| Soybean[Glycine Max（L.）Merr.]is native to China and has abundant germplasm resources.Due to its rich protein and oil content,it has become one of the most important crops in China.As the fourth largest nutrient element,sulfur element plays an irreplaceable and important role in soybean growth and development.However,the treatment of atmospheric SO2 pollution and the gradual decrease of the available sulfur content in the soil have resulted in a low sulfur content that can be absorbed and utilized by soybean growth.Incomplete nutrient application is a major cause of soil sulfur deficiency.However,excessive fertilizer application will not only cause soil compaction but also increase planting costs,and cannot solve the problem of soybean sulfur deficiency from the root cause.One of the most important reasons is that the absorption and utilization efficiency of sulfur element in soybean is low.Therefore,it is imperative to urgently study the mechanism of elemental sulfur metabolism in soybeans and cultivate soybean varieties for efficient sulfur utilization.In this experiment,184 recombination inbred line populations（RIL）composed of Kefeng No.1 and Nannong 1138-2 were used as the research subjects for group linkage analysis.An artificial climate room conducted a low-sulfur tolerance hydroponic experiment at the seedling stage of soybeans to investigate the phenotype traits related to low-sulfur tolerance,combined with SNP markers developed by the population for linkage analysis,to obtain important QTL linkage sites for low-sulfur tolerance,and to screen for related lowsulfur tolerance Candidate genes GmRAMP4,GmRPL12,GmNTF2.The three genes were analyzed for biological information,induced expression,tissue expression,and cloned genes for hair root experiment to conduct a preliminary study of gene function.The results obtained from the study provide relevant ideas for the subsequent research on soybean low-sulfur tolerance.The main findings are as follows:1.Measure the chlorophyll value of new leaves,chlorophyll value of old leaves,plant height,root length,dry weight above ground and dry weight below ground of 184 recombinant inbred line soybean seedlings,and link them analysis.The results showed that these six indicator traits all had high genetic variation and basically showed normal or nearnormal distribution,proving that soybean low sulfur tolerance is a quantitative trait controlled by multiple genes.Correlation analysis found that under normal sulfur environment,except for plant height and new leaf chlorophyll,root length and old leaf chlorophyll and plant height showed a negative correlation,the other indicators showed a positive correlation.After a lowsulfur treatment environment,these indicators are positively correlated.The results of analysis of variance showed that,except for the chlorophyll of the old leaves in the second environment and the dry weight of the ground under the first environment,the remaining indicators reached significant differences in the three environments.2.Using the phenotypic traits of sulfur-related indicators in three environments and the average value of three environments of 184 recombination inbred lineage materials,combined with the development of high-density SNP markers in the group for linkage analysis.The results showed that under the plant height trait,environment 1 and environment 2 co-located chromosome 1,Marker 37,and the contribution rates reached 3.2%and 2.00%,respectively.In the underground dry weight traits,environment 1 and environment 2 colocated 3 significant sites,mainly distributed on chromosomes 7,9 and 17.Among the three environment averages,three linkage sites were located together with environment 2 and were located on chromosome 14;one location was located on chromosome 17 with environment 1;Further in-depth mining of the data,in the environment 1,the linkage analysis of the six phenotype data under normal and low sulfur treatment was obtained:in the root length trait,the linkage is located at a significant site on chromosome 8 to contribute The rate is as high as 7.25%.3.Screen the candidate genes obtained by repeated mapping in multiple environments,and finally select the three genes GmRAMP4,GmRPL12,and GmNTF2 for the next functional verification.Interestingly,the amino acid sequences of these three genes all show a certain relationship with the ribosome.At the same time,the materials of Kefeng No.1 and Nannong 1138-2 were used to conduct low-sulfur induced expression analysis to detect whether the candidate genes responded to low-sulfur induction.The results showed that the three genes were differentially expressed in the two parental materials.Tissue expression results showed that all three genes had higher expression levels in roots and leaves,followed by pods and seeds,and the expression levels in stems were basically low.The overexpression and interference vectors were further constructed for Agrobacterium-mediated hairy root experiment to study the preliminary function of the gene.After overexpression and interference with GmRAMP4,the plant phenotype did not change.In the GmRPL12 gene that was overexpressed and interfered,the phenotype was significantly different.That is,after overexpression,the plant after low sulfur induction treatment had a significant increase in the amount of root hair.The hair root content decreased after low sulfur induction treatment.The related phenotype,sulfur content and fluorescence quantitative expression in plants further verified that GmRPL12 gene is a positive regulation gene.In GmNTF2,the overexpression and interference vectors were also constructed and transferred to soybean plants.An interesting phenomenon was found:the cotyledon nodes of the overexpression plants did not emit hairy roots,but the hairy roots normally grew in the interference plants.After the low-sulfur induction of the interfering plant,the hair roots of the interfering plant were significantly larger than the no-load control.The phenotype,sulfur content in vivo and fluorescence quantitative results showed that the GmNTF2 gene was a negative regulatory gene. |
PDF Full Text Request