The Study Of Zinc Finger Gene Family And Gene Function Analysis

Tomato(Solanum lycopersicum)is one of the most important vegetable crops worldwide,which is also important in the vegetable industry of China.Meanwhile,tomato serves as a model species for fundamental research.Therefore,it is meaningful to reveal the mechanisms underlying plant development and abiotic-stress tolerance in tomato,which is helpful both for basic research and genetic improvement.Previously,a microarry analysis regarding drought stress has been performed on the two drought-tolerant introgression lines(IL)derived from wild species(S.pennellii,LA0716)and their recurrent parent cultivated tomato M82(S.lycopersicum)in our laboratory.In the differentially-expressed gene set,a few Double B-BOX(DBB)zinc finger genes have been identified,which belongs to the BBX gene family.BBX genes of Arabidopsis and rice had been reported,however,this family remains to be exploited in tomato.In the present study,another zinc finger gene named as SlZF3 was also investigated in detail.SIZF3 has been shown to be induced by salt stress in our previous microarray study.Dwarf phenotype and stress tolerancewere observed in SlZF3-overexpressing transgenic lines.In this study,we aimed at a systematic analysis of the BBX zinc finger gene family in tomato,and also elucidation of the functions and mechanisms of SlZF3 in tomato development and plant stress tolerance.The main results were as follows:1.Genome-wide analysis of tomato B-BOX gene familyThe B-BOX(BBX)proteins encode a class of zinc-finger transcription factors possessing one or two B-BOX domains and in some cases an additional CCT(CO,CO-like,and TOC1)domain.They play important roles in regulating plant growth,development and stress response.Nevertheless,no systematic study of BBX genes has been undertaken in tomato(Solanum lycopersicum).In this study,a systematic analysis was carried out on the 29 BBX genes of tomato,including their structures,conserved domains,phylogenetic relationships,subcellular localizations,and promoter cis-regulatory elements.Meanwhile,their tissue expression profiles and expression patterns under various hormones and stress treatments were also investigated in detail.Tomato BBX genes can be divided into five subfamilies,and twelve of them were found to be segmentally duplicated.Real-time quantitative PCR analysis showed that most BBX genes exhibited different temporal and spatial expression patterns.For example,SIBBX24 was constitutively expressed with high abundance in nearly all tissues studied,and eight genes(SIBBX4,5,6,11,16,18,19,and 22)showed relatively higher expression levels in vegetative tissues and non-ripening fruits.The expression of most BBX genes can be induced by drought,polyethylene glycol-6000 or heat stress.Some BBX genes were induced strongly by phytohormones such as abscisic acid,gibberellic acid or ethephon.The majority of tomato BBX proteins was predicted to be located in nuclei,and the transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that all the seven BBX members tested(SlBBX5,7,15,17,20,22 and 24)were localized in nucleus.Our analysis of tomato BBX genes on the genome scale would provide valuable information for future functional characterization of specific genes in this family.2.Mechanism underlying the dwarfism of tomato lines overexpressing SIZF3Previously,SIZF3 has been isolated from to be a salt-inducible gene from a microarray analysis.SlZF3 encoded a ZAT 12-like C2H2-type zinc finger,which possessed an EAR repression domain and an NLS signal peptide.Transient expression assay using Arabidopsis mesophyll protoplasts demonstrated that SlZF3 was localized in nucleus.When SIZF3 was overexpressed,it was found that the hypocotyl was significant shorter in the transgenic lines than in untransformed control.The transgenic lines showed dwarf phenotype with smaller and thicker leaves during the whole life cycle.Paraffin section demonstrated that cell elongation of the internode was inhibited in the transgenic lines.And the dwarf phenotype could be recovered by application of exogenous GA3.Gene expression analysis revealed that the expression of key genes in GA biosynthesis pathway were changed.The transcript level of SlCPS,SlKS,SlKO,SlKAO and SlGA20oxs(2,3 and 4)was significantly down-regulated,while it was upregulated for GA2oxs genes(2,3 and 5).These changes migh result in reduced level of gebberellin in the transgenic plants,which ultimately leads to the dwarf phenotype.3.Regulatory mechanism of SlZF3 in AsA accumulation and stress toleranceAscorbic acid(AsA)is an important antioxidant and plays key roles in scavenging reactive oxygen species(ROS),thus to enhance plant tolerance to various stresses.Although AsA biosynthesis and catabolism has been extensively investigated,the information regarding AsA regulation is still limited.In this study,we characterized zinc finger protein as a novel regulator of AsA synthesis that targets GDP-mannose pyrophosphorylase.SIZF3 was induced rapidly by NaCl and drought stress.Protein interaction analysis demonstrated that SlZF3 acted as a competitor against VTC1 to bind the COP9 subunit CSN5B and can positively regulate AsA biosynthesis in both Arabidopsis and tomato.The EAR-motif of SlZF3 was not necessary for the interaction between SlZF3 and CSN5B in yeast and plant cells,however,it was important for the stability of SlZF3.As a result,overexpression of SIZF3 increased AsA level significantly in Arabidopsis and tomato.Consequently,salt and drought tolerance of the plants was improved via the enhanced ROS-scavenging pathway mediated by AsA.These findings may provide an new avenue for genetically improve the AsA content and plant stress tolerance.