Molecular Cloning And Functional Analysis Of GmTIR And GmPK1b Gene From Soybean

Soybean [Glycine max (L.) Merr] is the major oil crop in the world. Disease, salt damage and drought are major factors limiting the yield and quality of soybean. To adapt biotic and abiotic stress, plants have developed a series of defense mechanisms in the long process of evolution. It’s great significance of searching and utilization of soybean stress-responsive gene resources.With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in crops has been an important aspect of modern agricultural research.In this study, we characterized expression patterns of GmTIR and GmPK1b gene from soybean, and further researched their functions. It was useful for providing stress tolerance of soybean. The idiographic experiment results are following:1. Gene isolation and sequence analysisIn this study, we have isolated GmTIR gene from soybean by using homology cloning technique. The GmTIR contained a771bp open reading frame, encoding a257amino acid protein. The phylogenetic analysis indicated that GmTIR protein had more close relationship to N protein from tobacco than others.2. Expression patterns analysis of GmTIR gene under various stress treatmentsReal-time Quantitative PCR was used to analyze the expression patterns of GmTIR in soybean. Tissue-specific expression patterns analysis of GmTIR gene showed that GmTIR gene was all detected in different tissues, but the levels of transcript were different. The gene was constitutively expressed in soybean, strongly in leaves and roots, weakly in stems, flowers and immature green beans (the bean of15days after flowering). Biotic stress responses analysis revealed that GmTIR was involved in response to Phytophthora sojae. Abiotic stress responses analysis revealed that GmTIR was involved in response to NaCl and PEG6000stresses, and weakly response to cold stress. The specific expression patterns of GmTIR gene in soybean after treatment with exogenous hormone, indicated that the expression of GmTIR gene was intensively induced by SA, MeJA and ABA, and insensitive to ACC.3. Functional analysis of GmTIR transgenic Arabidopsis for stress toleranceUnder salt stress treatment, the germination rate of GmTIR transgenic Arabidopsis was obviously higher than that of wild-type Arabidopsis. The germination rate of wild-type Arabidopsis was32.58%, but that of GmTIR transgenic Arabidopsis was61.47%and55.03%, respectively. The root length of wild-type Arabidopsis was3.71mm, but that of GmTIR transgenic Arabidopsis was7.64mm and8.28mm, respectively. The proline rate of wild-type Arabidopsis was3.06mg/g, but that of GmTIR transgenic Arabidopsis was5.41mg/g and6.89mg/g, respectively. Under drought stress treatment, the germination rate of GmTIR transgenic Arabidopsis was obviously higher than that of wild-type Arabidopsis. The germination rate of wild-type Arabidopsis was46.07%, but that of GmTIR transgenic Arabidopsis was74.78%and77.12%, respectively. The root length of wild-type Arabidopsis was22.26mm, but that of GmTIR transgenic Arabidopsis was19.71mm and21.51mm, respectively. The proline rate of wild-type Arabidopsis was2.94mg/g, but that of GmTIR transgenic Arabidopsis was4.19mg/g and4.07mg/g, respectively. The above results proved that GmTIR over-expression increased salt and drought stress tolerance of transgenic Arabidopsis.4. Expression patterns analysis of GmPKlb gene under various stress treatmentsReal-time Quantitative PCR was used to analyze the expression patterns of GmPK1b in soybean. Biotic stress responses analysis revealed that GmPK1b was involved in response to Phytophthora sojae. The specific expression patterns of GmPK1b gene in soybean after treatment with exogenous hormone, indicated that the expression of GmPK1b gene was induced by ACC, and insensitive to S A and MeJA.5. Functional analysis of GmPK1b transgenic Arabidopsis for stress toleranceUnder salt stress treatment, the germination rate of GmPK1b transgenic Arabidopsis was obviously higher than that of wild-type Arabidopsis. The germination rate of wild-type Arabidopsis was32.58%, but that of GmPKlb transgenic Arabidopsis was67.90%and69.08%, respectively. The root length of wild-type Arabidopsis was4.53mm, but that of GmPK1b transgenic Arabidopsis was6.63mm and7.51mm. respectively. The proline rate of wild-type Arabidopsis was3.06mg/g, but that of GmPK1b transgenic Arabidopsis was6.28mg/g and6.88mg/g, respectively. Under drought stress treatment, the germination rate of GmPK1b transgenic Arabidopsis was obviously higher than that of wild-type Arabidopsis. The germination rate of wild-type Arabidopsis was46.07%, but that of GmPK1b transgenic Arabidopsis was84.70%and75.95%, respectively. The root length of wild-type Arabidopsis was15.52mm, but that of GmPK1b transgenic Arabidopsis was21.80mm and27.76mm, respectively. The proline rate of wild-type Arabidopsis was2.94mg/g, but that of GmPK1b transgenic Arabidopsis was6.10mg/g and7.49mg/g, respectively. These results showed that GmPK1b over-expression inereased salt and drought stress tolerance of transgenic Arabidopsis.In conclusion, over-expression of GmTIR and GmPK1b in transgenic Arabidopsis thaliana increased the environmental stress tolerance compared with the control. This Indicates that GmTIR and GmPK1b are good candidate for improving crop performance under environmental stresses.