Molecular Cloning And Characterization Of The Arginase Gene In Atropa Belladonna

Tropane alkaloids（TAs）are a class of small nitrogen-containing organic compounds extracted from plants of the Solanaceae family and include hyoscyamine,anisodamine and scopolamine.Hyoscyamine and scopolamine are in clinical use as anticholinergic drugs.Due to the extremely low content of TAs in wild TA-producing plants,the contradiction between supply and demand of TAs has become increasingly prominent.Therefore,molecular biotechnology methods are currently being developed to break the specific rate-limiting step of the TAs biosynthetic pathway,thereby significantly increasing the content of the targeted products of hyoscyamine and scopolamine.Functional identification of genes for TAs biosynthesis pathways is the foundation for this research.The Atropa belladonna is an important commercial source of TAs in the Chinese Pharmacopoeia.At present,some progress has been made in the analysis of the TA biosynthesis pathway using A.belladonna.However,some steps remain unclear.Current studies indicate that the biosynthesis of TAs begins with the decarboxylation of ornithine and arginine.Most studies on putrescine biosynthesis focus on the evaluation of arginine decarboxylase and ornithine decarboxylase,while research on arginase in TA biosynthesis has rarely been reported,which severely limits our understanding of the biosynthesis mechanism of TAs.This work uses A.belladonna to research the arginase gene.The work is outlined below.According to the A.belladonna transcriptome database,an arginase gene （AbARG）was obtained with a coding region of 1014 bp.The sequence encodes a 338amino acid polypeptide with a molecular weight of 37 kDa and an isoelectric point of5.9.Amino acid sequence alignment showed that AbARG has high similarity to plant arginases,including 96.2%amino acid similarity with tobacco,88.5%with tomato,and88.3%with grape.The amino acid sequence similarity between AbARG and animal and microbial arginine acids is extremely low;the similarity with human arginase is only16.1%,and the similarity with Bacillus subtilis is only 20%.Phylogenetic analysis also showed that AbARG is closely related to tobacco arginase.On the whole,arginases in the plant kingdom are far from being evolutionarily related to arginases from microorganisms and animals.The tissue expression pattern of AbARG was analyzed by real-time PCR and was different from the reported tissue expression pattern of known genes of the TA biosynthetic pathway,which encoded putrescine N-methyltransferase（PMT）and hyoscyamine 6-β-hydroxylase（H6H）and had root specificity,while AbARG was predominantly expressed in the root and stem.Similar expression levels of the different genes were found in leaves.AbARG was expressed in Escherichia coli and the recombinant protein was purified using a nickel column.The recombinant protein was identified using arginine as a substrate.Thin-layer chromatography（TLC）experiments indicated that AbARG could catalyze the conversion of arginine to ornithine,that is,it had arginase activity.Enzyme kinetics showed that the AbARG Kcat was 51.18 s^-1,Km value was 45.9±3.25mM,and Vmax was 1189.52±83.75 nmol s^-1 mg^-1.In order to study the role of AbARG in TA biosynthesis,we constructed an AbARG overexpression vector and an interference vector using Agrobacterium rhizogenes C58C1-mediated transformation of A.belladonna explants,allowing the detection of the genes rolB and rolC in related hairy roots.The presence of the resistance gene NPTII was detected in the transgenic hairy roots,while the presence of NPTII was not detected in the control hair roots.The real-time fluorescence quantitative PCR showed that the expression of AbARG was significantly increased in hairy roots overexpressing AbARG,while the expression of AbARG was significantly decreased in hairy roots expressing an interfering construct,indicating that the ideal transgenic hairy roots had been obtained.Analysis of TAs in hairy roots showed that over-expression of AbARG had no significant effect on the content of hyoscyamine,anisodamine,or scopolamine in the roots of A.belladonna plants.In the hairy roots harboring the construct interfering with AbARG expression,the contents of hyoscyamine was decreased,indicating that the decrease in the expression of AbARG inhibited the biosynthesis of TAs.In summary,AbARG exhibits a high degree of amino acid similarity and an intimate evolutionary relationship with arginases of the plant kingdom,particularly those of tobacco.Unlike the tissue expression pattern for genes of the TA biosynthetic pathway,AbARG does not have tissue expression specificity.Enzymatic kinetic studies showed that AbARG can efficiently convert arginine to ornithine.Over expression of AbARG had no significant effect on the biosynthesis of TAs in A.belladonna hairy roots,but a decrease in AbARG expression inhibited TA biosynthesis in A.belladonna hairy roots.Taken together,these data indicated that AbARG plays an important role in the biosynthesis of TAs,and this study was important theoretical significance for the analysis of the biosynthesis mechanism of TAs.