Development Of An Effective System Of Virus Induced Gene Silencing By Barley Stripe Mosaic Virus (BSMV) In Wheat Spikes

Wheat is one of the most important food crops.The yield and quality of wheat directly affect national economy and people's living level. The research on the functional identification of genes related to important traits of wheat spike is of great significance in improving the yield and quality of wheat. As the bottleneck of wheat genetic transformation is still not solved, many research methods of functional gene identification, such as gene knockout, antisense RNA technology, transgenic overe-xpression and T-DNA insertion mutant library, can hardly be widely applied in wheat. In recent years, a newly-developed method of functional gene identification, virus induced gene silencing (VIGS), has become more and more popular for its simplicity, rapidity, efficiency and high-throughput. Since the method was firstly succeeded in silencing one plant endogenous gene -phytoene desatarase (PDS) with recombinant of tobacco mosaic virus (TMV) in Nicotiana benthamiana in 1995, VIGS vectors derived from 15 kinds of virus have been developed and applied successfully. But so far, only the recombinant barely strip mosic virus( BSMV) has been successfully used in inducing gene silencing in wheat. Since the system of BSMV induced gene silencing in wheat was first successfully developed by Scofield, this technology has been widely used in disease resistant gene identification in wheat seedlings, and has achieved some good results. However, the system of BSMV induced gene silencing in wheat ears has not been established.In the present study, Xiaoyan 6 was used as plant materials, recombinant BSMV used as vexctor.The recombinant BSMV carrying the fragment of wheat PDS gene were used to transfected wheat in two-leaf stage, four-leaf stage and booting stage, respectively, to induce endogenous PDS gene silencing in wheat. This purpose of this study is to develop an effective methodologies of BSMV induced gene silencing in wheat spike, providing a simple, fast and effective technical support for the research on functional identification of genes related to important traits of wheat spike. The main results obtained are as follows:1. By inoculating newly expanded leaves of wheat seedlings or flagleaves of wheat at booting stage with the recombinant BSMVcarrying PDS gene frangment from wheat, the endogenous PDS gene of wheat in leaves or ears were successfully silenced2. No evident diference in phenotypic changes was observed between inoculating the recombinant BSMV onto newly expanded leaves at two-leaf satge and at four-leaf stage of wheat, indicating that the effect of BSMV inducing endogenous PDS gene silencing was not infulenced by the growth period of wheat seedling.3. When inoculating the newly expanded leaf of wheat seedling with the recombinant BSMV, the symptoms of virus transfection were observed on the first leaf above the noculated one 7-8 days after inoculation. Photobleaching spots ( phenotype of endogenous PDS gene silenced ) was observed on the second leaf above the noculated one 13 days after inoculation, and then the most large area of photobleaching was observed 22 days after inoculation4. When inoculating the flagleaves of wheat at booting stage with the recombinant BSMV, the phenotype of endogenous PDS gene silencing in wheat heads was observed from 8 to 20 days after inoculation. The time process of the endogenous PDS gene silencing in wheat heads/spikes was similar to that in leaves, but the phenotype of endogenous PDS gene silencing in wheat heads/spikes was not the same as that in leaves.5. The quantity of the endogenous PDS gene tanscripts mRNA in wheat leaves and heads/spikes with photobleaching spots was detected by Real-Time PCR. The result showed that the quantity of the mRNA significantly declined on the 8 -10 days after inoculation, and reached the lowest on the 15 -17 days after inoculation, and then increased gradually, recovering to normal level on the 30 days after inoculation. In additions, the results indicated that the time when the most effective endogenous PDS gene silencing was observed in transfected wheat was not consistent with the time when that was detected by Real-time PCR, the former occurred 5 days earlier than the latter.In generat, the system of BSMV induced endogenous gene silencing in wheat ears has been developed in the present study. The efficient system of the endogenous genes silencing induced by BSMV in wheat heads/spikes will provide a fast and effective method for functional identification of genes wheat heads/spikes.