Cloning and Drought Stress Function Analysis of Pathogenesis-related Proteins GbPR10 Gene in Sea-island Cotton (Gossypium barbadense)
LIU Jian-Guang1,*, DOU Hai-Kuan1,*, ZHAO Gui-Yuan1, GENG Zhao1, HAN Shuo2, AN Ze-Tong1, ZHANG Han-Shuang1,**, WANG Yong-Qiang1,**
1 National Cotton Improvement Center Hebei Branch, Institute of Cotton, Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Cotton in Huanghuaiha Semiarid Area, Ministry of Agriculture, Shijiazhuang 050051, China; 2 Insititue of Open Education, Xingtai Open University, Xingtai 054000, China
Abstract：The pathogenesis-related protein 10 (PR10) gene plays an important role in plants when resistance to biological and abiotic stresses. In this study, a PR10 gene was cloned from island cotton (Gossypium barbadense) 'Pima90-53' and named GbPR10 (GenBank No. MT612460) based on the transcriptome data of drought stress; To reveal tissue expression specificity, exogenous hormones induction, expression characteristics under different stress for GbPR10 gene by qPCR; Meanwhile the promoter of GbPR10 gene was cloned and analyzed; GbPR10 gene overexpression vector PCAMBIA 1301-GbPR10 was transformed into Arabidopsis thaliana to analyze the tolerance of over expression of GbPR10 gene in drought stress conditions. The results showed that the open reading frame of GbPR10 gene was 486 bp, encoding 161 amino acids, and contained a Bet_v1-like domain with P-loop conserved domain, which belonged to the Bet_v1 family of path-related proteins. The sequence blast analysis showed that the cloned GbPR10 was located on chromosome D02 and exist a homologous gene on chomesome A02 with the similarity of 96.07%. Phylogenetic tree analysis with the reported PR10 sequence showed that GbPR10 was closer with GbPR10-5 and NtPR10. Protein phosphorylation site prediction analysis showed that GbPR10 contains 8 serine phosphorylation sites, 4 threonine phosphorylation sites and 3 tyrosine phosphorylation sites. Tissue specific expression results showed that GbPR10 gene was predominance expressed in seedling and bolling roots, especially in 4 leaf age, which the expression level in 4 leaf age root was more than 1 000 folds than that in leaf. Exogenous hormones induction expression of GbPR10 showed that GbPR10 was significant up-regulated by exogenous ABA (abscisic acid), ET (ethylene), MeJA (salicylic acid) and SA (salicylic acid), and meanwhile GbPR10 was also up-regulated under 20% PEG6000 (polyethylene glycol 6000) as well as 200 mmol/L NaCl stress treament. All these induction expression of GbPR10 indicated that GbPR10 may be involved in abiotic stress responses. The GbPR10 promoter fragment of 2 176 bp was obtained using promoter sequence analysis tools (BDGP, FPROM), and the promoter sequence analysis using New PLACE online tool found that the GbPR10 promoter contains many hormone response, defense respones and abiotic stress reponse elements including ET response element AGCBOX, ABA response element DPBFCOREDCDC3, MYB1AT, MYB2AT, MYCCONSENSUSAT, and several abiotic related regulatory elements CBFHV, MYB1AT, MYB2AT, MYCCONSENSUSAT, WRKY71OS. The result further indicated that GbPR10 could be involved in abiotic stress responses. Over-expression of GbPR10 analysis under drought stress showed that the root length of GbPR10 transgenic A. thaliana lines with 10 d seeding was significantly higher than that of wild type under the PEG MS medium (P<0.05), and under natural drought condition, the drought tolerance of transgenic A. thaliana with GbPR10 gene was significantly higher than wild type lines (P<0.05). In conclusion, GbPR10 gene plays an important role in cotton drought response and this study could provide a theoretical reference for the molecular mechanism of cotton drought resistance.
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