葡萄CR4类受体激酶基因家族的鉴定及表达分析

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

【目的】对葡萄属欧亚种群黑比诺葡萄(Vitis vinifera L.‘Pinot Noir’)和东亚种群山葡萄(V.amurensis)基因组中CRINKLY4类受体激酶(CR4)家族成员进行生物信息学分析及VvCR4在不同组织及胁迫下的表达分析,为CR4基因功能研究和应用提供理论依据。【方法】运用生物信息学工具对2种葡萄基因组中CR4基因家族成员进行鉴定及分析,利用基因芯片数据分析欧亚种葡萄该家族在不同组织中的表达情况,并通过qRT-PCR分析黑比诺葡萄CR4成员在不同非生物胁迫下的表达模式。【结果】共鉴定出5个VvCR4成员和4个VaCR4成员,分为3个亚组,同一种葡萄CR4基因结构高度保守,所有成员启动子区均存在多种植物激素和逆境胁迫相关的响应元件。VvCR4在不同时期的花与果实发育中表达量相对较高;qRT-PCR表明VvCR4成员对MJ、SA、ABA、4℃的胁迫响应明显,大多为上调表达且出现表达高峰的时间点因基因和胁迫的差异而不同。【结论】VvCR4成员在MJ处理下呈显著正调控模式,可能与MJ信号转导密切相关,在SA、ABA、Flg22与低温胁迫诱导下,VvCR4.1、VvCR4.2、VvCR4.3、VvCR4.4都有不同程度的响应,VvCR4.5响应不明显。

【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 CR4family members of grapevine was carried out with bioinformatics, combining Arabidopsis thaliana(AtCR4s) 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 sequence 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 analyzed by MEME database. The cis-acting elements of promoter sequences were predicted by PlantCARE, which were then visualized with TBtools. And the chromosomal location information of CR4family 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. amurensis, 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-1MJ, 100 μmol · L-1SA and 1 μmol · L-1Flg22, at low temperature(4 ℃), were employed to analyze the expression of VvCR4 with quantitative PCR(qRT-PCR).【Results】In this experiment, 5 of VvCR4 and 4 of VaCR4 were identified from two species of Pinot Noir and V. amurensis, respectively. 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 2304to 2640 bp, the protein length ranged from 767 to 879 aa, and the molecular weight ranged from 82.54to 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 membrane, extracellular cells and cytoplasm. Gene structure analysis showed that all of VvCR4s had no intron, while the three members of VaCR4 family only had one intron each. In the multiple sequence alignment, 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. Phylogenetic 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 orthologous 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 development 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 response 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 expression of VvCR4 was tissue specific and high in each development stage of flower and pericarp.