Identification and bioinformatics analysis of CR4 receptor-like kinase gene family in grapevine

Abstract:【Objective】Bioinformatics was used to analyze receptor- like kinase CRINKLY4 (CR4) genes of Vitis vinifera L.‘Pinot Noir’and V. amurensis, and to further analyze the expression patterns of the Pinot Noir family members in different tissues and response to abiotic stress and hormones, so as to provide some basis for the functional study and application of CR4.【Methods】Identification of CR4 family members of grapevine was carried out with bioinformatics, combining Arabidopsis thaliana (At-CR4s) related data with Pfam, and SMART software was used to verify gene structure, and then to screen and identify the genomic data of CR4 from two species of grapevine. And the ProtParam tool of ExPASy software was employed to analyze physicochemical properties of CR4 proteins, including the number of amino acids, molecular weight, isoelectric point, aliphatic index and instability index. Then CELLO V2.5 was used to predict the subcellular structure localization. The phylogenetic tree of CR4 of different species was generated with neighbor-joining method using MEGA-X software. Multiple se-quence alignment maps of CR4 members of two grapevine species was made by using DNAMAN. The intron and exon compositions of CR4 members were analyzed by GSDS 2.0. Protein motif was ana-lyzed by MEME database. The cis- acting elements of promoter sequences were predicted by Plant-CARE, which were then visualized with TBtools. And the chromosomal location information of CR4 family of two species of grapevine also was visualized with TBtools. In addition, MCScanX was used to detect the collinearity of different species CR4 (A. thaliana and Pinot Noir, A. thaliana and V. amuren-sis, Pinot Noir and V. amurensis). The expression data of different grapevine tissues were downloaded from the GEO database and different tissues including bud, flesh, flower, leaf, pericarp, petals, pollen, rachis, root, seed, skin, stem, tendril and carpel. Finally, the grapevine cells treated with 100 μmol · L-1ABA, 100 μmol·L^-1 MJ, 100 μmol·L^-1 SA and 1 μmol·L^-1 Flg22, at low temperature (4 ℃), were em-ployed to analyze the expression of VvCR4 with quantitative PCR (qRT-PCR).【Results】In this experi-ment, 5 of VvCR4 and 4 of VaCR4 were identified from two species of Pinot Noir and V. amurensis, re-spectively. All members of VvCR4 and VaCR4 were hydrophobic proteins with isoelectric points < 7and were acidic proteins. The length of coding sequence (CDS) of VvCR4 members ranged from 2304 to 2640 bp, the protein length ranged from 767 to 879 aa, and the molecular weight ranged from 82.54 to 96.35 ku, which were distributed on 5 chromosomes and most of VvCR4s were distributed at both ends of the chromosome, and also the VvCR4 members were mainly located in the plasma, membrane and nucleus. The length of VaCR4s encoded with CDS was between 1842 and 2241 bp, protein length between 613 and 746 aa, molecular weight between 65.95 and 79.20 ku, and VaCR4.1, VaCR4.2 and VaCR4.3 were unevenly located on 3 chromosomes, and only VaCR4.4 was located on Scaffold_1136.The prediction results of subcellular localization showed that VaCR4s were located in the plasma mem-brane, extracellular cells and cytoplasm. Gene structure analysis showed that all of VvCR4s had no in-tron, while the three members of VaCR4 family only had one intron each. In the multiple sequence align-ment, the amino acid sequences of the two species grapevine were similar and highly conserved at the C-terminal, and glycine and cysteine appeared frequently in many absolutely conserved sites. Phyloge- netic analysis divided the CR4 genes of Arabidopsis thaliana, Malus domestica, Solanum lycopersicum,Musa acuminata, Populus trichocarpa, Oryza sativa and two species of grapevine into three subgroups,and the quantity and distribution of conserved motifs in the same subgroup of grapevine were similar. 3pairs of orthologous genes were found between A. thaliana and Pinot Noir, Pinot Noir and V. amurensis,respectively. Only 2 pairs of orthologous genes were observed between A. thaliana and V. amurensis,The Ka/Ks (ratio of non-synonymous substitution rate to synonymous substitution rate) values of all or-thologous genes were less than 1, so VvCR4 and VaCR4 were subjected to strong purification selection after genome differentiation, which may be due to the relatively conserved function of these genes. The results of VvCR4 in different tissues showed that the expression level of them was higher in each devel-opment stage of flower and pericarp than that in other tissues, so the expression of VvCR4 was tissue specific. Cis-acting elements of various plant hormones and stress response, such as MJ, ABA, SA and low temperature, were found in the CR4 promoter region of the two grapevine species. qRT-PCR results showed that the response degree of VvCR4 members under different abiotic stress were different, and the expression trend of VvCR4 members at different times was different. VvCR4 members had strong re-sponse to MJ, ABA and 4 ℃ stress, besides the expressions trend of VvCR4.5 was down-regulated, and the other VvCR4s were up-regulated.【Conclusion】The VvCR4 was more conserved than the VaCR4 in phylogenetic evolution, and the two grapevine species had a range of potential mechanisms in response to abiotic stress, especially closely related to hormonal response, e.g., MJ and ABA. Moreover, the ex-pression of VvCR4 was tissue specific and high in each development stage of flower and pericarp.