Gene Cloning And Functional Characterization Of Maize MYC2 As The Regulator In JA Signaling

Maize(Zea mays)is one of the five major food crops in our country.And which also plays an important role in China's agricultural production and economic development.During growth and development,corn sometimes suffering pests and diseases,that will not only lead to a decrease in yield,but also affect quality of grains.MYC2 belongs to a large family of bHLH transcription factors and which is a key regulator of the jasmonic acid signal pathway,which is involved in the regulation of a large number of jasmonate-responsive genes.MYC2 can bind to the JAZ protein when without JA-IIe and be located upstream of the JA signal pathway,which can participate in a variety of biological and abiotic stress response processes in plants in depened on JA signal pathway.At present,MYC2 is widely studied in the model plant Arabidopsis.And which also have been researched in rice,tomato,potato,paclitaxel,tobacco,bananas,apples,but only a few research literature.There is no one MYC gene has been reported in corn.In this study,a bHLH transcription factor with the highest similarity to AtMYC2 was selected by evolutionary analysis,namely ZmMYC2,and the next step is to study it.The expression patterns of ZmMYC2 under multiple biological stress,abiotic stress and hormone treatment were detected by qRT-PCR.At the same time,the interaction between ZmMYC2 and JAZ protein which are jasmonic acid signal inhibition factors was analyzed by Yeast two-hybrid assays.Furthermore,we transfered ZmMYC2 into arabidopsis thaliana,and explored the biological function of ZmMYC2 in the growth,development and stress response.The main results are as follows:1.We found ZmMYC2 by evolutionary analysis between the bHLH family genes in the maize genome and AtMYC2,and ZmMYC2 has the closest evolutionary relationship with AtMYC2.Phylogenetic analysis of ZmMYC2 and MYC2 reported in other plants revealed that ZmMYC2 has the closest evolutionary relationship with OsMYC2.ZmMYC2 shares the conserved amino acid sequences and conserved domains with other characterized MYC2,suggesting that ZmMYC2 might has the conserved function of MYC2 family.2.qRT-PCR technique was used to analyze the expression of ZmMYC2 under different treatments.The results showed that MeJA treatment could induce ZmMYC2 expression rapidly and massively in maize leaves,indicating that ZmMYC2 may be involved in the regulation of JA signal.At the same time,in the roots,the combined with MeJA and EP for treatment,pathogen infectionand and wound can also induce ZmMYC2 expression;however,the expression of ZmMYC2 was significantly inhibited under the treatment of SA and GA.In the roots,ABA,PEG6000,and NaCl treatment induced the expression of ZmMYC2.The results indicated that ZmMYC2 may participate in the response of maize to various biological and abiotic stresses by regulating the JA signal and its interaction with other hormones.3.Yeast two-hybrid experiments demonstrated that ZmMYC2 was able to interact with ZmJAZ14,ZmJAZ17,AtJAZ1 and AtJAZ9.This result indicated that ZmMYC2 is able to play roles in JA signaling through interaction with JAZ.4.ZmMYC2 was overexpressed in Col-0 and myc234.The obtained transgenic Arabidopsis lines were treated with MeJA for root growth inhibition assays.We found that the Arabidopsis root was significantly shorter then Col-0 when overexpressing ZmMYC2 in wild type.While myc234 was not sensitive to JA,root growth was not affected by MeJA treatment,but complementary expression of ZmMYC2 restored its sensitivity to JA,and root growth was significantly inhibited.These results indicate that ZmMYC2 can regulate growth of roots by depending on the JA signal path.5.Further observations of anthocyanin accumulation in transgenic Arabidopsis lines,we found that MeJA did not induce the accumulation of anthocyanin in myc234,whereas the complementary expression of ZmMYC2 restored the accumulation of anthocyanin.This result indicates ZmMYC2 can regulate the accumulation of anthocyanin by depending on the JA signal path.6.We inoculate mycelium of Botrytis Cinerea to isolated leaves of 6 genotype Arabidopsis lines.Leaf lesions were significantly smaller than wild type when ZmMYC2 were overexpred in Col-0,indicating that ZmMYC2 enhanced Arabidopsis resistance to Botrytis Cinerea.However,no significant difference in lesion area was observed between ZmMYC2-OE/myc234 and myc234,which may be due to the fact that ZmMYC2 was not sufficient to compensate for the loss of resistance to the mold caused by missing myc2/3/4 gene.Conclusions: In this study,we cloned the ZmMYC2 gene from maize and analyzed its function.It was found that ZmMYC2 could be induced by JA and responded to a variety of biological and abiotic stress and hormone treatment.In addition,ZmMYC2 interacts with ZmJAZ14 and ZmJAZ17 whom are inhibitors in JA signal.Through overexpression of ZmMYC2 in Arabidopsis thaliana,ZmMYC2 was found to be involved in JA-mediated root growth inhibition,anthocyanin accumulation,and pathogen resistance in Arabidopsis.This study laid a foundation for the further analysis of the molecular mechanism of ZmMYC2,who involved in the regulation of JA-mediated growth and defense responses in maize.