| Soybean is an important grain and commercial crop in China.In the process of soybean planting,weed damage is the main adverse factor affecting the yield and quality of soybean,which can reduce yield by 20%～50%in severe cases.Therefore,weed prevention and control is an important measure to ensure high yield and benefit in soybean.At present,chemical weeding is the main method of weed control in soybean production.Dicamba is an endogenous conduction hormone herbicide with high efficiency,low toxicity,strong herbicidal effect,rapid and moderate efficacy,and has great application potential.Due to the sensitivity of cultivated soybean varieties to dicamba,there are no reports on the development and utilization of dicamba resistant soybean materials in China.In the present study,an optimizing dicamba monooxygenase(dmo)gene vector system provided by Professor Zhong-ping Lin’s laboratory in Beijing university was used for soybean transformation,and a soybean variety W82 was selected as the receptor to conduct genetic transformation of soybean cotyledon section method mediated by agrobacterium tume faciens to create transgenic soybeans with the target dmo gene.The herbicide toelrance and molecular character of the transgenic soybean lines was also investigated to reveal the degradation capacity of dmo gene after codon optimization,and the results may provide useful information and germplasm for transgenic breeding of herbicide tolerance in soybean.The main research results are as follows:1.Creation of three soybean transgenic lines with overexpression of dmo gene and high resistance to dicamba.Agrobacterium-mediated soybean cotyledon node transformation was used to carry out genetic transformation of W82,and 42 T1 generation regenerated plants were identified for glufosinate resistance and PCR amplification of dmo/bar gene.A total of 12 glufosinate-resistant plants containing the target gene were screened out.It was Seven lines with dicamba tolerance were identified after spraying 280 g.a.i.ha’1 dicamba.Furthermore,the plants of T2and T3 generations were sprayed with 280 and 560 g.a.i.ha-1 dicamba,respectively,and the three highly tolerant lines W1-1,W2-2 and W3-1 were obtained.Among them,W2-2 could be tolerant to 10 times(2800 g.a.i.ha-1)of dicamba dose for field production.qRT-PCR results showed that dmo target gene were inserted as single copies in W2-2 and W3-1 lines,while two copies in W1-1 line.The insertion position of exogenous gene dmo in the W2-2 transformation event was determined to be between 11244873 bp and 11244980 bp on chromosome 2 by whole-genome sequencing method.2.Physiological characteristics of the transgenic soybean under dicamba treatment.After dicamba treatment,different responses between W82 and W2-2 plants were found in the contents of chlorophyll,H2O2,MDA and the activities of oxidase SOD,POD and CAT.The values of chlorophyll content in leaves of transgenic W2-2 lines and control W82 showed a decreasing trend,with significant differences of 6.7%and 76%,respectively.The activity of oxidase CAT,POD and SOD in plant leaves showed an increasing trend in the transgenic line W2-2,which was 1.18,1.02 and 1.12 times as much as that before treatment,respectively,while a decreasing trend with 0.6,0.8 and 0.9 times of that before treatment in the control W82 respectively.The content of H2O2 and MDA in plants increased as a whole in W2-2 and W82 after treatment,and the increase of W82 was significantly higher than that of W2-2 in the whole process.3.Response characteristics of related genes in transgenic soybean leaves treated with dicamba.qRT-PCR analysis of various genes in leaves of the transgenic line W2-2 and control W82 treated with 280 g.a.i.ha-1 dicamba was performed.It was found that expression levels of auxin metabolism pathway genes(GmIAA27,GmGH3.1 and GmGHS.2)and ABA metabolic pathway gene GmArCED1 in transgenic line W2-2 remain unchanged at 6 h point after treatment with dicamba,but the levels were up-regulated expression in leaves of W82,reaching a significant difference.Ethylene metabolic pathway gene GmACS1 was temporarily up-regulated in transgenic line W2-2,but its expression level returned to normal after that.The expression of GmACS1 gene in W82 was increased,which was significantly different from that before treatment.4.Under low light condition,dmo gene participated in auxin metabolism regulation and influenced leaf morphogenesis.The plants of transgenic lines and W82 were treated with different light conditions of 15,000Lx and 2000Lx for 15d.It showed that the first trifoliate leaf of W82 plants showed shrinkage and incomplete expansion phenotype under 2000 lx light,while the transgenic lines had normal leaf morphology.Through qRT-PCR analyses of dmo and auxin,ethylene and ABA metabolic pathway genes in transgenic lines and W82 under two different light conditions,it was found that low light environment induced up-regulation of dmo gene expression,and auxin metabolic pathway was also involved in response.The expression level of dmo gene in transgenic lines increased by 200 times compared with that of W82 under low light condition,which was significantly different.At the same time,under low light conditions,the expressions of the auxin metabolism genes GmIAA27 and GmGH3.1 in transgenic lines were up-regulated 0.4 times as those in the control W82,and there were significant differences in the expressions between the transgenic lines and W82.Meanwhile,the gene expression of the key gene GmNCED1 in the ABA metabolic pathway was very low and close to zero in the W82 and transgenic lines under low light.Gene expression level of ethylene metabolic pathway GmACS1 was similar in transgenic lines and W82 plants under low light condition,which was not significantly different. |
