小麦TaVIP1家族基因克隆、分子特性及功能分析

植物VIP1蛋白(Vir E2 interacting protein 1)与农杆菌侵染植物后T-DNA在细胞内的转运有关,影响植物转化效率,但还未见有关小麦中VIP1基因研究的报道。本研究利用in silico技术从普通小麦中克隆了TaVIP1家族基因,该家族基因全部由4个外显子构成,编码产物相似性高,与拟南芥AtVIP1蛋白质序列相似性仅为50.7%~51.4%。Southern杂交结果表明,TaVIP1基因在小麦基因组中存在3个拷贝。根据小麦3个TaVIP1基因的差异序列设计特异引物,以四倍体小麦Langdon与中国春D组染色体代换系为材料进行PCR检测,结合生物信息学分析,将小麦3个TaVIP1基因分别定位在4AL、4BS和4DS染色体上。亚细胞定位分析表明,TaVIP1蛋白分布于细胞膜、细胞质和细胞核中。农杆菌转化烟草,过表达TaVIP1基因降低了烟草转化效率。小麦TaVIP1基因编码的氨基酸序列与拟南芥AtVIP1基因及烟草NtVIP1基因编码的氨基酸序列相似性很低,这可能是小麦农杆菌转化效率低的原因之一。

Cloning,Molecular Characterization,and Functional Analysis of Wheat TaVIP1 Genes

Plant VIP1 (VirE2 interacting protein 1) is involved in the transportation of T-DNA in plant cells after Agrobacterium infection, affecting plant transformation efficiency. However, the function of TaVIP1 is unknown. Using in silico technique, a family of TaVIP1 genes were successfully cloned from common wheat in this study. The gene family encompasses four exons and shares high similarity among its members while only 50.7%–51.4% similarity to the AtVIP1 in Arabidopsis. Southern blotting assay showed that the TaVIP1 had three allelic copies in wheat genome. The three copies of TaVIP1 were further assigned to wheat chromosomes 4AL, 4BS, and 4DS according to Blastn in the IWGSC (International Wheat Genome Sequencing Consortium) database, and experimentally confirmed by PCR using specific primers to each TaVIP1 allele based on their DNA sequences and Langdon durum disomic substitution lines. Subcellular localization analysis showed that TaVIP1 proteins were located in cell membrane, cytoplasm and nucleus. Over-expression of TaVIP1 in tobacco obviously reduced Agrobacterium-mediated transformation efficiency. The amino acid sequence encoded by TaVIP1 only had less similarity to the corresponding amino acid sequence encoded by AtVIP1 in Arabidopsis or NtVIP1 in tobacco. This might be a reason of the low transformation efficiency of wheat mediated by Agrobacterium.