Genetic Transformation Of RNAi Vector Of ARF Gene And Its Effects On Physiological Characteristics Of Microtuber In Potato

ADP ribosylation factor (ARF) is a member of the ras superfamily of genes,which are about the size of20kDa guanine nucleotide-binding protein, belongs toARF subfamily of small G protein superfamily. ARF as the cofactor of cholera toxincatalyzing Gs protein, was firstly discovered, purified and named ARF. In this paper,ARF gene of RNA interference expression vectors driven by the constitutiveexpression promoter CaMV35S were introduced into potato cultivar ‘Gannongshu2’by Agrobacterium-mediated transformation method, and obtained transgenic potato.And measured physiological indicators of transgenic microtuber induced from thetransgenic plants, in order to study the impact of ARF gene on potato’s physiologicalcharacteristics, thus providing a theoretical basis for further research the roles of ARFgene in the potato tuber. The main results are as follows:1. Sterile plantlets of potato cultivar ‘Gannongshu2’ as donor material, to inducemicrotuber. Expression vector pHellsgatearf1genetic transformation using ARFgene of RNA interference expression vectors driven by the constitutive expressionpromoter CaMV35S, The results from selection of the transformed plants on theculture media containing kanmycin showed that obtaining56transgenic plants.2. Specific primers were designed for transgenic plants obtained by PCRassaying. The PCR detection of nptⅡ gene, constitutive expression promoter CaMV35S and interference ARF gene fragment, the analysis of real time fluorescencequantitative polymerase chain reaction (qRT-PCR) showed that the ARF geneexpression in the transgenic plants. The results show that the ARF gene expressiondeclined at varying degrees in the transgenic lines Z-1、Z-2、Z-5、Z-9、Z-11、Z-12,and interference degree of gene expression reached35.23%-93.22%, the interferencedegree of gene expression reached92.53%,93.22%in the transgenic lines Z-11andZ-12respectively.3. The result from assay of starch, soluble sugar, protein, sucrose and glucosecontent of microtuber induce from the transgenic plants showed that the starch contentof the transgenic plants increased by4.29%-34.27%, the protein content increased by1.47%-7.35%, the soluble sugar content increased by1.44%-17.39%, and the sucrosecontent increased by11.53%-53.84%compared with control, however, the glucosecontent of the transgenic plants decreased6.06%-21.21%compared with control.