The Function Research Of Soybean GmeR Gene

One of my research fellows in our lab has sucessfully cloned in soybean the GmeR gene and itspromoter, which encodes a protein with bioactivities of ser/ala glyoxylate apotransaminase., The GmeRgene is mainly expressed in leaves of soybean. The GMER protein is shown to be located in peroxisomeaccording to the in-situ hybridization, and palys an important role in photorespiratory process,as thekey enzyme that controls the actvition of outburst of reactive oxygen species in plant.Since we knowthat GmeR gene is not a R gene,but a gene encoding protein with bioactivities of ser/ala glyoxylateapotransaminase..The research of the correlation between GmeR gene and downy mildew resistance canlead us to have a better understanding of how the gene that encodes enzym functions in the immunresponses of a plant, as well as the mechanism of how plant responses to pathogens attacks. In myresearch, what I have conducted include the following parts:1.The identification and analysis of the genetic background of different soybean varieties and theirresistance to downy mildew.To identify the correlation between GmeR gene and the resistance of soybean to downy mildew, Ianalyzed and identified genetic background and the resistance to downy mildew of16different soybeanvarieties I have chosen from the repository of Soybean seed,including Wuxing1,Amsoy, Binhai13-1,Kefeng1,Qihuang25, Zhongpin95-5383, Jinshanpu,Wenfeng7, Guanshuitieyingqing,Yudou22,Tiefeng18, Dongnong49, Tiefeng31, kefeng14,Jilinxiaoli1,Caidou2.It turns out that four of them arehighly resistance varieties, including: Jilinxiaolii1,Yudou22,Dongnong49,Guanshuitiejiaqin; oneresistant breed, Amsoy;two medium resistant varieties, Tiefeng18,Tiefeng31and one mediumlysusceptible one, Jingshanpu.The varieties I have chosn are respectve since their genetic background andresistance to downy mildew are different from one another.2.Semi-Quantitative RT-PCR analysis of the different expression level of GmeR in differentsoybean varieties.The result of Semi-Quantitative RT-PCR indicates that the transcriptional expression of GmeR indifferent varieties differs apparently from one another, and GmeR gene is related to the host’s resistanceto downy mildew. Taking the mediumly susceptible soybean variety-Jingshanpu, as the control, we cansee highly resistant soybean varieties, Jinlixiaoli1, Yudou22and Dongnong49, is respectively3.1,2.7and2.4times that in Jingshanpu. Analysis of variance showed significant differences. In resistantvariety Amsoy2.2times that in Jingshanpu. Analysis of variance showed significant differences.Differences of in other varieties are also apparent in other varieties.3.Real-time Quantitative PCR analysis of the different expression level of GmeR in differentsoybean varieties as well as in different growth stage of soybeanThe result of RT-QPCR shows that the transcriptional expression of GmeR in highly resistantsoybean varieties, Jinlixiaoli1, Yudou22and Dongnong49are all around27.5,3.1times that inJingshanpu. Analysis of variance showed significant differences. The result of RT-QPCR is consistent with that of Semi-Quantitative RT-PCR. The the transcriptional expression of GmeR in resistant varietyAmsoy and is23.14，and it is13.15in mediumly resistant breed Tiefeng31, both of which are lowerthan that in the three highly resistant varieties mentioned above. It can be concluded that the expressionof GmeR differs from one another, which is be positively correlated with host’s resistance to downymildew. The RT-QPCR analysis of in the second/third leaves developed at different growth stage ofsoybean shows that that the expression of GmeR increases as the plant grows, which shares the the samechange trend with the photorespiratory process in soybean. It is a futher evidence for the involvement ofGMER protein in the photorespiratory process of soybean.4.Northern blot analysis of the different expression level of GmeR in different soybean varietiesNorthern Blot has been conducted to varify this conclution. It suggests that the expression of GmeRin Jilinxiaoli1is4.549times that in Jingshanpu, which is consistant with the results ofSemi-Quantitative RT-PCR and Real-time QPCR.5.The influence of GmeR gene on transgenetic tobacco and soybean.In order to test the influence of GmeR gene on the resistance of soybean to downy mildew, I firstlyestablished sense and antisense plant expression vector of GmeR, pCGt2301and pCGr2301,andtumefacted the vector into soybean variety Jinlinxiaoli1through Agrobacterium, obtaining5and4transgenetic breeds respectively. Meanwhile, through my study of the proper concentration ofkanamycin uesed in the resistance screening of transgenetic soybean in the tissure culture, I believe thesuit concentration shall be50mg/l.To test whether function of GmeR gene remains the same in other plant species, I tumefacted thepCGt2301/pCGr2301vector into the tabacco variety, Honghua, through Agrobacterium, obtaining7and4transgenetic breeds respectively.The target sequences has been successfully inergrated into thegenome of tabacco, evidenced by gus staining and PCR analysis.Generally speaking, according to my research, GmeR gene is further proved to be related with thesoybean’s resistance to downy mildew，and its expression level in the different soybean varities ispositively correlated with their resistance to downy mildew. It is of importance for the breeding ofsoybean with high resistance to downy mildew through genetic engineering.