Transcriptional Profiling Of Jacalin-Related Lectin Genes And Functional Analysis Of TaJRL2.1 In Wheat(Triticum Aestivum)

Plant lectins superfamily includes proteins as storage substances or as functional molecules exhibiting biological properties related to protection of plant tissue against pests and pathogens and to tolerance to abiotic stresses. Jacalin-related lectins (JRLs) are carbohydrate-binding proteins widely present in plants and have one or more domains with sequences similar to the jacalin protein isolated from jackfruit (Artocarpus integrifolia). However, JRLs’ functions are still poorly understood in wheat (Triticum aestivum).67 expressed wheat JRL genes have been identified through bioinformatics analysis previously. To further understand the assignment of these genes, sequence alignments were performed by searching against chromosome-based survey sequences of Chinese Spring using 95% or 99% overlap percent identity cutoff, and 65 genes were assigned to chromosome groups or specific chromosomes. Quantitative RT-PCR revealed that the most TaJRLs have tissue specificity, low basal expression level and can be induced by one or more biotic stresses (Blumeria graminis f. sp. Tritici and Fusarium grminearum inoculation), abiotic stresses (low temperature, salt and drought) and hormonal treatments (SA,JA,and ABA).TaJRL2.1 and TaJRL2.2 shared a high sequence similarity up to 97%, but the expressions among tissues were obviously different. Subcellular localization using onion epidermal cells indicated that both of the two encoded proteins were expressed throughout the cells. The two genes were localized on chromosome 1AL and 1B centromeric region, respectively. Quantitative RT-PCR revealed that the transcript of TaJRL2.1 was abundant in young spikes and could be induced by MeJA and F.grminearum treatment; TaJRL2.2 was mainly expressed in root and was inhibited by MeJA treatment. All these results suggested that functional differentiation might already occurred between TaJRL2.1 and TaJRL2.2.To test whether TaJRL2.1 protein had the general characteristics of lectin proteins, TaJRL2.1 coding sequence has been fused into the prokaryotic expression vector for obtaining the recombinant protein. The purified TaJRL2.1 recombinant protein could agglutinate rabbit erythrocytes like other lectins. However, the agglutination activity was not inhibited by glucose, mannose, N-acetylglucosamine, N-acetylgalactosamine, galactose and lactose, indicating that none of the six carbohydrates can interact with His-TaJRL2.1 specifically.To investigate the function of TaJRL2.1 in wheat scab resistance, we inserted Actin1::TaJRL2.1::Ter expression cassette into stable transformation vector pUCBS which had reconstructed in advance. Three TaJRL2.1 overexpressed transgenic plants were regenerated by gene gun mediated transformation of scab susceptible cultivar PH691 and moderately resistant cultivar Y158 callus and confirmed by PCR selection, GUS staining and QRT-PCR verification. Over-expression of TaJRL2.1 enhanced resistance to F. graminearum in two generations of transgenic plants, but the result of His-TaJRL2.1 anti-fungal evaluation showed that TaJRL2.1 recombinant protein did not obviously inhibit the hyphal growth of F. graminearum.To explore the resistance mechanism of TaJRL2.1, we screened the TaJRL2.1 interacting proteins and analyzed the expression profiling of TaJRL2.1 transgenic plants. Using Yeast Two-Hybrid System, we found no specific self interaction capability of TaJRL2.1. pGBKT7-TaJRL2.1 was used as the "bait" to screen against a cDNA library construced from the Wangshuibai spikes after F. grminearum inoculation,43 proteins were identified as the candidate interacting proteins, including inorganic pyrophosphatase, MATH domain-containing protein, protein phosphatase 2C, ubiquitin, ribulose bisphosphate carboxylase/oxygenase activase B, glycolate oxidase, annexin, pollen-specific protein SF3-like, plastid-lipid-associated protein and some proteins related to resistance such as EREBP, WRKY, NAC, bZIP transcription factors critical in the regulation of plant stress response. Affymetrix GeneChip Wheat Genome Array was used to identify differentially expressed genes in PH691 and two independent PH69l-TaJRL2.1-OX transgenic plants. A total of 246 genes were found to be more than 2 fold change expressed in both transgenic plants, among which 203 genes were upregulated,43 genes were downregulated.The expression tendency of 5 selected differentially expressed genes by experimental quantitative RT-PCR was in agreement with the microarray results, indicating that most of the data from the microarray data were reliable. These differentially expressed genes included defence and stress tolerance related genes (29.3%), and also involved in signaling (8.1%), transcription regulation (12.6%), and genes associated with metabolism and detoxification (9.3%). KEGG annotation showed that lignin biosythesis pathway was activated by the overexpression of TaJRL2.1. Upregulated expression of some resistance genes or genes participating in defense responses were consistent with the phenomenon of enhanced disease resistance in transgenic plants.