小麦TaVIP1家族基因克隆、分子特性及功能分析

摘要

Plant VIP1 (VirE2 interacting protein 1) is involved in the transportation of T-DNA in plant cells afterAgrobacterium infection, affecting plant transformation efficiency. However, the function ofTaVIP1 is unknown. Using in silico technique, a family ofTaVIP1 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 shares 50.7%–51.4% similarity to the AtVIP1 inArabidopsis. Southern blot-ting assay showed that theTaVIP1 had three allelic copies in wheat genome. The three copies ofTaVIP1 were further assigned to wheat chromosomes 4AL, 4BS, and 4DS according to Blastn in the IWGSC (International Wheat Genome Sequencing Consor-tium) database, and experimentally confirmed by PCR using specific primers to eachTaVIP1 allele based on their DNA sequences and Langdon-Chinese Spring disomic substitution lines. Subcellular localization analysis showed that TaVIP1 proteins were lo-cated in cell membrane, cytoplasm, and nucleus. Over-expression of TaVIP1 in tobacco obviously reducedAgrobacte-rium-mediated transformation efficiency. The amino acid sequence encoded byTaVIP1 only had low similarity to the correspond-ing amino acid sequence encoded byAtVIP1 inArabidopsis orNtVIP1 in tobacco. This might be a reason of the low transforma-tion efficiency of wheat mediated byAgrobacterium.%植物VIP1蛋白(VirE2 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。