CRISPR/cas9 System Based Functional Characterization Of SlMAPK6 Gene In ’Dwarf Tomato’

Tomato(Solanum lycopersicum),which belongs to the genus Solanaceae,it is an important horticultural fruit and vegetable in China.Because of its rich nutritional elements,tomato has high economic value.However,no matter open-air cultivation or protected cultivation,the effects of biotic and abiotic stresses seriously threaten tomato production.Among them,tomato virus diseases,such as Tomato yellow leaf curl virus(TYLCV),occur in a large area in China,which seriously restricts the safety of tomato production.Mitogen-activated protein kinases(MAPK)is ubiquitous and highly conserved in eukaryotes.It belongs to serine(Ser)/threonine(Thr)protein kinases,Tomato MAPK cascade signal system consists of three levels: MAPKKK、MAPKK and MAPK,which is one of the important pathways in eukaryotic signal transduction network and plays an important role in regulating plant disease resistance and stress resistance,MAPK is in the downstream of the three-stage signal system,which is very important in the process of three-stage signal transmission.At present,a total of 16 MAPK genes have been identified in tomato,which can be divided into four subgroups: subgroup A,subgroup B,subgroup C,and subgroup D.Among them,the MAPK of the subgroup A,such as MAPK1/2/3,is relatively thorough,but there is little research on the function of MAPK6 in subgroup B.In this study,’dwarf tomato’ was used as experimental material to explore the function and role of SlMAPK6 in tomato growth,development and the regulation of plant resistance to TYLCV immunity by means of biochemistry,molecular biology and bioinformatics.The main results are as follows:(1)The full-length cDNA sequence of SlMAPK6 gene was obtained from dwarf tomato by homologous cloning technique and analyzed by bioinformatics.Biological information analysis showed that there were abundant phosphorylation sites in SlMAPK6 protein sequence.It is speculated that SlMAPK6 may activate downstream proteins through serine phosphate site and threonine phosphate site,and construct phylogenetic tree by constructing MAPK protein amino acid sequence of Arabidopsis thaliana and tomato.It was found that compared with Arabidopsis thaliana,SlMAPK6 and At MAPK4 have the closest genetic relationship.It is speculated that SlMAPK6 is similar to At MAPK4 in function and participates in negative immune regulation in tomato.Realtime fluorescence quantitative PCR was used to detect the tissue-specific expression of SlMAPK6 gene in dwarf tomato seedlings,and the expression patterns of SlMAPK6 gene under signal induction(salicylic acid、2,1,3-benzothiadiazole)、abiotic stress(drought、salt)and biotic stress(TYLCV、Botrytiscinerea).The results showed that the expression of S1MAPK6 was higher in stem,leaf and flower,but relatively low in root,inflorescence and fruit,More importantly,SlMAPK6 participates in the induced expression of signal induction(salicylic acid、2,1,3-benzothiadiazole)、abiotic stress(drought、salt)and biotic stress(TYLCV、Botrytiscinerea).(2)Using CRISPR/Cas9 technology to design 3 target sites for targeted editing of SlMAPK6,and the mutants CRISPR-3 and CRISPR-7 were obtained.The results of PCR and sequencing showed that the SlMAPK6 knockout vector was successfully constructed.Through kanamycin gene screening,target site amplification,sequencing,and off-target effect analysis of the T1 generation plants.the results showed that the ’dwarf tomato’ plants were mutated successfully and did not miss the target by Sanger sequencing.Through phenotypic observation and measurement,compared with the wild-type control,all the mutant plants showed a more developed root system and more lateral branches.The expression of genes related to lateral branch development was detected by real-time fluorescence quantitative PCR.The results showed that the expression of genes related to lateral branch growth in the mutant was significantly higher than that in the wild type,while the expression of SlCDD7 and SlCDD8 related genes in the mutant was significantly lower than that in the wild type.It is suggested that SlMAPK6 may regulate the morphogenesis of tomato plants by regulating the monogrolactone pathway.(3)SlMAPK6 knockout mutants CRISPR-3,CRISPR-7 and wild-type tomato were Inoculated with TYLCV infectious clones,compare of TYLCV virus content in knockout mutants CRISPR-3、CRISPR-7 and wild-type tomato by phenotypic observation,and the real-time fluorescent quantitative PCR technology at 21 days after inoculate TYLCV infectious.The results showed that wild-type tomato showed more serious virus expression than mutants after inoculation with virus.the relative content of virus in Knockout mutants CRISPR-3 and CRISPR-7 was significantly lower than that in wild-type,indicating that the disease resistance of the knockout mutants was higher than that of wild type.In order to explore the antiviral mechanism of the SlMAPK6 knockout mutants,the expression of genes related to salicylic acid(SA)and jasmonate(JA)signal pathway and the accumulation of reactive oxygen species(ROS)in knockout mutants CRISPR-3、CRISPR-7 and wild-type tomato were detected.The results show that SlMAPK6 may be involved in the regulation of SA/JA defense pathway and reactive oxygen pathway to regulate plant disease resistance.