The Construction Of Ethylene Responsive Transcriptional Factor MdERF98 Expression Vector,Apple Callus Transformation And Preliminary Function Characterization

Ethylene was an important plant hormone,and it was of extremely important and special interest in apple growth and development because of its critical role in controlling fruit ripening.Meanwhile,ethylene was also an important element participating in disease resistance and other biotic or abiotic resistance.Ethylene response factor(ERF)is a kind of transcription factors(TF)specially exist in plants,which was involved in regulating plant growth and development,resistance and the responses to ethylene and other stress related plant hormones.The previous research in our lab showed that the transcription of MdERF98 could be specifically induced by the apple ring rot casual fungi Botryosphaeria dothidea(Zhang,2015).However,the function of this gene and its role in disease resistance and the resistance agianst other adverse enviromental conditions had not been characterized.And the function of this gene aroused our strong interest.Therefore,in the research,we first did the bioinformatic analysis in which the 282 ERF members in apple genome was categorized according to their amino acid sequence and the phylogeny analysis were made;Secondly,MdERF98 were cloned into the binary expression vector and transformed the apple callus and the callus with MdERF98 overexpression were generated;And in the end,we investigated the salt resistance of apple callus with MdERF98 overexpressed.1.Bioinformatic and phelogeny analysis of the apple AP2/ERF gene family showed that MdERF98 had high protein similarity and genetic relationship with MDP0000312701,MDP0000286915,MDP0000187369,MDP0000880063,MDP0000155543 and MDP0000133854,and all the function of the subgroup genes was elusive.Furthur,it was shown that MdERF98 had low similarity with Pti4,the classical ERF transcription factor with clear characterized function in disease resistance.2.Using apple callus of cDNA as the template,we amplified MdERF98 from the apple genome which was subsequently cloned into the binary vector pCB302 and then transformed the apple callus ‘Wanglin'.The callus cells with MdERF98 overexpressed were successfully generated.3.MdERF98 overexpression significantly enhanced the salt resistance of apple callus.In the research,six concentrations of NaCl,i.e.0 mM?25 mM?50 mM?100 mM?150 mM and200 mM were set.Studies have shown that callus cells growth declined gradually with NaCl concentration increase.And MdERF98-overexpression ‘WangLin' callus showed stronger resistance to the salt growth inhibition compared with that of the wild type.4.Unlike in Arabidopsis,MdERF98 overexpression did not promote the synthesis of ascorbic acid.The ascorbic acid content in MdERF98-overexpressing apple cells did not show significant difference with that in wild type.Therefore,the enhanced resistance towards salt stress may not due to the up-regulated ascorbic acid biosynthesis and thus the enhanced antioxidant capability.MdERF98 may enhance the salt resistance of apple cells via other pathway.5.The water content of MdERF98-overexpressing apple callus and wild type was compared.Studies have shown that MdERF98-overexpressing apple callus has larger water content than that of wide type.The above results provided new information for understanding the function of MdERF98,which paved the way for further characterization of the function of this gene.