谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应

Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice

【目的】盐胁迫影响作物的产量和品质,而作物的耐盐性受特定基因的调控。在前期获得谷子类受体蛋白激酶基因SiRLK35过表达水稻株系的基础上,拟结合植株在盐胁迫下的表型,对部分盐胁迫指标及响应基因的表达模式进行检测和验证,解析谷子SiRLK35参与盐害响应的可能机制。【方法】选取谷子SiRLK35过表达水稻株系,分别为过表达株系(over-expression,OE)-1(OE-1)、OE-2和OE-3,以野生型中花11为对照,实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测各株系中SiRLK35的表达情况;分别用含0、150和200 mmol·L-1 NaCl的MS培养液处理三叶期的对照及转基因株系幼苗,观察其表型,统计150 mmol·L-1 NaCl处理3 d后苗长及根长;用150 mmol·L-1 NaCl处理四叶期幼苗,统计处理14 d后材料干重、死亡率和死叶率,对各材料的耐盐性进行评价;进一步挑选SiRLK35过表达程度高且耐盐表型好的OE-1株系,利用3,3'-二氨基联苯胺(3,3'-Diaminobenzidine,DAB)和氮蓝四唑(nitrotetrazolium blue chloride,NBT)染色检测其胁迫下过氧化物积累及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化物酶(peroxidase,POD)的活性;并对部分盐害响应基因的表达模式进行检测。【结果】谷子SiRLK35在株系OE-1中的相对表达量最高;150 mmol·L-1NaCl处理3 d幼苗后,中花11苗长和根长的生长受抑制程度均大于SiRLK35过表达株系,其中OE-1的受抑制程度最小;四叶期幼苗经150 mmol·L-1 NaCl处理14 d后,转基因株系地上部和地下部干重降低幅度均低于对照,且幼苗死亡率和死叶率均低于对照;盐胁迫下对照过氧化染色反应明显,O2-和H2O2的积累均高于过表达植株,SOD和POD抗氧化酶活性均高于对照;部分盐害响应基因在SiRLK35过表达株系中表现为上调,其中,OsLEA3在盐处理24 h的相对表达量是对照中的1.9倍。【结论】谷子SiRLK35异源转化水稻获得的过表达株系对盐胁迫具有一定的抗性,SiRLK35可通过调控抗氧化酶活性及相关信号途径,从而参与盐害响应。

[Objective]Salt stress affects the crop yield and quality, while the crop salt tolerance is regulated by specific genes. Using heterogeneous over-expressed rice lines (OE-1, OE-2, and OE-3) of foxtail millet receptor-like protein kinase gene SiRLK35, the possible mechanisms of SiRLK35 under salinity will be dissected on phenotypic, physiological and molecular levels. [Method] The expressions of SiRLK35 in OE-1, OE-2, and OE-3 were analyzed by qRT-PCR. The phenotypes of three-leaf stage seedlings treated with 0, 150 and 200 mmol L -1 NaCl were observed, and the lengths of seedlings and roots were measured after treated with 150 mmol L -1 NaCl for 3 days. The dry weights, death rate and dead leaf rate of four-leaf stage seedlings that treated with 150 mmol L -1 NaCl for 14 days were also measured. OE-1with the highest SiRLK35 expression was further used for DAB and NBT dyeing analysis. The activities of partial antioxidases, and expression patterns of marker genes were detected. [Result] There was the highest SiRLK35 expression level in OE-1. The growth of control and OE seedlings were all inhibited under 150 mmol L -1 NaCl. The decreased levels of dry weight, the death rate and dead leaf rate of OE rice were all lower than those of the control, together with the less accumulation of O 2- and H2O2, and the higher activities of antioxidases under salinity. Partial of salt responsive genes were up-regulated in SiRLK35 OE lines, especially the OsLEA3 was induced about 1.9 times higher after treated with NaCl for 24 h. [Conclusion] SiRLK35 OE rice lines have tolerance under salinity, and the gene SiRLK35 of foxtail millet could participate in salt response by regulation the activities of antioxidases and related signal pathways.