瞬时过表达ThCBL4基因提高刚毛柽柳耐盐能力

A Novel Calcineurin B-like Proteins Gene(ThCBL4) Improving the Salt Tolerance in Transient Overexpression Tamarix hispida

[目的]本研究拟从刚毛柽柳(Tamarix hispida)中克隆获得1个ThCBL4基因,研究该基因的耐盐功能。[方法]以"Calcineurin B-like protein"作为关键词,对实验室前期构建的刚毛柽柳转录组序列进行比对查找获得ThCBL4基因c DNA序列,并通过RT-PCR和测序验证克隆获得的ThCBL4基因序列。利用生物信息学工具进行序列分析。利用Real time RT-PCR分析了0.4 mol·L-1NaCl、20%(w/v)PEG6000和100μmol·L-1ABA胁迫处理后ThCBL4基因在刚毛柽柳根和叶中的表达情况。为了进一步分析ThCBL4基因的耐盐功能,构建了植物过表达载体pROKⅡ-ThCBL4,抑制表达载体p FGC5941-ThCBL4,并进行柽柳瞬时转化,同时以pROKII瞬时侵染柽柳作为对照。分析比较NaCl胁迫后瞬时转化柽柳POD、SOD酶活性、MDA含量和DAB、NBT及Evans blue染色情况,以综合评定ThCBL4基因的耐盐功能。[结果]ThCBL4基因c DNA长度为1 432 bp,编码区长度675 bp,编码224个氨基酸,编码蛋白分子量为25.57 k Da,理论等电点为5.0。3种不同环境处理后,ThCBL4基因在刚毛柽柳根中均为上调表达,在刚毛柽柳叶中均为下调表达。对盐胁迫后瞬时表达柽柳ThCBL4基因的表达分析显示,成功获得瞬时过表达(标记为OE)、抑制表达(标记为RNAi)转基因株系。进一步的生理指标和生理染色结果显示,盐胁迫下过表达ThCBL4植株的保护酶类SOD与POD活性明显高于对照和抑制表达株系。NBT和DAB染色结果显示,过表达植株的O·-2和H_2O_2含量明显低于对照,抑制表达植株的O·-2和H_2O_2含量明显高于对照;而Evans blue染色和MDA含量测定结果显示,与对照相比,过表达ThCBL4植株着色更浅、MDA含量更低,而抑制表达ThCBL4植株着色更深、MDA含量更高。[结论]ThCBL4基因可能参与了刚毛柽柳的耐盐胁迫应答,瞬时过表达ThCBL4基因通过提高植株的保护酶活性,降低活性氧氧化程度和细胞损伤的程度,从而提高了转基因刚毛柽柳的耐盐能力。

[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 andtransient 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 c DNA 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 H_2O_2 contents in overexpression plants were significantly lower than the control. On the contrary,the O_2-and H_2O_2 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.