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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (7): 1025-1032.doi: 10.3724/SP.J.1006.2020.94152

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and functional analysis of GmNRT1.2a and GmNRT1.2b in soybean

LI Guo-Ji**,ZHU Lin**,CAO Jin-Shan,WANG You-Ning()   

  1. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2019-10-09 Accepted:2020-03-24 Online:2020-07-12 Published:2020-04-27
  • Contact: You-Ning WANG E-mail:youningwang@mail.hzau.edu.cn
  • About author:** Contributed equally to this work
  • Supported by:
    National Transgenic Major Project of China(2018ZX0800919B);National Natural Science Foundation of China(31872873)

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Abstract:

Nitrate transporters (NRTs) have been found to be involved in nitrate uptake, transport and allocation in Arabidopsis. The role of GmNRT1.2s in soybean (Glycine max) symbiotic nitrogen fixation process has been speculated by bioinformatics analysis, however, its biological function has not been explored yet. In this study, we mainly focused on analyzing the expression pattern and biological function of GmNRT1.2a and GmNRT1.2b in soybean. The relative expression levels of GmNRT1.2a and GmNRT1.2b were higher in leaves, which induced by nitrate and up-regulated with increasing nitrate concentration. GmNRT1.2a and GmNRT1.2b expressions were also induced by rhizobial inoculation and nod factor (NF) treatment. Overexpression of GmNRT1.2a or GmNRT1.2b caused dramatic increment of nodule number. The results provide some data for further investigating the molecular mechanism of GmNRT1.2a and GmNRT1.2b in regulating the symbiotic nitrogen fixation process of soybean.

Key words: soybean, nodule, expression analysis, nodulation, symbiotic nitrogen fixation

Fig. 1

Construction of GmNRT1.2a and GmNRT1.2b overexpression vectors A: the coding sequence of GmNRT1.2a and GmNRT1.2b were obtained through PCR amplification. B: the partial vector map of pEGAD."

Fig. 2

Expression pattern of GmNRT1.2a and GmNRT1.2b in different developmental stages of soybean Mean values indexed with different letters are significantly different at P < 0.05. DAI: days after inoculation."

Fig. 3

Expression pattern of GmNRT1.2a and GmNRT1.2b under different concentrations of nitrate Soybeans were germinated in vermiculite at different nitrate concentrations (0N-0 mmol L-1, LN-0.25 mmol L-1, HN-15.75 mmol L-1). The roots were collected at 15 days after germination. The expression of GmNRT1.2a (A) and GmNRT1.2b (B) was analyzed by RT-qPCR. Bars with different lowercase letters in each figure are significantly different at P < 0.05."

Fig. 4

Expression patterns of GmNRT1.2a and GmNRT1.2b in response to rhizobium inoculation and nod factors treatment Values followed by different letters are significantly different at P < 0.05."

Fig. 5

Nodule number increased by 35S::GmNRT1.2a or 35S::GmNRT1.2b under low nitrate condition A, B: RT-qPCR analysis of GmNRT1.2a or GmNRT1.2b in single hairy root transformed with empty vector, 35S::GmNRT1.2a or 35S::GmNRT1.2b inoculated with Bradyrhizobium japonicum USDA110 at 10 and 28 days treatment. GmELF1b was used as an endogenous control for gene expression. Bars with different lowercase letters in each figure are significantly different at P < 0.05. C: the phenotype of nodule per hairy root transformed with empty vector (EV), 35S::GmNRT1.2a and 35S::GmNRT1.2b at 28 DAI. Bar = 5 mm. D: nodule number per hairy root transformed with empty vector (EV), 35S::GmNRT1.2a or 35S::GmNRT1.2b were counted at 28 DAI. The values followed by * and *** are significantly different at P < 0.05 and P < 0.001."

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