基于转录组测序的斑茅SaWRKY基因克隆与分析

Cloning and Analysis of SaWRKY from Erianthus arundinaceum Based on Transcriptome Sequencing

本研究以斑茅(Saccharum arundinaceum)野生种中抗旱性较强的‘斑茅90-14’和抗旱性较弱的‘斑茅82-53’为供试材料,利用转录组测序技术构建干旱处理后的斑茅差异基因表达谱,并筛选出了1个上调表达的WRKY基因。通过荧光定量PCR检测该WRKY转录因子基因在2个供试斑茅材料伸长期的表达模式,结果表明该基因均受干旱诱导上调。利用转录组数据得到的序列片段,结合RT-PCR技术克隆到该基因,命名为SaWRKY,NCBI登录号为MH745130.1;对其进行同源序列比对和系统进化树分析,结果表明:该基因总长度为1001 bp,其中蛋白质编码区(CDS)全长810 bp,编码269个氨基酸;该基因与禾本科同源基因整体相似度高。生物信息学分析结果显示,SaWRKY蛋白分子式为C1218H1936N378O382S12,分子量为28.37 kD,理论等电点为9.12,不稳定系数55.67,预测为不稳定蛋白。疏水性/亲水性预测表明SaWRKY蛋白为亲水蛋白。二级结构预测显示,SaWRKY氨基酸组成包含α螺旋(16.73%)、延伸主链(14.87%)、β转角(4.46%)和无规则卷曲(63.94%),属于不规则结构。本研究结果为进一步阐明SaWRKY基因在斑茅中响应干旱胁迫的生物学功能提供了理论依据。

In this study,taking the’90-14’with strong drought resistance and the’82-53’with weaker drought resistance as the test material and using transcriptome sequencing technologies to build the expression profile of differential gene of Erianthus arundinaceum after drought treatment,an up-regulated WRKY gene was screened through the analysis of gene expression profile.The expression pattern of the WRKY transcription factor was analyzed in 2 samples through the RT-qPCR technology.The results show that the gene is up-regulated by drought in2 materials.Using the sequence fragments obtained by transcriptome data,combining with the WRKY gene—SaWRKY,GenBank accesion number is MH745130.1,cloned by RT-PCR technology,and performing the homologous sequence comparison and phylogenetic tree analysis,the results show that the total length of the gene is 1001 bp,among this the total length of protein coding region(CDS)was 810 bp,encoding 269 amino acids.The gene is highly similar to the whole gramineous genes.Bioinformatics analysis showed that the molecular formula of SaWRKY protein was C1218 H1936 N378 O382 S12,the molecular weight was 28.37 kD,the theoretical isoelectric point was 9.12,the instability coefficient was 55.67,which was predicted to be unstable protein.Hydrophobic/hydrophilic prediction indicated that the SaWRKY protein was a hydrophilic protein.Protein transmembrane analysis revealed that SaWRKY was a non-transmembrane protein.The secondary structure prediction showed that the amino acid composition of SaWRKY includedα-helix(16.73%),extended chain(14.87%),β-sheet(4.46%),and irregular curl(63.94%),which belonged to an irregular structure.The results provided a thoeretical basis for further eclucidating the biological functions of SaWRKY gene in response to drought stress in Erianthus arundinaceum.