Isolation Of MYB Transcription Factors,GmMYB68 And GmMYB3? And Functional Research In Soybean (Glycine Max L.) Under Salt-Alkali Stress

Soybean(Glycine max L.)is an important source of edible oil and plant protein,and its growth and yield are affected by salinity stress?Soil salinity significantly reduces soybean production worldwide,the adverse effects in plants are directly proportional to the concentration of salt,and the increase in soil salinity could decrease soybean yield by up to 40%.During soybean growth and development,salt stress significantly reduces plant height and leaf size,decreases seed quality and chlorophyll content.Salt tolerance in plants is a complex trait regulated by multiple signal pathways.However,the mechanisms underlying photosynthesis inhibition under salt stress remain poorly understood.Therefore,identifying the regulators that are directly associated with salt tolerance might have a remarkable impact on soybean production.MYB(v-myb avian myeloblastosis viral oncogene homolog)transcription factor(TF)plays a crucial role in plant development,secondary metabolism,and abiotic stress responses.However,the function of MYB TFs about enhancing resistance in soybean is limited.In this paper,we isolated GmMYB68 and GmMYB3a from Jilin 32,a saline-tolerant soybean variety.Through the detection of transcription level and physiological index,the regulation mechanism was further analyzed,which created the conditions for the cultivation of salt tolerant varieties of soybean.The main experimental results as follows:1.Two MYB transcription factors,GmMYB68 and GmMYB3a were cloned from soybean Jilin32,Using the NCBI BLAST tool,full-length sequence of GmMYB3a ORF was obtained:total length of 672 bp,encoding a protein with 223 amino acids.The C1 motif(L1srGIDPxT/SHRxl/L)and a conserved repression-associated C2 motif(pdLNLD/ELxiG/S)(EAR motif)were identified at the C-terminus,which was presumed to play a transcriptional inhibition in the nucleus.The results of bioinformatics analysisshowed that both of them belonged to R2R3 MYB TFs,and then they had a high genetic similarity with the other MYB genes from various species.2.The Yeast one-hybrid vectors of GmMYB68 and GmMYB3a was constructed separately,and the transcription activation verification proved that they did not have transcription activation function.3.The expression patterns of GmMYB68 and GmMYB3a were analyzed by qRT-PCR.The results showed that GmMYB68 and GmMYB3a were expressed in different tissues,GmMYB68 were mainly expressed in flowers and hypocotyl,GmMYB3a had high expression in root and pod,both of them were in response to five abiotic stresses.They also responded to five abiotic stress treatments including low temperature,high salinity,drought,mechanical damage and ABA.4.The GmMYB68 and GmMYB3a were constructed in the plant expression vectors pTF101,a soybean variety "Williams82”,which was not saline-tolerant and had a mature genetic transformation system,was selected as the receptor material,and the genetic transformation was completed by using Agrobacterium-mediatedmethod,and the transformed plants were treated with salt and alkali stress.Compared with the wild type plants,the chlorophyll content,net photosynthetic rate(Pn),stomatal conductance(Gs)and transpiration rate(Tr)of transgenic plants under saline-alkali stress decreased,and the soluble sugar,proline content and carbon dioxide concentration(Ci)showed an upward trend,but the influences on the physiological indexes of adversity is different.In GmMYB68overexpression lines,transgenic plants subjected to Na2CO3 treatment accumulated 90.51-100.93%and 51.36-57.14%higher contents of soluble sugars and Pro,respectively,than transgenic plants that were not alkali stressed.These increases of soluble sugar and Pro contents in transgenic plants were significantly higher(t-test P<0.05)than in wild-type plants under the same stress conditions.We also further analyzed fructose-1,6-bisphosphatase(FBP)and pyrroline-5-carboxylate reductase(P5CR)genesby qRT-PCR,as these are related to soluble sugar and free Pro biosynthesis,respectively.Ten days after stress treatment,the chlorophyll content decreased in all the plants.However,when compared with well-watered plants,the chlorophyll content of wild-type plants decreased only when subjected to salt stress.GmMYB3a overexpression enhanced the sensitivity of soybean plants to both salt and alkali stresses.In addition,GmMYB3a affected only the free Pro content in transgenic soybean.The free Pro content was significantly lower in transgenic soybean plant than in wild-type plants.5.After the plant harvest,five agronomic characters,including plant height,branch number,single plant pod number,single plant number and hundred grain weight were analyzed statistically,and the overexpression of GmMYB68 had different effects on agronomic traits.Under salt stress,the effect was mainly to increase the number of grains per plant,but to increase the grain weight per plant under alkali stress,there was no significant difference between RNAi strain and wild type.The overexpression of GmMYB3a had no significant effect on agronomic characters.6.The transcription levels of stress-related genes in transgenic plants and wild-type plants were tested,the expression levels of these genes were changed under the treatment of salt or alkali.In GmMYB68 over-expression plants,stress-related genes such as GmABI5,DREB2,GmNHXl,PAL were significantly increased.Interestingly,the expression of two broad-spectrum disease resistance genes(GmNPRI-I and GmNPR1-2)was significantly higher in transgenic lines,suggesting that GmMYB68 may also participated the resistance responses to biotic stresses.Although the stress-related genes DREB,PAL,MYC,and AOS accumulated under salt-alkali conditions,their transcription level in GmMYB3a transgenic lines was significantly lower than that in wild-type plants.Under both normal and alkali conditions,GmMYB3a increased the transcript level of ABF4 in transgenic lines when compared with that in the wild-type plants.However,the transcript level of ABF4 in transgenic lines was low under salt stress condition.While GmABI5 expression was up-regulated in plants subjected to NaCl treatment,its expression in transgenic lines subjected to H2O and Na2CO3 treatments decreased significantly when compared with that in the wild-type plants under similar conditions.