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.

Double-mutant invasive mucinous adenocarcinoma of the lung in a 32-year-old male patient:A case report

BACKGROUND Invasive mucinous adenocarcinoma of the lung,formerly known as mucinous bronchioloalveolar carcinoma,is a rare category of lung tumors and radiologically characterized by dense pneumonic consolidation,ground-glass opacity,crazy paving,and nodules.However,early pleural effusion is uncommon in this malignancy.CASE SUMMARY The case of a 32-year-old male patient who visited our facility with symptoms of cough and gradually aggravated shortness of breath was reported.X-ray examination revealed a massive left hydrothorax.The patient underwent thoracocentesis,and pleural fluid tumor markers,including carcinoembryonic antigen,carbohydrate antigen 19-9,neuron-specific enolase,and cytokeratin 21-1 fragment,were significantly elevated.A similar tendency was observed among the serum tumor markers.After draining the pleural effusion,the patient underwent chest computed tomography,and no obvious mass was found in the lung.Thoracoscopy revealed that the left visceral pleura was covered with nodular,cauliflower-like protrusions of various sizes.These histopathological results suggested cancerous cells,and the immunohistochemical findings were consistent with mucinous adenocarcinoma of pulmonary origin.It tested positive for cytokeratin,cytokeratin 5/6,carcinoembryonic antigen,and thyroid transcription factor-1.CONCLUSION The patient was diagnosed with a rare case of lung mucinous adenocarcinoma.Subsequent genetic testing was positive for epidermal growth factor receptor-21 mutations and echinoderm microtubule-associated protein-like 4-lymphoma anaplastic kinase fusion.This prompted treatment with alfatinib and crizotinib.

ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in Arabidopsis

The phytohormone abscisic acid （ABA） plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been discovered, understanding of the ABA signaling pathway is still far from complete. In this work, we characterized the function of ROP11, a member of the plant-specific ROP small GTPases family, in the ABA signaling process. ROP11 is preferentially expressed in guard cells in all plant organs with stomata. Expression of a constitutively active ROP11 （CA-ROP11） suppresses ABA-mediated responses, whereas reduced expression of ROP11 or expression of its dominant-negative form （DN-ROP11） causes the opposite phenotypes. The affected ABA-mediated responses by ROP11 include seed germination, seedling growth, stomatal closure, induction of ABA-responsive genes, as well as plant response to drought stress. Furthermore, we showed that ROP11 and its closest-related family member, ROP10, act in parallel in mediating these responses. ABA treatment does not affect ROP11 transcription and protein abundance; however, it causes the accumulation of CA-ROP11 in the nucleus. These results demonstrated that ROP11 is a negative regulator of multiple ABA responses in Arabidopsis.