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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (5): 47-55    DOI: 10.13523/j.cb.20180507
    
Functional Analysis of Rpfan37 in the Symbiotic Nodulation Process of Robinia Pseudoacacia
Zhao FENG1,2,Min-xia CHOU2,**()
1 College of Medical Technology, Shaanxi University of Chinese Medicine, Xianyang 712046, China
2 State Key Laboratory of Crop Stress Biology in Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, China;
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Abstract  

Objective:To investigate the new gene Rpfan37, a homolog of PITP, in order to provide an idea for understanding the function of relative genes that involved in the symbiotic nodulation process.Methods:Through the previous study, target genes which were suspectly associated with symbiotic nodulation were screened out from the suppression substractive hybridization library of Robinia pseudoacacia interacting with symbiotic rhizobia. Using qRT-PCR to analyze the target gene expression level at different times and in different tissues. The RNAi recombinant plasmid was transformed into the plant root through K599 and verify the function of Rpfan37 after inoculation.Results:qRT-PCR analysis showed up-regulation characteristics of Rpfan37 in roots especially at 15 days post-inoculation (dpi), however, inoculation and non-inoculation treatment had no significant effect on the gene expression which sharply decreased in the matured nodule. Knockdown of Rpfan37 via RNA interference resulted in impaired development of both plant growth and nodule. Compared with empty vector plants, fresh weight, the root and stem length, nodule number per plant deceased dramatically in Rpfan37 RNAi plants. The root hair development of RNAi plant was abnormal and the number of root hair curling, ITs and nodule primordia were also significantly reduced in the Rpfan37 RNAi roots. Nodulation paraffin sections showed that the number of infected cells in RNAi plant nodules was significantly reduced compared with the control. Real-time PCR analysis of the expression levels of leghemoglobin gene indicated that nodule development and maturation was significantly blocked in the Rpfan37 RNAi roots.Conclusion:The related gene Rpfan37 found in Robinia pseudoacacia can participate in the symbiotic nodulation process. It provides a new theoretical basis for understanding the function of phosphatidylinositol transporter in symbiotic nodulation process.

Key words： Rpfan37      Symbiotic nodulation      RNA interference      Phosphatidylinositol transfer protein Robinia pseudoacacia     
Received: 02 February 2018      Published: 05 June 2018
ZTFLH:  Q945  
Corresponding Authors: Min-xia CHOU     E-mail: minxia95@163.com
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Zhao FENG
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Cite this article:

Zhao FENG,Min-xia CHOU. Functional Analysis of Rpfan37 in the Symbiotic Nodulation Process of Robinia Pseudoacacia. China Biotechnology, 2018, 38(5): 47-55.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180507     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I5/47

Primer name Primer sequence(5'-3') Use
f37-F TGAAATCAGGGCAGGTGT RT-PCR
f37-R GATAAGACTGGCAGGAAGAA RT-PCR
GUS-F CCCGTGAAATCAAAAAACTCG RT-PCR
GUS-R AAAGAAATCATGGAAGTAAGACTGC RT-PCR
Lb-F CTCAAGGCTCACGCTGAAAAGG qRT-PCR
Lb-R TTGCTCAATTCGTCGCTCCATT qRT-PCR
BP37-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCCA
GCATCTTCTCTACTTCC		 RNA干扰载体构建
RNA interference vector construct
BP37-R GGGGACCACTTTGTACAAGAAAGCTGGGTTTC
CAACCTCATTGTTTCTC		 RNA干扰载体构建
RNA interference vector construct
qfan37-F TGAAATCAGGGCAGGTGT qRT-PCR
qfan37-R GATAAGACTGGCAGGAAGAA qRT-PCR
Table 1 Primers used in this study
Fig.1 Relative expression levels of Rpfan37 in different developmental stages Data are presented as mean ± SEM, which were calculated over biological replicate (n=3) and technical replicate (n=3)
Fig.2 The identification of transgenic plants and expression analysis of GUS and target gene in RNAi hairy roots (a) GUS staining for identification of 8 day post-innoculation (dpi) RNAi roots Bar = 1cm (b) RT-PCR analysis of the expression levels of GUS gene during the nodulation in RNAi plants (c) RT-PCR analysis of the expression levels of target gene in 15 and 30dpi transformed roots of R. pseudoacacia in RNAi plants and control (d) qRT-PCR analysis of the expression levels of target gene in 15 and 30dpi transformed roots of in RNAi plants and control. Data were normalized to 18S rDNA expression Error bars indicate the standard errors, with 3 replicates. Different letters indicate statistical significance below a statistical P value of 0.05
Fig.3 Effects of Rpfan37 RNAi on growth phenotype,nodule morphology and nodulation number of Robinia pseudoacacia (a) Plants of Rpfan37 RNAi and control were grown in the medium with vermiculite and perlite for one week, and then they were inoculated with rhizobia. The plants at 15dpi were showed in the figure (b) The phenotype of nodules at 30dpi from control(i) and RNAi Bars = 1cm (c)-(f) The root and stem length, fresh weight and the nodule number of transgentic plant at 30dpi The number was scored at 30dpi. Data are means±SEM, n=40. Different letters indicate statistical significance below a statistical P value of 0.05
Fig. 4 The form of root hair and infection threads in the root hair at 8dpi The root hairs formation of control(i) (a) and RNAi (b). The form of infection threads in the root hair of control(i) (c) and RNAi (d); Bars=200μm The number was scored at 10dpi. Data are means ± SEM, n=40. Different letters indicate statistical significance below a statistical P value of 0.05
Fig.5 Effects of Rpfan37 RNAi on plant root hair curling, infection thread and primordia number The number was scored at 10dpi. Data are means ± SEM, n=40. Different letters indicate statistical significance below a statistical P value of 0.05
Fig.6 LM (light microscopy) micrographs of longitudinal section of the 30dpi R. pseudoacacia nodules (a)Control(i) (empty vector) (b) Rpfan37-RNAi (c) Magnification of nitrogen fixation zone in (a) (d) Magnification of nitrogen fixation zone in (b) Bars = 100μm
Fig. 7 Real-time PCR analysis of the expression levels of leghemoglobin genein 15 and 30dpi nodule Error bars indicate the standard errors, with 3 replicates. Different letters indicate statistical significance below a statistical P value of 0.05
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