Genome-wide identification and expression analysis of AcNINV family in pineapple

Abstract: 【Objective】Sucrose plays a crucial role in plant growth and development. The alkaline/neutral invertase (NINV) proteins irreversibly cleave sucrose into fructose and glucose. To date, the genome- wide identification, characterization, and expression profile analysis of the NINV gene families have been reported in many species but not in pineapple.【Methods】Genome sequence and annotation data were acquired from the pineapple genome database through the blastp against protein database and the tblastn against genome databases using the query sequences of Arabidopsis thaliana NINV proteins to identify the potential members of the NINV gene families in pineapple. The physicochemical characteristics, including molecular weight (MW), theoretical isoelectric points (pI), and instability index were obtained using the ExPASy tool. The CDS sequence and protein length were acquired from the pineapple genome database. The synteny analysis between different species and exon/intron structures were visualized using the TBtools. The conserved motifs of the AcNINV gene families were analyzed using the online MEME tool. The phylogenetic tree was constructed using the MEGA 5.0 through the neighbor-joining approach. The total RNA was isolated in accordance with the method described by Wuet al. RT-qPCR was performed using the LightCycler480 Ⅱ System (Roche, Switzerland) and the DyNAmo Flash SYBR Green qPCR kit (Thermo, USA). RT-qPCR was performed in a total volume of 10 μL, which contained 1 μL diluted cDNAs and 5 µL SYBR Green PCR Master Mix, and the primer final concentration was 250 nmol·L^-1 . The sucrose content was determined by HPLC.【Results】In this study, six NINV genes (AcNINV1-6) were identified. The six AcNINV genes were distributed on five different chromosomes (LG1, LG3, LG14, LG17, LG21). There were two genes on LG21: AcNINV5 and AcNINV6, while another AcNINV gene on the other chromosomes. AcNINV proteins had a length of 556-673 aa and MW between 63.03 and 74.80 ku. Only one collinear NINV gene pair between pineapple and A. thaliana, and eight collinear NINV gene pairs between pineapple and Oryza sativa were identified. The number of exons in the AcNINV ranged from 4 to 6, and the number of introns ranged from 3 to 5. The AcNINV1, 2, 4, 5 possessed 4 exons and 3 introns, the AcNINV3 and AcNINV6 had 6 exons and 5 introns. A total of 10 conserved motifs were predicted in the AcNINV genes. Among them, the AcNINV1, 2, 4, 5 contained 10 conserved motifs, but the AcNINV3, 6 only had 9 conserved motifs and lacked motif10. A phylogenetic tree was constructed by combining six AcNINV genes from pineapple with the amino acid sequences of the NINV gene family from three species, including Arabidopsis, rice, and cassava. Six AcNINV genes were clustered into two subfamilies, i.e., α and β. The AcNINV1, 2, 4, 5 belonged to clade β, the AcNINV3 and AcNINV6 belonged to clade α. The promoter cis-elements of the AcNINVs were examined using the Plant-CARE database. There were many cis-elements involved in light (MRE, ATC-motif, Box 4, L-box, GATA-motif, G-box, GT1-motif, TCCC-motif, 3-AF1 binding site, LAMP-element, Sp1, TCT-motif, GA-motif, chs-CMA1a, AT1-motif, AE-box), stress (TC-rich repeats, LTR, ARE, GC-motif) and hormones (ABRE, TGA-element, AuxRR-core, P-box, O2-site, TATCbox, TCA-element, TCA-element, CGTCA-motif, TGACG-motif) in the promoter region. The number of light responsive elements were the largest group, followed by the hormone responsive elements, stress responsive elements. The AcNINV5 had the most cis-elements in light response and hormone response. The AcNINV1 had the most cis-elements in stress response. The sucrose content increased first and then decreased. The sucrose content remained constant on the 20th-40th days after anthesis (DAA). Afterwards, the sucrose content extensively increased from 40 DAA to 80 DAA and reached the highest value on the 80 DAA, which was 126-fold higher than that at 20 DAA. The sucrose content from 80th DAA to 100th DAA extensively decreased but was still 12-fold higher than that on 20th DAA. The expression of the six AcNINV genes was studied using RT- qPCR during fruit development in pineapple. The expression was significant different among the different AcNINV gene members. The expression of the six AcNINV genes were presented three types. The first type was upregulated, such as the AcNINV2, 3, 6. The second type was downregulated first and then upregulate, but the amplitude of change was not significant, the AcNINV1, 5 belonged to this type. The third type was downregulated, and the AcNINV4 gene belonged to this type. The expression level of the AcNINV4 gene remained almost unchanged on the 20th-40th DAA, but it rapidly decreased from the 40th DAA to the 100th DAA. On the 20th DAA, the AcNINV4 gene expression was five times more than that of the 100th DAA. Meanwhile, RT-qPCR was used to clarify the expression profile of the AcNINV gene families in the different tissues. The AcNINV1 had the highest expression in the peduncle and the lowest expression in the core. The expression level of the AcNINV2, AcNINV3 and AcNINV6 were highest in the pericarp and lowest in the flesh. The expression level of the AcNINV4 and AcNINV5 was the highest in the peduncle and lowest in the flesh. Furthermore, the AcNINV2 gene had the highest expression in the peduncle, pericarp, and core. While the AcNINV6 gene had the highest expression level in the flesh.【Conclusion】A total of sixNINV genes (AcNINV1-6) were identified and they were distributed on 5 chromosomes in pineapple. The sucrose content increased with the ripening of pineapple fruit, and the AcNINV4 gene was downregulated during the ripening process of pineapple fruit. Therefore, it was speculated that AcNINV4 may be a hydrolytic enzyme gene that may catalyze the degradation of fruit sucrose.