| Tea plant[Camellia Sinensis(L.)O.Kuntzes]is one of the important industrial crops and export agricultural products in China.Since a long time,fungal diseases have been one of the principal causes of tea plant growth and yield,which results in the great economic losses,so how to control fungal diseases has been one of the concerned questions by the breeders.Controling the fungal diseases by applying pesticides are disadvantageous to the human health and the environment pollution.It is absolutely one of the promising way to insert the gene of disease-resistant in the tea plant through applying the genetic engineering method.β-1,3-glucanase is one kind of pathogensis-related-protien.It achieves the broad spectrum bacteriostasis effect through hydrolyzed the cell wall of fungus byβ-1,3-glucanase.As for the most of tea plant, however,β-1,3-glucanase enzyme is one kind of induction protein.Therefore,ifβ-1, 3-glueanase of tea can recombinate in tea itself and make it composition expression,it will inhibit the disease.Meanwhile,it certainly does not affect the quality of the tea.This paper studies the biology activeness and function ofβ-1,3-glucanase enzyme through expressing the enzyme in E.coil.Make a base for applying the biological techology to breed the tea cultivars and using the genetic engineering research to inhibit pathogen. The experiment results show that:1.Gather the samples of the same variety of tea in different place.The result of the enzyme's activity shows that the activity ofβ-1,3-glucanase in Anhui agricultural university is low while the sample from Shi Tai is higher.It means that the environment can influence the expression of enzyme.2.Compare the different part of tea plant in Anhui agricultural university,the result shows that,the activity of enzyme in tender leaf is higher.3.According to the sequence announced from the NCBI database,we design the primer.Selecting the sample of high activity,we amplified the Opening Reading Frame(ORF)by PCR.Then we express the enzyme by recombine technology.But as for some sequence in ORF complement to ribosome binding site,the gene is hard to express.It is designed the first time that make the restriction site sequence to take place of the complement sequence while the sequence of amino acid remain invariable.The expression of exogenous protein is significantly increased after transformation.The molecular weight of the protein induced by IPTG was about 75.8KD as expection. 4.The result of activity detection ofβ-1,3-glucanase by 3,5-dinitrosalicylic acid method shows that exogenous protein have the activity to hydrolysis laminarin.5.The result ofbacteriostasis shows that theβ-1,3-glucanase significant inhibits the young Trichoderma mycelium while it has a little effect to inhibit the strong growing ones.The emzyme is also inhibit the Collectotriehum camelliae Massee. |
