Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis
Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis
复制标题
巴西橡胶树三个多聚泛素启动子的鉴定及功能评价
作者：
Shichao Xin;Jinu Udayabhanu;Xuemei Dai;Yuwei Hua;Yueting Fan;Huasun Huang;Ti;ai Huang
橡胶树是一种重要的经济树种，是天然橡胶的唯一商业来源。用内源性多聚泛素启动子取代CaMV 35 S启动子可能是提高该物种遗传转化的可行途径。然而，尚未报道橡胶树中的内源性多聚泛素启动子。在这里，我们确定了三个橡胶多聚泛素基因HbUBI10.1，HbUBI10.2和HbUBI10.3，其编码的泛素单体具有几乎相同的氨基酸序列的AtUBQ 10。将这些HbUBI基因上游的基因组片段（包括特征前导内含子）扩增为推定的HbUBI启动子。计算机模拟分析表明，在这些HbUBI启动子中存在许多在强组成型多聚泛素启动子中保守的顺式作用元件。转录组学数据显示HbUBI10.1和HbUBI10.2在橡胶树植物中具有组成型表达。半定量RT-PCR结果表明，这3个HbUBI基因在转基因橡胶树叶片中的表达量均高于CaMV 35 S驱动的GUS基因。所有三种HbUBI启动子在瞬时和稳定转化测定中都表现出指导GFP表达的能力，尽管它们产生的原生质体转化效率低于CaMV 35 S启动子。这些HbUBI启动子将扩大用于在橡胶树基因工程中驱动转基因表达的启动子的可用性。
Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering.