Genetic Transformation of Chinese Cabbage (Brassica rapa pekinensis) with Floral-dip Method

[ Objective] This study is aimed to transform gus gene into Chinese cabbage （Brassica rapa pekinensis） by floral-dip method. [ Method] The Chinese cabbage was transformed by floral-dip method for the first time. Then the cabbage seeds were harvested and screened by hygromycin, and the plants with resistance were confirmed by histochemical GUS assay and PCR detection. [ Result] The target gene was successfully integrated into the Chinese cabbage genome, and the transformation rate was 0.1%. [ Conclusion ] This study optimized the genetic transformation system for Chinese cabbage, and laid the foundation for improving the means of genetic transformation of Chinese cabbage.

Cadmium induced a non-coding RNA microRNA535 mediates Cd accumulation in rice

Identifying key regulators related to cadmium(Cd)tolerance and accumulation is the main factor for genetic engineering to improve plants for bioremediation and ensure crop food safety.MicroRNAs(miRNAs),as fine-tuning regulators of genes,participate in various abiotic stress processes.MiR535 is an ancient conserved non-coding small RNA in land plants,positively responding to Cd stress.We investigated the effects of knocking out(mir535)and overexpressing miR535(mir535 and OE535)under Cd stress in rice plants in this study.The mir535 plants showed better Cd tolerance than wild type(WT),whereas the OE535 showed the opposite effect.Cd accumulated approximately 71.9%and 127%in the roots of mir535 and OE535 plants,respectively,compared to WT,after exposure to 2μmol/L Cd.In brown rice,the total Cd accumulation of OE535 and mir535 was about 78%greater and 35%lower than WT.When growing in 2 mg/kg Cd of soil,the Cd concentration was significantly lower in mir535 and higher in OE535 than in the WT;afterward,we further revealed the most possible target gene SQUAMOSA promoter binding-like transcription factor 7(SPL7)and it negatively regulates Nramp5 expression,which in turn regulates Cd metabolism.Therefore,the CRISPR/Cas9 technology may be a valuable strategy for creating new rice varieties to ensure food safety.

Comparative Proteomic Analysis of Arabidopsis Mature Pollen and Germinated Pollen

Proteomic analysis was applied to generating the map of Arabidopsis mature pollen proteins and analyzing the differentially expressed proteins that are potentially involved in the regulation of Arabidopsis pollen germination. By applying 2-D electrophoresis and silver staining, we resolved 499 and 494 protein spots from protein samples extracted from pollen grains and pollen tubes, respectively. Using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, we identified 189 distinct proteins from 213 protein spots expressed in mature pollen or pollen tubes, and 75 new identified proteins that had not been reported before in research into the Arabidopsis pollen proteome. Comparative analysis revealed that 40 protein spots exhibit reproducible significant changes between mature pollen and pollen tubes. And 21 proteins from 17 downregulated and six upregulated protein spots were identified. Functional category analysis indicated that these differentially expressed proteins mainly involved in signaling, cellular structure, transport, defense/stress responses, transcription, metabolism, and energy production. The patterns of changes at protein level suggested the important roles for energy metabolism-related proteins in pollen tube growth, accompanied by the activation of the stress response pathway and modifications to the cell wall.

A zinc finger protein, interacted with cyclophilin,affects root development via IAA pathway in rice

The plant hormone auxin plays a crucial role in lateral root development. To better understand the rnolecular mechanisms underlying lateral root formation, an auxin-responsive gene OsCYP2 （Oso2g0121300） was characterized from rice. Compared to the wild type, OsCYP2-RNAi （RNA interference） lines exhibited distinctive defects in lateral root development. Yeast two-hybrid and glutathione S-transferase puIl-down results confirmed that OsCYP2 interacted with a C2HC-type zinc finger protein （OsZFP, O501g0252900） which is located in the rice nucleus. T2OsZFP-RNAi lines had significantly fewer lateral roots than did wild-type plants, which suggests a role for OsCYP2 and OsZFP in regulating lateral root development.Quantitative real-time polymerase chain reaction showed that the expression of certain Aux/IAA （auxin/indole-3- acetic acid） genes was altered in OsCYP2- and OsZFP-RNAi lines in response to IAA. These findings imply that OsCYP2 and OsZFP participate in IAA signal pathways controlling lateral root development. More importantly, OslAA11 showed functional redundancy not only in OsCYP2-RNAi lines but also in OsZFP-RNAi lines, which provides important clues for the elucidation of mechanisms controlling lateral root development in response to auxin.

Quantitative detection of Cymbidium mosaic virus by real time PCR

The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction(real-time RT-PCR)was applied to quantitative detect a 764 bp nucleotide sequence containing total coat protein(cp)gene of Cymbidium mosaic virus(CyMV).The plasmid containing the target sequence was constructed to prepare the standard curve and detect the sensitivity.The standard curve was drawn based on the linear relationship between the logarithm(base 10)of the quantity of target sequence and cycle threshold[C(T)].While the concentration of plasmid DNA falling within the range of 2.6×10^(7)to 2.6×10^(2)copies per tube established a regression equation,y=-0.3583x+10.32,and related coefficient:r^(2)=0.995.The real-time RT-PCR assay for CyMV had a minimum detectable quantity of two copies per tube.The naturally infected samples of Phalaenopsis sp.and the artificially inoculated samples of Arachnis sp.with trace CyMV were quantitatively detected using this method.CyMVin the positive samples of Phalaenopsis sp.and Arachnis sp.was confirmed by DNA sequencing and cp gene homeology blast.The results showed that CyMV extracted from the leaves of orchid in Hangzhou,Zhejiang Province,China,could be derived from Kunming city(KM),Yunnan Province,China.This method characterized by high sensitivity,specificity,and precision is suitable for early diagnosis and quantitative detection of CyMV.

Advances in plant proteomics—key techniques of proteome

Following the completion of genome sequencing of model plants,such as rice(Oryza sativa L.)and Arabidopsis thaliana,the era of functional plant genomics has arrived which provides a solid basis for the develop-ment of plant proteomics.We review the background and concepts of proteomics,as well as the key techniques which include:(1)separation techniques such as 2-DE(two-dimensional electrophoresis),RP-HPLC(reverse phase high performance liquid chromatography)and SELDI(surface enhanced laser desorption/ionization)protein chip;(2)mass spectrometry such as MALDI-TOF-MS(matrix assisted laser desorption/ionization-time of flight-mass spectrometry)and ESI-MS/MS(electrospray ionization mass spectrometry/mass spectrometry);(3)Peptide sequence tags;(4)databases related to proteomics;(5)quantitative proteome;(6)TAP(tandem affinity purification)and(7)yeast two-hybrid system.In addition,the challenges and prospects of pro-teomics are also discussed.