Objective It is reported that the Calcineurin B-like proteins (CBLs) play important roles in the regulating plant growth and stress tolerant. In this study, a CBL gene (ThCBL4) was cloned from Tamarix hispida. And the salt tolerance function of ThCBL4 was studied.

Method The word "Calcineurin B-like protein" was used as a query to search against the functional annotation of non-redundant unigenes based on the previous constructed T. hispida transcriptomes for identification of the ThCBL4. The expression patters of ThCBL4 genes in the roots and leaves of T. hispida under 0.4 mol·L^-1 NaCl, 20%(w/v)PEG₆₀₀₀ and 100 μmol·L^-1 ABA treatment by using the real time RT-PCR. Furthermore, the plant overexpression vector and knockdown vector were constructed and transient transformed into T. hispida. At the same time, the vector pROKⅡ was also transient infected into T. hispida as a control. The MDA contents, POD, SOD activities and DAB, NBT and Evans blue staining under NaCl stress were measured and compared among the pROKⅡ-ThCBL4 transient transformed T. hispida, ThCBL4 knockdown transient transformed T. hispida and the control.

Result The cDNA length of ThCBL4 was 1 432 bp, containing a length of 675 bp open reading frame encoding 224 amino acids. The relative molecular weight of ThCBL4 protein was 25.57 KDa, with isoelectric point 5.0. The expressions of ThCBL4 in T. hispida were upregulated in roots and downregualted in the leaves under NaCl, PEG or ABA stress conditions. Furthermore, the relative expression levels of ThCBL4 among three transient expression lines was the highest in the overexpression lines while the lowest in the knockdown transient expression lines. The results of physiological indicators and physiological staining showed that the protective enzymes SOD and POD activity in ThCBL4 overexpression plants were significantly higher than the control and suppression of expression lines under salt stress. NBT and DAB staining showed that the O^2- and H₂O₂ contents in overexpression plants were significantly lower than the control. On the contrary, the O^2- and H₂O₂ contents in the inhibited expression lines were significantly higher than the control. At the same times, Evans blue staining and MDA assay results showed that the ThCBL4 overexpressing plants colored more shallow compared with the control, MDA contents is lower than the control, and colored deeper and higher content of MDA in the inhibited expression of ThCBL4 plants. These results showed that the protective enzyme activities of ThCBL4 transient overexpression lines were higher than the control, the active oxygen contents were lower, and the degree of cell injury were lighter.

Conclusion The transient overexpression of ThCBL4 lines increased the salt tolerance, and ThCBL4 gene may be involved in the response to salt stress in T. hispida. In the future, its function and the regulation mechanism need to be further studied.