Molecular Mechanism Of Variation Of SlPSY1 Cis-acting Element Changing Lycopene Content

Tomato is not only an important economic crop,but also a classic model crop for studying fruit development and plant-pathogen interaction mechanisms.Distant wild tomatoes have better stress resistance and is a natural resource of stress-resistant genes.After domestication,the fruit yield of tomato increased continuously,but the genetic diversity decreased in this process,resulting in a significant decline in stress resistance of cultivated tomatoes.The probability of improving the stress resistance of cultivated tomato by conventional hybrid method is low,and the rapid artificial domestication of wild tomato can be realized by using CRISPR/Cas9 technology.Lycopene is an important nutritional ingredient,which can resist oxidation,fight cancer and improve immunity.The fruits of distant wild tomatoes are mainly green with low lycopene content.Therefore,through the genetic improvement of wild tomatoes with natural stress resistance,the cultivation of materials with high lycopene content is the key to de novo domestication of wild tomatoes.However,the differences in lycopene biosynthesis pathway between wild and cultivated tomatoes are still unclear,which seriously limits the precise "domestication" of wild tomato.PSY1 is a key enzyme gene in lycopene biosynthesis pathway,and its expression affects lycopene synthesis.Fruit ripening regulator RIN can directly regulate the expression of PSY1 and affect lycopene synthesis.The purpose of this study is to explore the molecular mechanism of PSY1 cis-acting element variation in the regulation of PSY1 expression and lycopene content in different germplasm.The main results are as follows:(1)Through the determination of lycopene content in different tomato germplasm,it was found that the lycopene content in distant wild tomatoes was lower than that in currant and cultivated tomatoes.After further verification by multi-tissue RNA-Seq data analysis and q RTPCR,the expression pattern of lycopene synthesis pathway related genes in wild and cultivated tomato was obtained: in cultivated tomato M82,the expression of upstream genes of lycopene synthesis pathway was high,while the expression of downstream genes is low.And these are opposite in distant wild tomato LA0716;(2)Expression analysis revealed that RIN transcription factor was induced to express during fruit ripening of different tomato germplasms.Furthermore,the RIN protein sequences of different tomato germplasms were compared,and the important protein functional regions of RIN were found to be highly conserved.Transient transformation experiment in tobacco showed no difference in the transcipitional activation ability of RIN between M82 and LA0716.Therefore,the difference in PSY1 expression in different tomato germplasm was mainly caused by the variation of cis-acting elements in the promoter region;(3)The important functional regions of PSY1 protein are highly conserved in different tomato germplasms,so by editing the cis-acting elements,it is theoretically possible to activate the expression and function of PSY1 in wild tomato;(4)By analyzing the binding sites of RIN in the promoter region of PSY1,multiple binding regions of RIN were found within 8 Kb upstream of the PSY1 promoter.Based on the comparative genomics analysis and the sequencing of candidate binding motifs,it was found that there were significant sequence differences in motif rbm4,rbm6,rbm9 and rbm10 between cultivated and wild tomatoes.It is speculated that the variation of these regulatory elements led to changes in the regulation of PSY1 expression in wild and cultivated tomatoes;(5)Three slpsy1-rbm4-cr homozygous mutants have been obtained by directional editing of Sl PSY1 cis-acting elements by CRISPR/Cas9 technology.The q RT-PCR and lycopene content of the mutant were determined,and it was found that the expression of Sl PSY1 in the mutant fruits was down-regulated at the color-breaking and full-ripening stage,while the Sl PSY1 expression was up-regulated at the ripening stage,and the lycopene content in the four developmental stages was lower,indicating that rbm4 only played a role in the color-breaking and full-ripening stage of the fruit.In this study,the variation of the PSY1 cis-acting element in cultivated and wild tomatoes leads to the difference in the regulation of PSY1 expression by RIN,which in turn affects the lycopene synthesis pathway.These results provide a theoretical basis for further de novo domestication of wild tomatoes,breeding new germplasm with high lycopene content and maintaining the stress and disease resistance of wild tomatoes.