Optimization And Application Of The MMDa V Gene Silencing System In Mulberry

Mulberry（Morus alba L.）is a perennial dicotyledonous woody plant in the Moraceae.Mulberry is the host plant for silkworm（Bombyx mori）,and it is useful in improving air quality,water retention,soil retention and sand prevention.Mulberry is also a traditional medicinal plant in China.Its fruits,roots,stems,leaves and by-products have high medicinal value.However,there is relatively little basic research on mulberry and few studies on gene function.This lack of data has limited the comprehensive utilization of mulberry.Transgenic technology is an important technique used to study gene function.In1985,it was first demonstrated that mulberry can be infected by Agrobacterium.Since then,transgenic research has failed to produce a stable mulberry transgenic system for mulberry,hindering basic research.Therefore,it is important to develop new methods of gene function research for mulberry.Virus-induced gene silencing（VIGS）can induce the silencing of a target gene by a viral vector carrying a partial c DNA fragment of a plant functional gene.Silencing can indicate the function of a gene through phenotype changes or physiological indicators.VIGS technology was successfully established in 1995 and proved to be simple,rapid,and easily implemented.VIGS is now widely used to study plant gene function,antivirus resistance,insect resistance,stress resistance,growth and development,and secondary metabolism.However,the efficiency of virus-induced gene silencing is affected by many factors including the plant growth temperature,the length and direction of the target gene c DNA fragment inserted into the silencing vector and the stability of the inserted fragment.By building based on this study,mulberry mosaic dwarf-associated geminivirus（MMDa V）silent system,some of the factors affecting the efficiency of gene silencing were optimized,and the function of the PDS gene in mulberry was verified.This system was used to explore the functions of two differentially expressed genes,NAC72 and MST4,in Botrytis cinerea.The main results were as follows:1.MMDa V was inoculated in mulberry and cowpea,and virus molecular detection was carried out on non-inoculated parts.MMDa V was found to replicate and transfer in different parts of mulberry and cowpea.MMDa V was also inoculated into 12 mulberry germplasm resources and the infection rate of MMDa V was the highest in M.alba.The virus molecules could replicate and transfer to different parts of the mulberry tree at 24°C and OD₆₀₀ 0.3without loss of the inserted fragment.The inoculated plants grew well under these conditions.In the experiment evaluating PDS gene silencing using the MMDa V silencing vector,two albino plants were obtained from each mulberry,but the silencing efficiency was low.2.To develop an explore a more efficient silencing system,the MMDa V heterogenously assisted 2m DNA1 silencing vector was used to silence the mulberry PDS gene.The detection of virus molecules of mix-inoculated of M.alba and cowpea showed that both could replicate in the inoculated plants and transfer to different plant parts.Insert fragment loss did not occur.Then,a partial c DNA fragment of PDS gene was inserted into the 2m DNA1 silencing vector to construct two silencing vectors,and mixed with MMDa V to inoculate above-mentioned three mulberry resources.Among plants from the three mulberry resources inoculated with the 248-bp silencing vector,11,6,and 7 plants with albino phenotype were obtained with a silencing efficiency of 7.05%,4.19%and5.01%,respectively.Four albino plants and two albino plants were obtained in plants inoculated with a 298-bp inserted fragment silencing vector,and silencing efficiency was4.12%and 1.38%,respectively.Compared with MMDa V alone,the silencing efficiency was significantly improved.3.To verify that this system can be used to study mulberry gene function,the MMDa V hetero-assisted 2m DNA1 silencing vector was used to silence two differentially expressed genes,NAC72 and MST4,in response to Botrytis cinerea.The silenced plants were grafted by B.cinerea.In the NAC72 and MST4 silenced plants of Morus notabilis,the area of disease spots was significantly reduced,while in the other two NAC72silenced plants,the area of disease spots was also significantly reduced.The area of disease spots in MST4 gene silenced plants was similar to that of the wild type.These results showed that the silencing of NAC72 gene might promote the expression of the resistance gene and enhance mulberry resistance to B.cinerea.This study provides a reference gene for the response of mulberry to the infection of B.cinerea.The silencing system could be used to quickly identify the gene function and provide a novel research method for gene function studies of mulberry.