以耐盐碱野生大豆(Glycine soja L.G07256)为材料,采用同源克隆方法和RT-PCR技术获得一个TIFY类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明,与其他物种相比,GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高,达到56%;序列分析表明GsTIFY11b蛋白除具有TIFY保守结构域外,还具有一个N端保守结构域和一个C端保守的Jas结构域;实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达;将GsTIFY11b转化拟南芥来验证其耐盐碱功能,获得两个转基因纯合体株系,盐碱胁迫分析结果表明,GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性,并且与野生型相比,转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明,GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明,GsTIFY11b定位于细胞核中。上述结果表明,该基因在细胞核中起着转录调节子的作用,可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。
bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses.In this study,two AtbZIP1 mutant Arabidopsis (bzip1) were used with T-DNA inserted into two different sites,designated as SALK-556773 and SALK-660942,in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites.PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression,while T-DNA insertion in 3'-UTR couldn't.The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants' drought resistance,while in 3'-UTR couldn't.The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.
MicroRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). Regulation is accomplished when the 22-25 nucleotide miRNAs bind to complementary sequences in the 3′-untranslated regions (UTR). One barrier to miRNA research is to find target genes. Although computational target predictions have shed light on important aspects of microRNA target recognition, questions remain concerning the rates of false positives. In addition, we do not completely understand how microRNAs can recognize and regulate their targets. As such, experimental approaches are required, which can reflect in vivo processes, eliminating false positive predictions and allow for an unbiased study of microRNA target recognition. In this review, we summarized experimental approaches that have been described for the identification and validation of mRNA targets associated with specific miRNAs.
Luo XiaoBai XiCai HuaJi WeiLiu XinTang Li-liZhu Yan-ming
