Studies On Improving Tomato Firmness Via CRISPR/Cas9 Mediated Mutagenesis Of Polygalacturonase Gene

Tomato(Solanum lycopersicum),as one of the most significant commercial vegetable crops grown in the world,has many nutritive compounds for human health.However,the fruit softening is the most common defect consideration of commercial importance and have tremendous associated costs in terms of fruit quality,transportation and storage.It is very urgent to carry out some related research to change this status.The softening process of tomato fruit is very complicated,which mainly involves a series of biochemical and physiological processes of tomato fruit cell wall.In these reactions,polygalacturonase(PG)has been proved to be closely related to the softening of tomato fruit.Previous researches indicated that PG is a cell wall protein,its main function is degrading the main components of cell wall,which leads to the breakdown of cell structure,resulting in softening of tomato fruit.Thus,one of the most effective approaches to reduce the loss is to cultivate anti-softening lines.Previous studies showed that transgenic antisense RNA fragments and RNA interference could decrease the activity of PG gene,which could play a certain role in preventing fruit softening.However,the random integration of exogenous transgenes into the tomato genome may cause unstable and/or off-target effects and the food safety of genetically modified food is also controversial.This project intends to use the third genome editing approach,CRISPR(Clustered regulatory interspaced short palindromic repeat)technology,to transform tomato.The new tomato materials with PG gene edited and delayed fruit softening were screened and identified.At the same time,the non-transgenic gene-edited homozygotes were obtained by offspring isolation.Our results were listed as followed.Firstly,the construction of CPISPR vector.Tomato PG gene was selected firstly as candidates by genome sequence and c DNA sequence analysis.There are 5 target sites,SP1,SP2,SP3,SP4 and SP5,were selected from the first,second and third exon of PG gene.CRISPR vector containing SP1,SP2,SP3,SP4 and SP5 were named pg1,pg2,pg3,pg4 and pg5,respectively.In order to evaluate gene editing efficacy of different target site,the corresponding vector was transformed to Agrobacterium rhizogenes K599 and infecting 2-3 leaf tomato seedlings.In terms of the editing efficiency,the ratio of SP1 gene editing is relatively high and reached to 75%.Secondly,the construction of tomato agrobacterium-mediated genetic transformation system.Through the selection of tomato explants and the matching of adventitious bud induction medium,we studied which type of tomato explants could obtain high efficient adventitious bud induction in a short time.Our results demonstrated that tomato cotyledon can be used as explant.The media for bud induction is MS+0.1 mg/L IAA+2 mg/L ZT and the media for root induction is MS+0.1 mg/L IAA.The parameters of tomato transformation are Agrobacterium tumefacien EHA105(OD600=0.6)infecting for 10 min(the rate of budding reached to 51 %)and coculturing for 2 d.Thirdly,off-target analysis.In order to guarantee the specificity of CRISPR/Cas9 in tomato,we analyzed the potential off-target effects of the editing of Sl PG based on the prediction of the web tool CRISOR(http://crispor.tefor.net/).The one most likely off-target sites(AGCTTTGGAGCTAAA*GGTGATGG)of SP1(AGCTTTGGAGCTAAG*GGTGATGG)target sites of Sl PG were selected and examined by site-specific genomic PCR and sequencing.The examined potential off-target site only possessed mismatches of 1bp(marked with asterisk on base upper right corner)compared with the on-target guide sequences.In this study,no mutations were observed in the examined potential off-target sites.Fourthly,gene analysis of Transgenic T2 generation.In the T2 generation,the homozygous slpg-T2#2(1bp insertion),slpg-T2#3(4bp deletion)and slpg-T2#5(10bp deletion)lines were isolated and grown under nature conditions.To assess the potential roles of Sl PG throughout tomato fruit development,we conducted detailed quantitative real-time PCR(q RT-PCR)to examine its transcription in fruit development section.Our results showed that Sl PG transcripts were significantly decreased at PK(pink)section when compared with wide type plants.Fifthly,phenotype analysis of Transgenic T2 generation.We next measured the fruit firmness at four different ripening stages(BR(breaker),PK,LR(light red),and RR(red ripe)).Our results demonstrated that in line slpg-T2#2,slpg-T2#3,and slpg-T2#5,the values of fruit firmness were high than WT lines.After the harvest of fruit at RR stage,WT and mutant fruits,storing at the same environment,were selected for the firmness test.The results also showed homozygous slpg mutants delayed fruit softening.Sixthly,analysis of non-transgenic lines.To obtain tomato lines homozygous for endogenous SlPG mutations but without any transgenic element of the Sl PG-sg RNA/Cas9 vectors,‘transgene-free’ plants were sought via a PCR strategy that used four sets of primer pairs spanning four distinct regions in the TDNA of sg RNA/Cas9 vectors and one region in the vector backbone.In this assay,we found that 8,2,and2 out of 20 T3 slpg mutants from homozygous slpg-T2#2,slpg-T2#3,and slpg-T2#5 were ‘transgene-free’and that their offspring plants were all ‘transgene-free’ homozygous Sl PG mutants,respectively.Lastly,morphological characteristics of homozygous slpg mutants.To assess the consequences of the site-directed mutagenesis in the targeted loci,we examined the morphological characteristics of the plant body and fruits in all three slpg mutants and WT plants and detected no morphological differences.In addition,some of agronomic characteristics of all slpg mutants and WT plants were also evaluated.Results indicates that the growth and development of all slpg mutants and WT plants are consistent and are not affected by gene editing.