The LIM protein family of wheat is characterized by the existence of one or several double zinc finger domains, namely LIM domain, which is a protein interaction domain. When there is only one LIM conserved domain, the family genes usually contain one or two UIMS or one DUF3633 conserved domain [9, 10]. At present, LIM gene family has been identified in many species such as Arabidopsis thaliana and rice[8]. In this study, a total of 29 members of TaLIM gene were identified from the whole genome database of wheat, all of which were higher than the LIM family members in the above species. This may be because wheat is AABBDD (allotetraploid) and has three partially homologous genomes, so it has more homologous genes.
In this study, through the analysis of the physical and chemical properties and cis-acting elements of the whole genome of wheat, 29 LIM gene family members were identified. Referring to the names of LIM family members of rice, Arabidopsis and corn, 29 LIM members of wheat were named TaLIM01 ཞTaLIM29, respectively. The length and the number of amino acid residues of members of TaLIM protein family are quite different, which is similar to that of radish and other animals and plants [12]. Gene structure analysis showed that the number of exons in subfamily ⅰ and ⅲ was the same, while the number of exons in subfamily ⅱ was quite different among genes.
A total of 80 cis-acting elements were identified from the promoter sequence of 2000 bp upstream of TaLIMs gene, of which 47 were found in most members, including light, anaerobic induction, auxin, gibberellin, low temperature and other responsive elements, among which the light-responsive cis-acting elements were the most, which was consistent with the characteristics of cis-acting elements of other wheat families, which also implied that LIM gene might play a role in the process of light induction in some periods, such as the embryogenesis of isolated microspores [12].
29 TaLIMs family members are unevenly distributed on 18 chromosomes, and different members have homology, which indicates that LIM gene has been replicated or recombined during the evolution of wheat. In the process of plant gene family evolution, replication events play an extremely important role in the amplification of family members [21]. Gene replication in plants can be divided into two types: fragment repeat events and tandem repeat events[22, 23]. In this study, no tandem repeats were found, with a total of 89.6% of genes having fragment repeats, which indicates that one of the important reasons for the amplification of TaLIMs family members is the occurrence of fragment duplication, which is similar to that of corn, rice and other species [24]. By calculating the Ka/Ks values, it was found that the Ka values of all genes ranged from 0.002 to 0.149, the Ks values ranged from 0.040 to 0.923, and the Ka/Ks values of all genes were less than 1, which proved that the homologous genes in this family were influenced by the existence of purification selection in the evolution process [25].
Previous studies have shown that the subcellular localization of LIM protein in plants can be found in both nucleus and cytoplasm. LIM protein located in nucleus is mainly involved in gene transcription regulation, while LIM protein located in cytoplasm is involved in cytoskeleton formation as a regulatory actin [26, 27]. The protein families of tobacco, sunflower and Arabidopsis show simultaneous localization of nucleoplasm [28]. In this study, the subcellular localization of proteins encoded by 29 genes of TalimsFamily was predicted, and it was found that 26 members except TaLIM09, TaLIM10 and TaLIM15 were all distributed in the nucleoplasm and mainly expressed in the nucleus. TaLIM09 was the only gene with distribution in vesicles and plasma membranes, and TaLIM26 and TaLIM28 were distributed in cytoskeleton. It is predicted that it is mainly involved in the formation of cytoskeleton actin and plays an important role in cytoplasmic flow, which indicates that this family gene may regulate plant development and defend against external stress by regulating transcription level and other developmental processes. At the same time, some studies have shown that the localization of LIM proteins in different subcellulars may mean that their functions are also quite different, but this conclusion has not been confirmed yet [7].
The early results showed that gene expression patterns with two LIM domains can be divided into two subfamilies according to the specificity of tissue or organ expression. WLIM subfamily members can be expressed in many tissues of plants, while PLIM subfamily members have pollen specificity or pollen preferential expression patterns [7]. LIM protein is divided into four subfamilies in grape, and two subfamilies in tomato, Arabidopsis and rice [8, 13, 20]. In this study, TaLIMs protein was divided into three subfamilies. It can be seen that in different species, the number of subfamilies is different, and the classification of subfamilies is less, which indicates that LIM protein family is highly conservative in all species. According to the qRT-PCR results, TaLIMs family was expressed in different degrees in all tissues. The first subfamily TaLIM02, TaLIM03 and TaLIM08 have significant specific expression in pollen, which indicates that TaLIMs may be involved in the development of different organs, indicating that this family gene may control cell proliferation and pollen growth; Members of the second subfamily are expressed in many tissues of plants, and the results of phylogenetic analysis show that this is consistent with the research results in Arabidopsis thaliana [8]; The third subfamily is a member with close homologous relationship, and has different expressions in the tissues. Compared with Arabidopsis thaliana, the genetic relationship between wheat LIM protein and rice LIM protein is closer. And most members of each subfamily contain the same conserved domain. It can be divided into two cases: the sequence containing only one zinc finger structure and the sequence containing two consecutive zinc finger structures, so it is inferred that they have similar functions. LIM family genes not only play an important role in lignin biosynthesis, pollen tube growth, plant tissue and organ growth and plant morphogenesis, but also play an important role in ABA, PEG, cold and salt stress [13, 29–31]. The results have confirmed that PtWLIM1a, PtWLIM1b and PtβLIM1a genes in poplar may be involved in general cell functions, such as xylem development and salt tolerance. PtGLIM1a may be highly expressed in xylem tissues, such as leaves and other organs with vascular tissues, and AtWLIM1 and OsWLIM1 are on the same evolutionary branch as PtβLIM1a, PtWLIM1b and PtGLIM1a, and their parental relationship is close [8, 13]. In this study, the genes of wheat subfamily ⅱ are in the same evolutionary branch as AtWLIM1 and OsWLIM1. According to qRT-PCR analysis, TaLIM16 and TaLIM27 of wheat subfamily Ⅱ are expressed in different situations after being induced by ABA, PEG, cold and salt. Therefore, it is speculated that TaLIM16 and TaLIM27 are involved in the regulation of salt stress and other important cellular functions. TaLIM20 and TaLIM26 were significantly up-regulated after ABA treatment, suggesting that TaLIM20 and TaLIM26 may be involved in xylem development. Under different stress conditions, TaLIMs showed different expression patterns. Six genes were upregulated after ABA treatment and salt stress, and two genes were upregulated after drought stress. TaLIM02 was significantly upregulated compared with the control under four stress conditions. TaLIM07 and TaLIM16 were significantly down-regulated compared with the control under four stresses. Therefore, it is speculated that TaLIMs may be involved in regulating the stress response process of wheat, such as low temperature and drought, and play an important role in plant response to abiotic stress.