The Expression Of Related Genes CkERF2 And CkCOV1 In Leaf Vascular Development During Responding Drought In Caragana Korshinskii

Caragana korshinskii,which is mainly distributed in arid and semi-arid areas across the Loess Plateau,has shown a high degree of drought adaptability at the morphological,physiological and molecular levels.At present,there are many studies on morphology and physiology,and few studies on molecules.Research shows the vascular bundles and veins of Caragana korshinskii were more developed with the increasing drought.Based on this phenomenon,in order to provide a theoretical basis for clarifying the molecular mechanism of vascular tissue in Caragana korshinskii under drought stress,we study the some functions of CkERF2 and CkCOV1 at the molecular level for developing vascular structure of Caragana korshinskii under drought stress.The main experimental results showed that:(1)The total RNA was extracted from Caragana korshinskii leave samples and the first strand cDNA was obtained by reverse transcription.The CkERF2 gene was cloned by RT-PCR method.The sequencing results indicated that the CkERF2 gene sequence was 756 bp,encoding 252 amino acids.Blastn alignment showed that the homology of the gene between the different species was high,and the three-dimensional structure of the AP2 domain was consistent with that in Arabidopsis with NCBI Online Database.(2)The immunogen is obtained by purification of the fusion protein produced by prokaryotic expression.The rabbits were immunized with that immunogen to obtain polyclonal antibodies.Western Blotting test showed that the polyclonal antibody is specific because the obtained polyclonal antibody can specifically react with the prokaryotic expression protein and no signal was detected in areas other than the size of the prokaryotic protein.(3)Caragana korshinskii was subjected to gradient drought treatment,and leaves were used as experimental materials.In this study,qRT-PCR and Western Blotting techniques were used to detect the relative expression of CkERF2 at both transcription level and protein level.The results showed that with the increase of drought stress,the expression of CkERF2 inCaragana korshinskii leaves had an increasing trend.This indicated that CkERF2 in Caragana korshinskii is responding to drought stress at both transcription level and protein level.(4)The recombinant vector pCAMBIA1302-CkERF2 was constructed by using GFP green fluorescent protein in pCAMBIA1302 vector,and the CkERF2 and GFP green fluorescent protein were fused and transformed into the tobacco leaves of 30 d seedling.The main veins of the tobacco leaves were crosscut and observed under a fluorescence microscope.The results showed that the CkERF2 protein was located in the xylem of vascular tissue,suggesting that CkERF2 might play an important role in regulating the differentiation and development of xylem.(5)The pGBKT7-CkERF2 recombinant vector and pGADT7 empty vector were transformed into yeast competent cells and coated on the nutrient deficient solid medium.The growth status of the colony was observed.The results of yeast self-activation experiments showed that yeast transformed with pGBKT7-CkERF2 and pGADT7 vectors could grow well on both the three and four deficient medium,and the color reaction could occur in the four deficient medium containing X-gal.The result suggests that CkERF2 has a transcriptional self-activating activity and provides a theoretical basis for the subsequent searching for CkERF2 interacting proteins.(6)As the method of cloning CkERF2,the CkCOV1 gene was cloned from Caragana korshinskii leaves.The CkCOV1 is a hydrophobic protein and its gene sequence has 946 bp,of which the open reading frame is 777 bp.It encodes 258 amino acids with a molecular weight of about 28.84 KD.Its isoelectric point is 5.46.The cluster tree was constructed using bioinformatics software MEGA 5 to analyze the genetic relationship of the gene.Quantitative analysis of gene expression level of Caragana korshinskii leaves after drought stress was performed by qRT-PCR method.The results showed that the expression of the gene in Caragana korshinskii leaves showed a decreasing trend with increasing drought.This means that the expression of CkCOV1 gene may be related to the development of vascular system in Caragana korshinskii under the dry aggravation.In conclusion,CkERF2 and CkCOV1 gene play an important role in response to drought and strengthen leaf vascular tissue respectively through positive and negative regulation.Especially,CkERF2 gene plays a key role in promoting differentiation and development of xylem.