A novel method for quickly cloning genes with multiple DNA fragments--one step cloning technique using isoschizomer-heterotail restriction endonuclease (IHRE) is described. Up to six DNA segments are ligated by using only one restriction endonuclease in this method. Comparing with routine method,it is simple, fast, economical and generates products with higher purity and achievement. Light chain of human enterokinase, DNA multi-epitope vaccine to HSV2 have been designed and successfully constructed via this method.

Objective To compare and analyze MRI characteristics of medical patellofemoral ligament(MPFL) injury patterns in adolescents and adults after acute lateral patellar dislocation.Methods MR images of 42 adolescents and 45 adults after acute lateral patellar dislocation were retrospectively reviewed.Routine MR scanning was performed in axial,sagittal and coronal planes,including T1 WI,T2 WI and fat saturation sequence.The x2-tests were performed to analyze the MPFL injury patterns between adolescent group and adult group.Results The prevalence rate of MPFL injury was 97.6％ (41/42) in adolescent group after acute lateral patellar dislocation,including 57.1％ (24/42) of complete tear and 40.5％ (17/42) of partial tear.The prevalence rate of MPFL injury was 100.0％ (45/45) in adult group,including 64.4％ (29/45) of complete tear and 35.6％ (16/45) of partial tear.There were no statistical difference between adolescents and adults in prevalence rate of MPFL injury,complete MPFL tear,partial MPFL tear (x2 =1.084,0.486,0.223 ; P ＞ 0.05).The prevalence rate of MPFL injury at femoral attachment,patellar attachment and midsubstance were 31.0％ (13/42),78.6％ (33/42) and 26.2％ (11/42) respectively in adolescent group,whereas they were 64.4％ (29/45),40.0％ (18/45) and 15.6％ (7/45) respectively in adult group.It occurred at multiple sites in 33.3％ (14/42) of adolescents,and in 15.6％ (7/45) of adults.There were significant differences between adolescents and adults in the prevalence rate of MPFL tear at femoral attachments and patellar attachments (x2 =9.759,13.324 ; P ＜ 0.05),but there was no statistical difference at its midsubstance (x2 =1.497,P ＞ 0.05).Although not statistically significant (x2 =3.749,P ＞ 0.05),MPFL tear at multiple locations were seen more in adolescents than in adults (33.3％ vs.15.6％).Conclusion The injury degree and distribution of MPFL are similar in adolescents and adults after acute lateral patellar dislocation,but there is significant difference in the site of MPFL injury distribution between adolescents and adults.MPFL is most easily injured at femoral attachment,secondly at patellar attachment in adults,while MPFL is most easily injured at patellar attachment,secondly at femoral attachment in adolescents.Adolescents are more likely to have multiple-site MPFL injury than adults.

ObjectiveTo study the changing characteristics of secondary metabolic compounds accumulated in Dendrobium nobile stems at different growth years， a simulated wild stone plant， in order to provide a theoretical basis for rational planning of the harvesting period of D. nobile. MethodUltra-high performance liquid chromatography-mass spectrometry（UPLC-MS/MS） was used to detect and analyze the secondary metabolites in the stems of 1-year-old， 2-year-old， and 3-year-old D. nobile. The mass spectrometry data were processed using Analyst 1.6.3 software， and all samples were subjected to principal component analysis（PCA）， cluster heat map analysis， partial least squares-discriminant analysis（PLS-DA）， and differential secondary metabolites were screened based on variable importance in projection（VIP） values>1， fold change（FC）≥2 and FC≤0.5. Then differential secondary metabolites were identified based on relative molecular weight， fragmentation ions and mass spectrometry database， and enriched pathways were identified based on the Kyoto Encyclopedia of Genes and Genomes（KEGG） database. ResultA total of 1 317 secondary metabolites were identified in the stems of D. nobile at three growth stages， with flavonoids， phenolic acids， alkaloids and terpenoids accounting for 76.55% of the total. Compared with the 1-year-old stems of D. nobile， 289 differential secondary metabolites were identified in the 2-year-old stems， of which 255 were up-regulated and 34 were down-regulated， 682 differential secondary metabolites were identified in the 3-year-old stems， of which 502 were up-regulated and 180 were down-regulated. Compared to the 2-year-old stems， the 3-year-old stems had 602 differential secondary metabolites， with 405 up-regulated and 197 down-regulated. As the growth stage of D. nobile increased， the top 10 up-regulated differential metabolites mainly included flavonoids， phenolic acids， phenylpropanoids and terpenoids， such as kaempferol derivatives， asperulosidic acid， apigenin derivatives， chrysoeriol derivatives， isorhamnetin derivatives， taxifolin derivatives， quercetin derivatives. KEGG enrichment analysis showed significant enrichment of secondary metabolites in the flavonoid biosynthesis， flavone， and flavonol biosynthesis， secondary metabolite biosynthesis， and phenylpropanoid biosynthesis pathways with the increase of growth years. ConclusionWith the increase of the growth years， the levels of secondary metabolites such as flavonoids， phenolic acids， phenylpropanoids and terpenoids in the wild-grown D. nobile have been significantly enhanced. In practical production， grading based on different growth years can be carried out to improve the medicinal and economic values of D. nobile.