Study On Tomato Breeding For Broad-spectrum Resistance To Geminiviruses Based On CRISPR/Cas9 System

Tomato is a kind of important characteristic economy crops in Xinjiang.It is very suitable for the growth of tomato in Xinjiang.However,with the increasingly deepening of world trade,tomatoes are facing increasingly severe the dangers of all kinds of plant geminivirus.However,the existing technology and methods is difficult to realize broad-spectrum resistance to geminivirus in tomato.Therefore it has important practical significance to develop broad-spectrum geminivirus-resistant tomato.In recent years,CRISPR/cas9 genome editing technology has developed rapidly and has been used in plant antiviral engineering.Based on the working principle of the CRISPR/Cas9 genome editing system,to achieve broad-spectrum antiviral breeding,there must be conservative target sequences on different viral genome sequences that satisfy the requirements of guide RNA design.Therefore,in this study,by collecting and analyzing the genome sequences of 80 types of geminiviruses that can infect tomato,we found that there are some highly conserved sequences in the nucleotide sequence of Rep(Replication-initiation protein,Rep).According to the design principle of guide RNA:A guide RNA with one PAM site and a guide RNA with two consecutive PAM sites were designed as the target sequences of the CRISPR/Cas9 system.The system was transformed into Micro-Tom tomato and then Agrobacterium-mediated inoculation of TYLCV infected clone inoculation experiments to study whether the two target sequences designed based on the conserved sequence of the Rep can truly achieve a broad spectrum antiviral effect.The results are as follows:1.Two guide RNAs designed with the conserved sequences of the geminiviruses Rep were used as target sequences of the CRISPR/Cas9 system,and corresponding CRISPR/Cas9 gene editing vectors were constructed,and the effectiveness of the target sequences against viruses was verified by transient transform ation of Nicotiana benthamiana.2.The CRISPR/Cas9 gene editing vector with the above two target sequences was transferred into tomato,and the positive transgenic tomato was subjected to TYLCV-SH2 infectious clone virus inoculation experiment,and the antiviral effect of transgenic tomato was detected by qPCR method.The result shows that the transgenic tomato materials with single and double PAM target sequence editing vectors did not show obvious disease resistance.It shows that the target sequence of single PAM can not achieve antiviral effect,and the target sequence of double PAM can not achieve antiviral effect may be that the double PAM target sequence and the target sequence of the TYLCV-SH2 infectious clone have two base mismatch.This may affect the CRISPR/Cas9 system combined with the target sequence,which affects the antiviral effect,so a subsequent site-directed mutation experiment was performed.3.Mismatched bases in the double PAM target sequence of the TYLCV-SH2 infectious clone were mutated to the double PAM target sequence that we designed earlier by site-directed mutation PCR.Transgenic tomatoes containing only double PAM target sequence editing vectors were selected as a test material,a mutant TYLCV-mSH2 infectious clone virus inoculation experiment was performed,and the antiviral effect was detected by qPCR,the results showed that compared with the Cas9-MCS transgenic tomato materials,target sequence contains double PAM material of transgenic tomatoes did not show obvious disease resistance.In conclusion,our results show that the two target sequences designed based on the conserved sequence of the Rep could not be used in tomato geminivirus-resistant breeding research based on the CRISPR/Cas9 system.In order to achieve a broad-spectrum virus resistance,other strategies may be needed.