| Gibberellin plays an important role in regulating plant growth and development throughout the life cycle of plant.The mutants related to gibberellin biosynthesis and signal transduction have contributed to the breeding of high-yield rice and wheat varieties in the“green revolution”in the last century.Tomato is one of the most important vegetable crops wide-cultivated around the world,and it is also an important model plant used for the study of plant growth and fruit ripening.And the study on the mechanism of tomato growth and development could offer the scientific basis for promoting plant growth and improving fruit yield and quality.In the present,we have known the basic mechanism of gibberellin modulated tomato plant development by the study the function of genes related to gibberellin pathway.However,there are still some genes involved in gibberellin signal pathway are unclarified for their function yet.In this study,the SlPRE2 which was predicted to be involved in tomato fruit development process and gibberellin signaling pathway was selected via a genome-wide gene analysis.The biological function of SlPRE2 was investigated from the molecular level by means of gene transfer,real-time quantitative PCR,microtechnique,and yeast-two hybrid.Meanwhile,the mutagenesis of SlPRO gene based on proTALEN mutant was performed using the CRISPR/Cas9 system,and the biological feature such as the gibberellin sensitivity was characterized for the dwarf mutant generated.The main results were shown as below:?1?Five SlPREs genes which are homologous to AtPREs were identified and named SlPRE1-SlPRE5.Multiple sequence alignment and phylogenetic analysis showed that the SlPREs belonged to the atypical bHLH and had high identity with AtPREs.The expression profile of SlPREs in multiple tissues was predicted based on the RNA-seq data resource and the SlPRE2 showed specific high expression in immature green tomato fruit.The SlPRE2 specific RNAi and overexpression vectors were constructed and the SlPRE2 RNAi and overexpression transgenic plants were generated via an Agrobacterium-mediated transformation.The expression of SlPRE2 in these transgenic lines was investigated by the quantitative PCR.?2?The SlPRE2 RNAi lines showed smaller fruit with thinner pericarp,smaller placenta and seed size compared with the wild-type.The tissue sections analysis showed that SlPRE2 RNAi fruit had smaller mesocarp cell size than the wild-type.With a further quantitative PCR investigation of the cell expansion and division related genes,we found that the expression of SlXTH2 and SlXTH5 which were involved in cell expansion were significantly reduced compared with the wild-type.These data indicated that the altered expression of SlPRE2 could affect tomato fruit development process.?3?Overexpression of SlPRE2 produces tomato with longer internode,rolling leaf with reduced chlorophyll content and smaller stomata aperture.The SlPRE2overexpression lines also had lighter green unripened fruit with lower chlorophyll content and lighter red ripening fruit with lower carotenoid and lycopene content compared with the wild-type.The reduced expression of genes involved in chlorophyll metabolism,chloroplast development and carotenoid metabolism also supported the phenotype in SlPRE2 overexpression lines.The abnormal chloroplast development was revealed in SlPRE2 overexpression fruit pericarp via the transmission electron microscope analysis.Meanwhile,they had longer hypocotyl than wild-type.These data suggested that the overexpression of SlPRE2 could affect plant architecture and pigment metabolism.?4?The expression of SlPRE2 is inducible by exogenous GA3 treatment,which suggested an underlying relationship between GA pathway and the SlPRE2.The altered expression of GA metabolic genes in SlPRE2 RNAi 10DPA fruit revealed that the expression of SlPRE2 could influence GA metabolism process in fruit.Meanwhile,the application of GA3 and PAC to SlPRE2 RNAi and overexpression plants showed that these transgenic plants had inversed sensitivity to treatment,which indicated that the expression of SlPRE2 could raise plant sensitivity to GA and resistance to PAC.Furthermore,SlPRE2 overexpression plant leaves had the changed expression of GA metabolism genes.These data indicated that the SlPRE2 participated in gibberellin responsiveness and regulated the expression of gibberellin metabolism genes.?5?The CRISPR/Cas9 vectors specifically targeting the proTALEN1 and proTALEN7mutation sites were constructed and transferred to tomato via the Agrobacterium-mediated transformation.Thirteen transgenic plants obtained from the tissue culture generated dwarf plant screening and following PCR detection of NPT?selection marker.Two fertile lines numbered 8 and 9 were further analyzed by target site sequencing and PCR product restriction enzyme digestion.These two dwarf lines were all heterozygous with an insertion and a deletion in the target site.The alleles PROGF8 and PROGF9 all had a single base insertion in proTALEN1 and proTALEN7 and restored the reading frame.And alleles PROLF8 and PROLF9 had six bases and one base deletion in proTALEN1 and proTALEN7 respectively and did not restore the reading frame.?6?The predicted molecular models of PROGF8 and PRO showed that the PROGF8protein had a longer disordered sequence between LExLE and TVHYNP motif and had weakened residue interaction between its LExLE and GID1,which likely weaken the stable interaction between PROGF8 and gibberellin receptor GID1.The growth of PROGF8 dwarf plant treated with GA3 and PAC revealed that PROGF8 mutant had significantly reduced sensitivity to GA3.The pollen germination and genetic cross analysis showed that PROGF8 had defective pollen development and reduced pollen activity.The reduced germination activity of 4-month stored seeds and fresh seeds of PROGF8 indicated that PROGF8 repressed tomato seed germination and acted as a semi-dominant allele for fresh seeds germination.In summary,five SlPREs genes were identified in this study,and we focused on the function of SlPRE2 in tomato fruit development,plant architecture,and gibberellin signal pathway.In addition,we obtained a dwarf PROGF8 mutant with significantly reduced GA sensitivity,pollen and seed germination activity via the CRISPR/Cas9system.The study on these two genes laid the foundation for further illuminating the mechanism of gibberellin in regulating tomato growth and development. |
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