Rapid Function Validation Of GmNAC08,GmNAC06 And GmNAC15 By Soybean Hairy Root

Obiect: Soybean [Glycine max(L.)merrill.] is an important oilseed plant and salt stress is one of the most important factors limiting the yield.Understanding the molecular adaptation mechanisms of salt stress is very important for enhancing salt tolerance of soybean.Plant stress responses are modulated by multiple regulatory networks,in which transcription factors(TFs)play crucial roles.NAC [No apical meristem(NAM),Arabidopsis transcription activation factor(ATAF),Cup-shaped cotyledon(CUC)] have been considered as plant-specific TFs,it has highly conserved DNA-binding domain in N-terminal,and highly variable C-terminal domain which contains the transcriptional activation domain.NAC TFs are not only involved in regulating plant development,but they are also response to biotic and abiotic stresses.Therefore,learning the function of NAC in response to salt stress would be useful for enhancing salt tolerance of soybean.Methods: In this research,we did not use the traditional genetic transformation method to study gene function.There are many difficulties to traditional genetic transformation,such as transformation efficiency is low,procedure is complex,plant is easy to pollution and need a lot of time.Agrobacterium rhizogenes(A.rhizogenes)-mediated transformation system of hairy root could solve these problem effectively,in addition to,it used to study the root specific genes.GmNAC08(Glyma08g18470.1),GmNAC06(Glyma06g21020.1)and GmNAC15(Glyma15g40510.1)gene were transformed into pCAMBIA3301 under the control of cauliflower mosaic virus 35 S promoter through In-Fusion cloning technology,meanwhile,we construct mutant expression vector of GmNAC08 and GmNAC06.Then they were introduced into A.rhizogenes strain K599 to generate composite plants.The composite plants consisting of wild-type leaves and transgenic hairy roots were used to analysis function of genes.Results and Conclusion:(1)GmNAC08: The transcription level of GmNAC08 in different soybean tissue was measured by qRT-PCR and RT-PCR.The transcription level was higher in root and cotyledon than other tissue.GmNAC08 in root is response to salt,drought and cold.GmNAC08 in leaf is response to salt,drought,exogenous ABA and cold.After exposure to different salt concentration gradient for four consecutive weeks,the expression level of GmNAC08 in root and leaf of soybean was measured by qRT-PCR and RT-PCR,suggesting that GmNAC08 might be response to salt stress at the fourth week.The composite was irrigated with 250 mM NaCl three times a week for two consecutive weeks,it show overexpression composite(OE)enhance salt tolerance of soybean,CRISPR/Cas9 composite(Mutant)decrease salt tolerance of soybean.That the OE,vector control composite(VC)and Mutant leaves were stained with DAB,NBT,trypan blue staining and chlorophyll content,electrolyte leakage were measured suggest the function of wild leaves were influenced by transgenic hairy roots in composite.We examine the transcription level of GmNAC08 in hairy root and leaf of OE by qRT-PCR and RT-PCR.It suggesting that overexpress GmNAC08 in hairy root couldn’t influnce on the expression level of GmNAC08 in leaf.MDA content,glycine betaine content and overexpression of GmNAC08 in hairy root could up-regulate the expression level of the salt stress tolerance gene including GmWRKY54,GmERF3,GmREF7 and GmMYB177 under normal growing conditions,which played important role in response to salt tolerance.Transient expression of Nicotiana benthamiana indicate it is a target gene of miR164 a.(2)GmNAC06: The transcription level of GmNAC06 in different soybean tissue was measured by qRT-PCR and RT-PCR.The transcription level was higher in root and cotyledon than other tissue.GmNAC06 in root is response to salt and cold.GmNAC06 in leaf is response to salt,drought,exogenous ABA and cold.After exposure to different salt concentration gradient for four consecutive weeks,the expression level of GmNAC06 in root and leaf of soybean was measured by qRT-PCR and RT-PCR,suggesting that GmNAC06 might be response to salt stress at the fourth week.The composite was irrigated with 250 mM NaCl three times a week for two consecutive weeks,it show OE enhance salt tolerance of soybean,Mutant decrease salt tolerance of soybean.The OE,VC and Mutant leaves were stained with DAB and NBT solution to specifically detect the accumulated of H2O2 and O2-.These data suggest that any injury caused by reactive oxygen species(ROS)may be effectively alleviated by leaf of OE,however,the injury caused by ROS may be even worse in Mutant than VC.Trypan blue staining showed that salt stress resulted in more serious cell death in the Mutant leaf.It may be related to accumulation of ROS.The chlorophyll content and electrolyte leakage suggest the function of wild leaves were influenced by transgenic hairy roots in composite.The transcription level of GmNAC06 in hairy root and leaf of one-month-old OE infected with A.rhizogenes K599 was detected by qRT-PCR and RT-PCR.It suggesting that overexpress GmNAC06 in hairy root couldn’t influnce on the expression level of GmNAC06 in leaf.MDA content suggest that ROS may be effectively alleviated by overexpression of GmNAC06 in hairy root.Glycine betaine content suggest that GmNAC06 could accumulate osmotic regulation substances so that enhance salt tolerance of composite.In addition,overexpression of GmNAC06 in hairy root could up-regulate the expression level of the salt stress tolerance gene including GmUBC2 and GmHKT1 under normal growing conditions.In conclusion,our data indicate that overexpression of GmNAC06 in hairy root could enhance salt tolerance of composite.(3)GmNAC15: The transcription level of GmNAC15 in different soybean tissue was measured by qRT-PCR and RT-PCR.The transcription level was higher in root and cotyledon than other tissue.GmNAC15 in root and leaf is response to salt,drought,exogenous ABA and cold.The composite was irrigated with 250 mM NaCl three times a week for two consecutive weeks,it show OE enhance salt tolerance.That the OE and VC leaves were stained with DAB,NBT,trypan blue staining and chlorophyll content were measured suggest the function of wild leaf was influenced by transgenic hairy root in composite.The transcription level of GmNAC15 in hairy root and leaf of OE was examined by qRT-PCR and RT-PCR.It suggesting that overexpression of GmNAC15 in hairy root couldn’t influnce on the expression level of GmNAC15 in leaf.MDA content,glycine betaine content and overexpression of GmNAC15 in hairy roots could up-regulate the expression level of the salt stress tolerance gene including GmERF3 and GmWRKY54 under normal growing conditions indicate that overexpression of GmNAC15 in hairy root could enhance salt tolerance of composite.In conclusion,our data indicate that overexpression of GmNAC08,GmNAC06 and GmNAC15 in hairy root enhance salt tolerance of composite.Transgenic hairy root could influence on the function of leaf.These results suggest that GmNAC08,GmNAC06 and GmNAC15 likely function as a positive regulator of salt tolerance.