羊草14-3-3蛋白基因的克隆及其在Na_2CO_3胁迫下的表达分析

Cloning of Lc14-3-3 Protein gene and Its Expression Analysis under Na_2CO_3 Stress from Leymus chinensis

为了获得与盐碱胁迫相关的14-3-3蛋白基因的表达模式,基于羊草14-3-3蛋白的EST序列,通过RACE方法从羊草中克隆得到的Lc14-3-3基因,并利用生物信息学分析获得了其基因信息,利用荧光定量PCR(qRT-PCR)分析获得了羊草植株在Na_2CO_3胁迫下Lc14-3-3基因的表达模式。结果表明:Lc14-3-3基因编码区全长为786 bp,编码261个氨基酸,推测蛋白质分子量为29.26 k D,理论等电点(p I)为4.83,与已知的单子叶植物来源的同类基因同源性较高,相似性为88%~100%。q RT-PCR表达模式分析表明,羊草14-3-3基因在无胁迫处理情况下有本底水平表达。在200 mmol/L的Na_2CO_3胁迫不同时间后,羊草叶片中Lc14-3-3基因的表达量在6~24 h之间表达量明显上升,但胁迫持续48 h时,Lc14-3-3表达量降至较低水平。但随着胁迫时间增加Lc14-3-3表达量骤增,96 h后高于对照组10倍左右。羊草根部的Lc14-3-3基因表达量在胁迫处理后6~24 h间为无胁迫条件的2倍,在胁迫处理48 h后下降,随着胁迫时间增加,96 h表达量为对照组11倍左右。此结果为进一步研究抗盐碱基因提供理论基础。

In order to obtain the expression pattern of the 14-3-3 protein gene associated with saline-alkaline stress. Based on the 14-3-3 protein EST sequence of Leymus chinensis, a Lc14-3-3 gene was cloned from L. chinensis by RACE. And the genetic information was obtained by bioinformatics analysis, the expression pattern of Lc14-3-3 gene in Leymus chinensis plants under Na2CO3 stress was obtained by fluorescence quantitative PCR （qRT-PCR）. The results indicated that the full length of the coding region ofLc14-3-3 gene was 786 bp, encoding 261 amino acids. The molecular weight of the protein was 29.26 kD and theoretical pI was 4.83. Homology analysis showed that the deduced amino acid sequence of Lc14-3-3 shared 88%-100% homology with 14-3-3 from monocotyledon, qRT-PCR expression pattern analysis showed that the 14-3-3 gene of Leymus chinensis was expressed at the background level without stress. With the treatment of 200 mmol/L Na2CO3 at different time, the expression of Lc14-3-3 increased between 6-24 h in Leymus chinensis leaves, and then dropped after 48 h, the expression of Lc14-3-3 was reduced to a low level. With the extension of Na2CO3 stress time, the expression of Lc14-3-3 was increased dramatically. After treated for 96 hours showed ten times peak compared with control.The results of qRT-PCR analysis showed that the expression level of Lc14-3-3 gene in Leymus chinensis root treated for 6-24 h after 200 mmol/L Na2CO3 was two times compared with control. The expression ofLc14-3-3 decreased to a low level after treatment for 48 h. As the increasing of stress time, the expression group at 96 h was 11 times higher than that of the control group. The results provided a theoretical basis for the further study of saline-alkaline genes.