| Water scarcity and soil salinization are global ecological problems,which have a tremendous negative impact on agricultural production via limiting the growth and development of plants.So far,about 7.6%of the world's soil has salinization and 43%of the soil in the arid or semi-arid state.The area of saline-alkali land in China is 27 million hm2,accounting for about 10%of the total cultivated land area,and is still increasing year by year.Moreover,drought,the most serious climate disaster in our country,is seriously endangering China's food and ecological security.As the population increases,the area of arable land declines day by day and it is particularly important to develop and utilize salinized and arid lands.Application of genetic engineering to cultivate new varieties of salt-tolerant and drought-resistant crops is an important way of using salt and dry land.Soybean(Glycine max(L.)Merill)is an important edible cash crop,which is rich in protein,high-quality vegetable oils,minerals,and vitamins.In 1984,Lauchli et al.utilized the classification of plant salt stress tolerance under high salt conditions and found that soybean was a salt-sensitive sweet soil crop.At the same time,the study found that the soybean yield was easily affected by climate disasters such as drought.Therefore,molecular mechanism study of salt tolerance,drought resistance,identification of the relevant genes is of great significance.Transcription factors(TFs)can directly regulate the expression of stress-resistant genes in plants and participate in a variety of physiological and biochemical processes,thereby enhancing the plant's comprehensive resistance.Currently,about 12%of the genes in the soybean genome have been found to be members of the transcription factor family.NAC transcription factor family and the MYB transcription factor family are two large families of transcription factors in plants.In recent years,studies in the Arabidopsis have found that NAC and MYB transcription factors play important roles in abiotic stress response.There are 226 NAC transcription factors and 206 MYB transcription factors in soybean.However,due to the large and complex genome of soybean,there are very limited researchers on the response of NAC and MYB transcription factors to salt and drought stress.Membrane-bound transcription factors(MTFs)contain a transmembrane domain that can be directly integrated into the intracellular membrane structure(eg,the plasma membrane,the endoplasmic reticulum,etc.).When plants develop to a specific growth stage or stimulated by changes in the external environment,the membrane-bound transcription factors are released from the membrane and transported to the nucleus.Up to date,no studies have been reported on the evolution and function of soybean NAC membrane-bound transcription factors(GmNTLs).In our lab,a NAC transcription factor gene GmSIN1 and an MYB transcription factor gene GmMYB84 were cloned based on expression profile analysis of soybean salt-tolerant variety Sheng Dou 9 under salt and non-salt conditions.The biological characteristics of these two genes were analyzed and the preliminary study on their resistance was carried out.On the other hand,15 GmNTLs were isolated and cloned by searching and identifying the whole genome of soybean in the early stage and their sequence characteristics and expression characteristics were analyzed preliminarily.Based on the above results,in this study,the molecular mechanism of GmSIN1 in response to soybean salt stress was further studied and how GmMYB84 participated in the response to drought stress of soybean was explored.In addition,the evolution and function of the GmNTLs family were identified and the membrane release mechanisms of their family genes were explored.The main results are as follows:1)35Spro:GmSIN1 transgenic soybeans exhibited significantly increased salt tolerance:By comparing soybean yields in saline and non-salt soybeans,35Spro:GmSIN1 transgenic soybean lines were found to achieve higher yields than the control.The important role of GmSINl in soybean salt tolerance was confirmed by overexpressing soybean field data.By comparing the relative elongation of soybean root under salt and non-salt conditions,it was found that the relative elongation of 35Spro:GmSINl was larger than that of wild-type,while the root elongation of 35Spro:GmSIN1 RNAi was smaller.By comparing the RNA-seq expression profiles of wild-type and 35Spro:GmSIN1 transgenic lines under salt and non-salinity,we found that GmSIN1 was involved in the regulation of ABA and ROS biosynthesis-related genes.Meanwhile,EMSA,ChIP and other experiments proved that GmST1 can directly bind to the cis-acting element upstream of GmNCED3-1/-2 and GmRBOHB-1/-2/-3.Promoter-MEME analysis of target genes regulated by GmST1 in RNA-seq was screened,and a new specifical GmSIN1 binding cis-acting element was identified and named as SIN IBM.The results showed that GmSIN1 was one of the most important transcription factors in response to salt stress,and mediated the early response of salt stress transcriptome in ABA-dependent manner.By directly regulating a variety of downstream genes,GmSIN1 balanced hormone levels such as ABA and ROS production and improved the salt tolerance of soybean.2)By searching and identifying the whole genome of soybean,15 NAC membrane-bound transcription factors in soybean were found.The 15 GmNTLs genes were cloned and analyzed for their tissue-specific and abiotic stress response patterns.Using subcellular localization in Arabidopsis thaliana protoplasts,all 15 GmNTLs were identified to locate on the ER.At the same time,the membrane release patterns of GmNTL1,GmNTL2,GmNTL3,GmNTL4 and GmNTL10 were analyzed using protoplasts of Arabidopsis thaliana.The Membrane release of GmNTL1,GmNTL2,GmNTL3,and GmNTL4 were triggered by H2O2.The Membrane release of GmNTL1,GmNTL3,GmNTL4,and GmNTL10 were triggered by cold.The p35S:GmNTL1,p35S:GmNTL11,p35S:GmNTL1-?C,p35S:GmNTL11-?C transgenic lines were constructed.Compared to the wild type,p35S:GmNTL1,p35S:GmNTL11,p35S:GmNTL1-?C,p35S:GmNTL11-?C exhibited significantly decreased sensitivity to NaCl,ABA,mannitol,and H2O2.3)The drought-resistant ability of 35Spro:GmMYB84 transgenic soybean lines was significantly enhanced.In soybean,the GmMYB84 transcription factor bind directly to the cis-acting element upstream of the GmRbohBl/2 up-regulates their expression,resulting in more accumulation of H2O2 in over-expressing plants compared to the wild-type under drought stress.This is the main cause for 35Spro:GmMYB84 plants showing strong drought resistance in early drought stress.With the increase of drought stress time,35Spro:GmMYB84 plants rapidly up-regulated the expression of peroxidase-related genes and activated the H2O2 scavenging mechanism in vivo to avoid oxidative stress injury caused by excessive accumulation of H2O2.Highly efficient ROS generation and scavenging were important for plant drought stress tolerance.The results demonstrated that GmMYB84,an R2R3-MYB transcription factor gene,played an important role in drought stress tolerance via ROS generation and scavenging. |
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