Construction of Arabidopsis thaliana ugt84a2/ugt84a4 Double Mutants Mediated by CRISPR/Cas9

[Objectives]The CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)gene editing technology is the third generation of"genome fixed-point editing technology"following the"zinc finger endonuclease(ZFN)"and"transcription activator effector nuclease(TALEN)".Glucotransferase genes UGT84A2 and UGT84A4,can simultaneously convert hydroxycinnamate into 1-O-β-glucose esters as isozymes.The CRISPR/Cas9 technology was used to construct double mutants of Arabidopsis thaliana ugt84a2/ugt84a4.[Methods]A CRISPR/Cas9 double mutant expression vector was constructed using UGT84A2 and UGT84A4 as the target genes.The Agrobacterium-mediated dip dyeing method was used to transform wild-type A.thaliana,and the CRISPR/Cas9system was used to target and knock out A.thaliana UGT84A2 and UGT84A4 genes.[Results]The descendants of A.thaliana with the UGT84A2/UGT84A4 gene were sequenced and analyzed.Thirteen positively transformed plants obtained were analyzed according to the sequencing results,and the ugt84a2/ugt84a4 double mutants were screened.[Conclusions]This study provides a reference for the functional study of UGT84A2 and UGT84A4 isoenzyme genes in other species,as well as strong theoretical and method support for accelerating the development and utilization of UGT84A2/UGT84A4 functional gene resources.

Sex-specific responses of tree-ring growth to climate in the dioecious tree Populus cathayana

Aims Radial growth in response to climate has been reported in many trees,but the sex-specific responses of tree-ring growth associated with altitude in dioecious trees are still poorly known.This study aims to examine whether(i)there are sex-related responses of tree-ring growth to climate in dioecious trees;(ii)these responses could be changed with altitude elevation.Methods The tree-ring width and basal area increment(BAI)were measured over the past 30 years(1982-2011),and the sexual differences in relationship between BAI and time span and correlations between ring width and climatic factors were investigated in Populus cathayana trees at two altitude sites(1,450 m and 1,750 m a.s.l.)in Xiaowutai Mountain,Hebei,north China.Important Findings The BAI was increased over the past 30 years.Trees at high-altitude sites had significantly lower mean ring width and mean BAI than those trees at low-altitude sites(P<0.001).In addition,sexual dif-ferences in tree-ring growth and its response to climate were more pronounced by altitude elevation.Male trees had a significantly larger mean ring width and mean BAI than did females at high-alti-tude sites,whereas no significant sexual differences in these traits were detected at low-altitude sites.Female trees were sensitive to previous October-November temperatures at high altitude but to current February and April precipitation at low altitude(P<0.05),whereas male trees were sensitive to current June temperature at high altitude but to January precipitation at low altitude(P<0.05).Our results indicated that the responses of tree-ring growth to cli-mate are sex dependent and can be changed with altitude elevation.