Cold tolerance of different walnut varieties

Abstract:【Objective】Flavonol biosynthesis pathway is an important component of the metabolic pathways and catalyzes the formation of flavonol. Flavonol synthase (FLS) plays an important role in flavonol synthesis and improves their resistance to drought and salt. The function of flavonol synthase (FLS) is of great significance to the fruit quality in apple. Therefore, the flavonol synthase gene was cloned from‘Gala'apple (Malus × domestica Borkh.) . and the expression and catalytic activity were studied.【Methods】The full-length of the gene Md FLS1 was cloned by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) . The sequences were obtained and analyzed by various bioinformatics methods. The Protparam online software was applied to analyze the nucleotides, amino acid physical and chemical characteristics, and protein affinity/hydrophobicity analysis. The protein conserved domain was predicted by the website (https://www.ncbi.nlm.nih.gov/) . The protein sec-ondary structure and tertiary structure analysis of Md FLS1 was completed by the SOPMA online software and SWISS MODEL online software, respectively. The protein signal peptide and transmembrane domain of Md FLS1 were predicted by Target P1.1 and TMHMM software, . 18 plant FLS genes nucleotide sequences were found in the Gen Bank database of NCBI. The phylogenetic tree of Md FLS1 protein and was constructed homologous sequence of different species by MEGA 7 software. The expression pattern of Md FLS1 was studied by q RT-PCR. Md FLS1 protein was obtained through prokaryotes by IPTG inducing, and its catalytic activity was determined by HPLC.【Results】Sequence analysis showed that the length of Md FLS1 gene contained 1 014 bp bases and encoded 337 amino acids. The molecular mass of Md FLS1 protein was 38.12 ku. And theoretical p I was 5.48. The glutamic acid (Glu) and valine acid (Val) were rich in amino acids. The unstable coefficient of the Md FLS1 protein was 38.94 in apple, and it belonged to the stable protein. The value of its grand average of hydropathicity (GRAVY) was-0.506, indicating that it was a hydrophilic protein. The Md FLS1 gene was located on chromosome8 and belonged to the family of alpha ketopamyl diacid dependent double adding enzyme, containing a heme oxygenase domain in the N-terminal. The tenth arginine had the lowest score-3.113 and was the strongest hydrophilic. The 176 th phenylalanine had the highest score 1.825 and was the strongest hydrophobic. The whole polypeptide chain was shown to be hydrophilic. SOPMA online software analysis showed that the two second structure of Md FLS1 protein was composed of 29.38% alpha helix, 24.33%elongation chain, 12.46% beta corner and 33.83% irregular crimp. Therefore, alpha helix and irregular crimp were the largest structural elements in the overall structure of protein Md FLS1, while the extended chain and the beta angle were relatively dispersed in the whole polypeptide chain. SWISS MODEL indicated that the protein Md FLS1 was mainly composed of alpha helix and irregular curl. The amino acid sequence analysis of Md FLS1 by Target P1.1 online tool showed that it did not contain signal peptide and transport peptide. The results of TMHMM online software analysis indicated that the whole Md FLS1 protein in apple was located outside the membrane, and there was no transmembrane domain.The online software predicted that the apple Md FLS1 was located in the cytoplasm. The amino acid sequence and phylogenetic tree comparison showed that FLS had a highly conserved amino acid sequence in different species. The results indicated that the protein Md FLS1 could be more closely related to sweet cherry, strawberry, peach, rose, and the similarity rates were 99.11%, 79.06%, 77.88%, and78.17%, respectively. The genetic relationship of Md FLS1 was farther to Arabidopsis, Camellia assamica, Acacia confusa Merr., and the similarly rates were less than 60%. Quantitative real-time PCR analysis demonstrated that the gene Md FLS1 was mainly expressed in leaves. Promoter cis-element prediction of apple Md FLS1 showed that there were many hormone responsive regulatory elements, such as ABA response element ABRE, salicylic acid responsive element TCA-element and gibberellin responsive element P-box; environmental factor response elements, such as light response elements of Box 4 and G-box; circadian rhythm regulation element circadian; abiotic stress response elements, such as drought responsive element HSE response element MBS and heat in the promoter of Md FLS1. These results indicated that Md FLS1 could respond to various environmental stress such as drought, salt stress, light, circadian rhythm and plant hormones, and affect plant growth and development. The protein MdFLS1 was obtained by prokaryotic induction. And the catalytic activity of Md FLS1 was verified by high performance liquid chromatography.【Conclusion】The biological information of the apple flavonol synthetase was preliminarily understood. The Md FLS1 might be involved in various hormonal signal responses and abiotic/biological stresses. The Md FLS1 in apple would respond to abiotic stressessuch as ABA, low temperature, high salinity and drought. The Md FLS1 protein had catalytic activity in vitro. The genetic transformation of the apple Md FLS1 gene into different plant materials is on the way.