Identification And Functional Analysis Of Tomato SlLCD Gene

Tomato is a kind of important economic crop in our country,which produces the most popular fruit and vegetable in the world.And in the meantime,it is also regarded as a useful model plant.The development of the fruits and seeds is an essential step during the growth and development of tomato,and this is also an important agronomic trait affecting the yield,quality,as well as the economic value of tomato.Hydrogen sulfide(H2S)is a newly found gasotranmitter in the last few years,and numerous studies have showed that H2 S having widely spread physiological functions in both animals and plants.In plants,the production of H2 S is mainly from the degradation of Cysteine under the catalysis of enzymes.Among the multiple enzymes found involved in the production of H2 S,L-cysteine desulfhydrase is regarded as an important one.In this study,we cloned the homologous gene of At LCD,which named Sl LCD.And the function of this gene was also explored through biochemical,molecular biological,genetic and the plant physiological means.The main results were as follows:1.The coding domain sequence(cds)of Sl LCD was obtained using PCR.The cds length of Sl LCD was 1365 bp,and the length of its encoded protein was 454 AA.The similarity between Sl LCD and At LCD was 81%,but the similarities between Sl LCD and the other H2 S generating enzymes were extremely low.The transcripts of Sl LCD were widely expressed in different tomato organs and tissues,with a preference in the carpels.2.The prokaryotic expression construct of Sl LCD was obtained and the fusion protein(Sl LCD-His)was purified successfully.The enzymatic analysis result showed that Sl LCD could catalyze the generation of H2 S from L-cysteine.However,compared with the activity of At LCD,the activity of Sl LCD was lower.3.To explore the function of Sl LCD,transgenic plants of Sl LCD-RNAiwere also created.The phenotype of these plants were analyzed.In the Sl LCD-RNAi transgenic plant,we found that in consist with the down-regulation of Sl LCD,the production of H2 S was also reduced.The Sl LCD-RNAi transgenic plants had defects in the formation of seeds,as in the fruits of these transgenic plants,there were no seeds or only a few seeds.The seed vigor of the transgenic seeds were also affected,as the germination rate of the Sl LCD-RNAi seeds were very low,and the down-regulation of Sl LCD was hard to be passed on to the offspring.T0 investigate the molecular mechanism under the phenotype,we analyzed the expression changes of Sl AGL6 in the Sl LCD-RNAi transgenic lines,and we found that Sl AGL6 was down-regulated in the transgenic plants,indicating that Sl LCD might affect the development of seeds in tomato in the same pathway of Sl AGL6.To validate the function of Sl LCD,the gene editing mutants based on the technology of CRISPR/Cas9 were also constructed for this gene.Through sequencing analysis,we found one homozygote and one bimorphic heterozygote.However,only about 50 AA in the N terminus of the Sl LCD protein was affected in the transgenic plants.This kind of mutation might not affect the function of SLLCD,as we notice no phenotype in these gene editing transgenic plants.4.To access whether Sl LCD affected the development of seeds through regulating the production of H2 S,we next performed an in vitro physiological analysis to detect the effect of H2 S and its scavenger(hypotaurine,HT)on the development of tomato seeds.The result showed that H2 S delayed the ripening of fruit,while HT accelerating this process.However,both these two treatments did not affect the formation of tomato seeds,indicating that Sl LCD might affect the formation of tomato seeds through some other pathway rather than by affecting the production of H2 S.In general,in this study,we found that Sl LCD affect the formation of tomato seeds through some unknown pathways which is related to the expression of Sl AGL6,but not through regulating the production of H2 S.This work explored the potential function of Sl LCD,and provided more valuablegene information to the improvement of the tomato germplasm.