Cloning and expression analysis of CpEXPA2 gene related to softening of papaya fruit

Abstract:【Objective】Expansins are a special group of plant cell wall proteins with no catalytic activity and are associated with several processes during plant growth and development. In particular, expansins play a role in fruit softening. Since a fruit-specific expansin gene Le EXPA1 was found from tomato fruit, ripening-related expansins had been identified in tomato, strawberry, banana, and a range of other fruit species. Papaya (Carica papaya L.) is an important fruit crop with high economic and nutritional value.‘Da Qing No. 10'is a red-fleshed papaya. The red-fleshed papaya softens faster than the yellow-fleshed papaya and has a shorter shelf life. However, some consumers prefer red-fleshed papaya. Therefore, it is of great importance to explore the roles of expansins in fruit softening of papaya. An expansin gene was cloned and its function and expression patterns in different tissues and different fruit development and ripening stages were analyzed in this study.【Methods】Nineteen expansin candidate genes were selected according to gene annotation.in the papaya transcriptome data.Then the heat map analysis was carried out using the relative expression of gene FPKM value and a gene related to the softening of papaya fruit was found. Total RNA was extracted from papaya pulp using General RNA the kit R1051 of Guangzhou Dongsheng Biotechnology Co. Ltd. Referring to BD Biosciences Clontech, Super SMARTTMPCR c DNA Synthesis Kit manual, double-stranded c DNA was synthesized by using papaya RNA (200 ng) as a template. And amplification was carried out as follows: initial denaturation at 94 ℃for 3 min, followed by 35 cycles of 94 ℃ (30 s) , 56.4 ℃ (30 s) , and 72 ℃ (2 min) , and then a final extension at 72 ℃ for 7 min. The PCR products were gel purified. The purified products were cloned into a p MD18-T vector (Ta Ka Ra) and transformed into E. coli DH5α competent cells, then the bacteria were spread onto LB Plates and incubated at 37 ℃ overnight. The positive clones were selected for PCR identification and sequenced by Shanghai Boshang Biotechnology Co. Ltd. Protein translation and homologous sequence analysis were taken by using DNAMAN software. Protein homology search was taken using NCBI Blastp. The NJ (neighbor-joining) phylogenetic tree was constructed by MEGA 5.0 software. Basic physicochemical properties of the amino acid were predicted by using online Prot Param.Signal peptide and transmembrane structure of amino acid were predicted using Signal P 4.1 Server and TMHMM online. The secondary structure of the amino acid sequence was analyzed using SOPM, and the tertiary structure of the amino acid sequence was analyzed using SWISS-MODEL online. The analysis of subcellular localization was performed using Cell-PLoc 2.0 online. Finally, the Real-Time quantitative PCR was performed using a CFX manager (Bio-Rad, USA) and the SYBR Premix Ex TaqTMkit (Takala, Japan) . RT-q PCR were used to analyze the quantitative expressions of Cp EXPA2 in different tissues and the peels and the fleshes of different maturity fruits of papaya. Amplification conditions were95 ℃ for 30 s, followed by 40 cycles of amplification (95 ℃ for 5 s, 60 ℃ for 30 s) and plate reading after each cycle. Relative expressions of all replicates of each sample were calculated using the 2-ΔΔCt method. Then, Excle 2003 software was used to analyze the results of the experiment.【Results】Cp EXPA2 gene contained a 780 bp open reading frame, which encoded 259 amino acids. The sequence structure analysis revealed that Cp EXPA2 gene contained a typical expansin structure features, belonging toα-expansin subfamily. The sequence alignment showed that the similarity between Cp EXPA2 and mountain papaya (ABF48653.1) , tomato (AAC64201.1) , strawberry (AAF21101.1) and Arabidopsis thaliana (AAB38073.1) homologues exhibited 66.15%, 64.86%, 66.80% and 65% identities in amino acid sequences, respectively. Meanwhile, the phylogenetic analysis illustrated that Cp EXPA2 was mostly closed to At EXPA6 (U30480) and At EXPA16 (NM＿115407) in Arabidopsis thaliana. The Prot Param tool predicted that Cp EXPA2 had a molecular weight of 28095.15 Ku, a theoretical p I of 9.53, and a formula of C1 255 H1 913 N351 O352 S17. Analysis of subcellular localization showed that Cp EXPA2 gene was located in cell wall in papaya. The secondary structure of Cp EXPA2 amino acids possessed α-helical (15.83%) , β-rotation (12.74%) , extension chains (25.87%) and irregular curls (45.56%) . The main component of the secondary structure of Cp EXPA2 protein was irregular curls. RT-q PCR revealed that the expression of Cp EXPA2 in different tissues exhibited different patterns, the expression level of Cp EXPA2 in fruit was higher than that in the leaf, flower and stem. In the fruits with different maturity, the expression of Cp EXPA2 in the peel was significantly higher than that in the pulp. The expression in thegreen period was very low, and the expression in the period of color break and the half yellow period increased sharply, and the expression in the full yellow period decreased. The fruit firmness of papaya decreased gradually with the maturity of fruit, and decreased rapidly in the color break stage and the fruit softened rapidly. As the firmness gradually decreased, papaya fruit softened and the expression of CpEXPA2 gene decreased relatively.【Conclusion】Cp EXPA2 gene may be associated with the ripening and softening of papaya fruit, and it may be a suitable candidate gene to manipulate fruit ripening in order to increase the shelf life of papaya. The study of this gene provides a new idea for the molecular regulation of papaya fruit softening.