| As an important oil, grain and forage crop, soybean is one of the agricultural prod-ucts with the largest demand-supply gap. Nowadays, soybean industry is facing huge challenge in China. However, the growth of soybean was often threatened by various diseases with soybean mosaic virus (SMV) being one of the most serious diseases in leaves of soybeans in the south of China. Currently the common methods included agricultural, biological and chemical control etc, but breeding SMV resistant varieties was the most direct and effective one. As a simple, rapid and effective means, genetic engineering technology can overcome the various defects in comparison with com-mon breeding, such as reproductive isolation and long duration.Based on a microarray analysis of gene expression differences between two con-trasting genotypes (SMV resistant and SMV susceptible) upon SMV inoculation, GmCnxl was selected as a candidate SMV resistant gene which related to the biosyn-thesis of molybdenum cofactor (Moco), with similar amino acid sequence compared with Arabidopsis thaliana Cnxl. Cofactor for nitrate reductase and xanthine dehy-drogenase(Cnxl) gene was a functional protein related to Moco biosynthesis playing roles in plant physiology and stress resistance. In this present study, an Agrobacte-rium-tumefaciens mediated gene transformation was conducted to transfer GmCnxl gene into soybean to demonstrate the function of GmCnxl and effect on SMV resis-tance. The main findings were summarized as follows:1. GmCnxl transgenic plants obtained by Agrobacterium-tumefaciens mediatedAn Agrobacterium-tumefaciens mediated gene transformation was conducted by using GmCnxl gene as the expression vector, bar gene as the selective marker gene, and cotyledo nary-node as the explants to get34putative transgenic soybean plants of4batches transformation, which was transferred113,194,181and238explants and was obtained2,20,6and6root plants. Rooting efficiency was1.77%,10.31%,3.31%and2.52%, respectively, with average being4.68%. 2. Identification of GmCnxl transgenic plantsThirty four To generation rooting plants were identified by coating leaves with Phosphinothricin, using bar protein quick dip stick and PCR analysis. The results in-dicated that27plants were positive. Southern blot for10independent T1transgenic plants selected from above showed that the imported interference fragment was one copy in all transgenic plants except two copies in line1.3. Functional analysis of GmCnxl geneThe determination about expression of GmCnxl gene for10T1transgenic plants by qRT-PCR showed that, the expression of GmCnxl gene increased from1.04to2.12fold in leaf and1.55to3.89fold in root of all transgenic positive plants in comparison to the non-transgenic plants, with line10and22exhibiting the highest expression le-vels among the different transgenic lines. These results indicated that the GmCnxl gene expression was more significantly upregulated in root than in leaf The determi-nation about Mo-enzymes activity in leaf for T1transgenic plants and non-transgenic plants showed that, for NR activity, the GmCnxl transgenic soybean plants produced high level (4.25μg g-1h-1) in line10, which were2.6-fold higher than these levels in the non-transgenic plants (1.62μg g-1h-1), while for AO activity, the level in the leaf was higher in GmCnxl transgenic soybean plants (30.00pmol L-1) in line10com-pared with the non-transgenic plants (7.71pmol L-1). Activity of XDH and SO showed no obvious relationship with the expression of GmCnxl. In addition, GmCnxl gene can also enhance the resistance of SMV, and different strains SMV (G1and G7) friction inoculation showed that, compared with non-transgenic soybean plants, the transgenic soybean plants showed great resistance to two kinds of SMV. DAS-ELISA analysis further revealed that, infection rate of GmCnxl transgenic plants were gener-ally lower than those of non-transgenic plants. |
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