Functional Analysis Of Chickpea NAC Transcription Factors CarNAC4 And CarNAC6

Drought has seriously affected the output of crops,threatening the sustainable development of traditional agriculture.NAC transcription factors(NAM-no apical meristem,ATAF-Arabidopsis transcription activation factor,and CUC-cup-shaped cotyledon)play an important role in plant growth and response to various stresses,especially in plants response to stress.However,the researches of NAC transcription factors mainly focused on model plants,such as rice and Arabidopsis thaliana,presently.Chickpea is the world's third largest legume crop.Due to growwth in arid and semi-arid regions perennially,chickpea has evolved a complete and efficient mechanism to adapt to drought stress.Therefore,it has great potential in digging out the resistance gene and analysis the molecular mechanism of plant response to stress.We has constructed cDNA library under drought stress with Xinjiang chickpea germplasm which has stress resistance,and cloned 6 genes(encoding NAC transcription factor),named as CarNACI to CarNAC6.The results showed that these 6 genes have typical NAC structures,and respond to stress and hormone treatment in different degree.We selected CarNAC4,CarNAC6,and ZF2(encoding ZF2 protein which interact with CarNAC4 protein)for further research.Main results of this study are as follows:1.Electrophoretic mobility shift assays(EMSA)verified that the purified CarNAC4 protein could specially bind with probes containing NACRS core sequence in vitro.Yeast-one-hybrid proved that CarNAC6 could specially bind with NACRS sequence and Core fragment contains NACRS cis-element in vivo.The results indicated that the DNA binding element which NAC transcription factor binding to were conserved,which lay a foundation for further study of the downstream genes regulated by CarNAC6.2.Transgenic Arabidopsis expressing CarNAC6 gene showed differences in hypocotyl length at seedling stage and flowering time compared with wild-type plants in terms of phenotype,while there was no condition that transgenic plants were dwarfed.Under drought treatment,the survival rates and proline content of transgenic Arabidopsis plants were significantly higher than that of wild-type.Under salt treatment,the inhibition of transgenic Arabidopsis root growth was lower than that of wild-type.while under ABA treatment,the inhibition of transgenic Arabidopsis root growth was higher than that of wild-type.These results proved that overexpression of CarNAC6 gene could enhance the drought tolerance of transgenic Arabidopsis,without changing the growth process of transgenic Arabidopsis,indicating CarNAC6 gene has great potential in plant genetic engineering.3.According to the cis-element of CarNAC4 gene promoter,we constructed plant expression vectors containing different 5' deletion fragment,and dis analysis via GUS transient expression system by Agrobacterium infecting tobacco leaves and GUS stable expression system of transgenic Arabidopsis.We found the core sequence which driving CarNAC4 gene expression existed the upstream 280 bp of the transcription initiation site GUS staining analysis of transgenic Arabidopsis containing full-length of CarNAC4-promoter in different periods and tissues showed that the mainly expression site were seedling leaves,mature leaves,flowers and pods.In addition the results of qRT-PCR indicated that the response patterns of different fragments to drought,salt and ABA treatments were different.4.Yeast-two-hybrid which CarNAC4 was used as a bait showed CarNAC4 could interact with itself and formed homodimer,as well as interact with CarNAC1 and formed heterodimer.Besides the part of C-terminal was as important as the N-terminal.We constructed mutants(changed or deleted the key amino acid residues),and proved the mytants couldn't form dimer anymore.These findings suggested the key amino acid residues played important role in the forming of CarNACs dimers.5.The interaction between CarNAC4 protein and ZF2 protein was confirmed by yeast-two-hybrid and bimolecular fluorescence complementation(BiFC).The subcellular localization of ZF2 protein showed that it was located in the nucleus,which was consistent with the location of the interaction.The expression pattern of ZF2 gene under different stress and exogenous hormone treatment showed ZF2 gene could response to drought,high salt,high and low temperature stress,ABA,IAA,MeJA,H2O2.What's more,the expression pattern of ZF2 gene under drought treatment was similar to that of CarNAC4 gene.Therefore,we hypothesized that CarNAC4 protein and ZF2 protein regulate plant response to drought stress through interaction.6.The phylogenetic tree showed that ZF2 has the highest homology with GmZAT 10-like and SCOF-1 protein.Multiple sequence alignment showed that ZF2 was a typical C2H2 type zinc finger protein,containing a NLS(B-box),leucine rich L-box,two conserved zinc finger domains and DLN-box/EAR-motif related with transcription inhibition or activation activity.Isolated and analyzed the cis-element of ZF2-promoter(2461 bp upstream),we found there were many cis-elements which had been confirmed response to stress and hormone-induced.GUS staining of transgenic Arabidopsis containing the ZF2-promoter showed the mainly expression site were seedling leaves and stem,mature leaves,flowers and pods.Transcriptional activation analysis showed that the full-length ZF2 and N-terminal without DLN had no transcriptional activation activity,while the DLN domain of ZF2 protein had transcriptional activation activity.Under Mannitol treatment,ZF2 transgenic Arabidopsis plants had higher root length and heavier fresh weight compared with wild-type.Under drought stress,the survival rates and proline content of transgenic Arabidopsis were significantly higher than that of wild-type.These results indicated over-expression of ZF2 gene could enhance the drought resistance of Arabidopsis plants,and proved ZF2 and CarNAC4 could participate in the response to drought stress.This study has analyzed the CarNAC4-promoter,homologous and heterologous dimers,protein interaction and binding activity,and function of transgenic Arabidopsis.We have studied the functional properties of 2 chickpea NAC transcription factors,analyzed molecular mechanisms of CarNAC4 and ZF2 in response to drought stress,and laid foundation for crop improvement using genetic engineering methods.