the intermediate sequence of Ct DHDPS gene.Then,the full-length c DNA sequence of Ct DHDPS gene was cloned from safflower seeds by using RT-PCR and RACE technology.(2)We analyzed the CtDHDPS gene by using bioinformatics method and studied the expression level of the gene.The results showed that the expression of the gene in the safflower seeds increased at first and then decreased,after that it increased and decreased at last.In the 12 th day after flowering,the amount of gene expression was the lowest.And in the 14 th day after flowering,the amount of gene expression was the highest.(3)We built the p Basta-Ct DHDPS plant expression vector by using molecular cloning and recombination.After that,we transferred Ct DHDPS gene into Arabidopsis thaliana by using Flora Dip.We used PCR method to detect 43 of the strains in resistant plants,after 1% agarose gel electrophoresis testing,and the result showed that the target gene has been transferred into Arabidopsis thaliana.(4)We determined the content of the total free amino acid in T3 generation seed of transgenic Arabidopsis thaliana.The result had shown that the content of the total free amino in the Ct DHDPS gene transgenic Arabidopsis thaliana was generally higher when compared with the wild-type Arabidopsis thaliana and the maximum difference could be reach 5.51 times.This result indicated that the Ct DHDPS gene could effectively regulate the expression of the total free amino acid.(5)We combined the Ct DHDPS gene with pEASY-E1 vector to construct the prokaryotic expression vector of p EASY-E1-Ct DHDPS.In our research,we observed 34.75 KD protein.It means that Ct DHDPS gene can be successfully expressed in E.coli.At the same time,the 379 and the 382 position of the Ct DHDPS gene were site directed mutagenesis,the mutant gene was successfully expressed in E.coli,and the expressed protein size was consistent with the Ct DHDPS protein size.The result of our experiment can be used in the future research of Ct DHDPS gene activity. |
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