小桐子磷酸烯醇式丙酮酸/磷酸转运体PPT1基因的克隆及低温表达分析

Cloning and chilling expression analysis of phosphoenolpyruvate/phosphate translocator PPT1 gene from Jatropha curcas

磷酸烯醇式丙酮酸/磷酸转运体(PPT)属于质体磷酸转运体(pPT)家族的成员,负责磷酸烯醇式丙酮酸与磷酸的反向运输,在维持叶绿体与细胞质碳代谢中发挥重要作用。本研究基于BLAST序列比对的方法,在全基因组水平对小桐子pPT基因家族进行鉴定与生物信息学分析。在此基础上,克隆到该家族PPT1基因的全长cDNA序列,命名为JcPPT1,利用qRT-PCR方法检测该基因在低温条件下的表达特性,同时,通过双酶切构建该基因的植物表达载体并在拟南芥原生质体中进行了表达与亚细胞定位分析。结果表明,小桐子pPT基因家族共鉴定到7个基因,聚类为GPT(JcGPT1、JcGPT2)、PPT(JcPPT1、JcPPT2)、TPT(JcTPT1、JcTPT2)及XPT(JcXPT)4个亚家族,基因结构分别包含5、9、12、1个外显子。蛋白质长度分布在392~440 aa之间,等电点都显碱性,二级结构都包含10段α-螺旋跨膜区域。表达分析显示,JcPPT1基因在小桐子叶片与根中都属于低温诱导表达基因,分别在低温胁迫12和0.5 h时达到最大表达量,较对照显著提高4.64倍(P<0.01)与5.91倍(P<0.01)。进一步构建了该基因绿色荧光蛋白GFP标记的植物表达载体pCambia1300-35S-JcPPT1-GFP,通过拟南芥原生质体转化与瞬时表达表明小桐子PPT1定位于叶绿体中。以上结果为开展小桐子PPT基因的功能分析以及其在叶绿体淀粉积累、逆境应答中的机制研究奠定了基础。

The phosphoenolpyruvate/phosphate translocator(PPT)is a member of the family of plastidic phosphate translocator(pPT),which catalyzes the reverse transport of phosphoenolpyruvate and phosphate,and plays an important role in maintaining carbon metabolism between chloroplast and cytoplasm.Based on the BLAST sequence alignment,the pPT gene family was identified from Jatropha curcas,and then the gene structure,phylogenetic relationship,and transmembrane region analysis were conducted using bioinformatics method.A PPT1 gene(named JcPPT1)was cloned,and the expression levels under chilling stress were detected by qRT-PCR technology.Plant expression vector of pCambia1300-35SJcPPT1-GFP was constructed by double-enzyme digestion and the subcellular localization analysis was performed in transgenic Arabidopsis thaliana protoplast.The results showed that 7 p PT genes were identified from J.curcas genome,which were clustered into 4 subfamilies of GPT(JcGPT1 and JcGPT2),PPT(JcPPT1 and JcPPT2),TPT(JcTPT1 and JcTPT2),and XPT(JcXPT),containing 5,9,12,and 1 exons in gene structure,respectively.The protein length of J.curcas p PT ranged from 392 to 440 aa,the isoelectric points all appear alkaline,and the secondary structure contains 10α-helical transmembrane regions.Meanwhile,JcPPT1 gene was remarkably cold-induced expression in J.curcas leaves and roots,which reached the highest expression levels after 12 h(4.64-fold,P0.01)and 0.5 h(5.91-fold,P0.01)chilling treatment,respectively.A pCambia1300-35S-Jc PPT1-GFP plant recombinant expression vector with green fluorescent protein(GFP)label was successfully constructed,and the transient expression in Arabidopsis protoplast showed that J.curcas PPT1 is localized in chloroplasts.In conclusion,this study lays a foundation for further studies on the gene function of PPT and the mechanisms underlying chloroplast starch accumulation and stress responses in J.curcas.