Studies On Construction Of RNAi Bivalent Antiviral Expression Vectors With Consensus Sequences Of ScMV And ScYLV And Genetic Transformation

Diseases of sugarcane yellow leaf virus (ScYLV) and sugarcane mosaic virus (ScMV) are wide-spread virus diseases around the world in the growth of sugarcane. Conventional control methods are very difficult to prevent and cure the diseases. Growth of sugarcane varieties infected the viruses is badly hindered, which resulting in less tonnage and sugar potential. It is difficult to control virus infection in sugarcane planting using chemical methods other than the environmental pollution. Therefore, the best choice against sugarcane virus diseases is to breed varieties with durable and stable resistance to virus disease. However it is very difficult to develop varieties due to the complex genetic background of sugarcane, which will take a long time up to twelve years to get one variety. What’s more, no natural sugarcane germplasm with traits resistance to ScYLV or ScMV. With the development of genomics, application of molecular biological methods, especially the RNA interference technology, makes it possible to create anti-viral sugarcane varieties by genetic engineering.Based on the viral structure and function characteristics of ScYLV and ScMV conserved sequences of their coat protein CP genes and the helper components protein HC-Pro gene of ScMV were selected as the interference sequence. RNAi bivalent antiviral interference sequences were combined by designing PCR primers with restriction enzyme Xba I and constructed into vector pHANNIBA to form ihpRNA structures. The RNAi bivalent antiviral interference structures were then inserted expression vector pART27. Gene transformation was performed both with Agrobacterium infection and gene gun bombardment.The main research achievements of this paper are as follows, (1) three expression vectors with RNAi bivalent antiviral interference sequences were constructed successfully which were named as pART27-MC1Y, pART27-MC2Y and pART27-MPY and three kinds of Agrobacterium engineering strains were obtained, (2) Two sugarcane varieties ROC22 and FN28 were tested with G418 in callus and differentiation stages and the screening concentrations were determined with 30 mg/L and 40 mg/L respectively. (3) 19 transgenic sugarcane lines were achieved by agrobacterium mediated transformation and ballistic bombardment, of which 6 plants were from Agrobacterium-mediated transformation(the ratio of MC1Y, MC2Y and MPY events were 2.069%,2.273% and 2.985% respectively), and the other 13 plants were obtained via ballistic bombardment(the ratio of MC1Y, MC2Y and MPY events were 5.263%,2.899% and 5.0% respectively).