Uncle Hu
Editor|Uncle Hu
preface
As a class of proteins that can specifically interact with cis-molecules, transcription factors play important functions in plant growth and development, morphological construction, and stress response.
NAC transcription factors are an important class of transcriptional regulatory proteins widely present in plants that regulate the expression of downstream genes by interacting with NACRS promoters.
NAC transcription factors play a key regulatory role in plant growth and development, including seed development, secondary cell wall formation, lateral root development, leaf senescence, fruit ripening and color formation.
Previous work found that NAC transcription factors have important regulatory roles in banana, persimmon, loquat, apple, peach, lychee, kiwifruit, pineapple and other fruits.
Pineapple, also known as "pineapple on the tree", is known as "equatorial apple" because of its luscious and sweet fruit, and is a "equatorial apple" with great development and utilization value.
Jackfruit is a fruit with respiratory change characteristics, which is prone to spoilage after harvest and is difficult to store for a long time, so it is of great significance to improve its postharvest shelf life.
NAC transcription factors are important transcription factors that regulate the ripening process of fruits, but their research in bromeliads has not been reported.
Therefore, we intend to analyze the functions of three NAC family members by RT-PCR, Real-timeqPCR and other methods to clarify the functions of NAC family members in the postharvest aging process of jackfruit, so as to clarify the functions of NAC family members in the postharvest aging process of jackfruit, and lay a foundation for in-depth understanding of jackfruit maturation mechanism and storage technology.
Materials and methods
In this study, the fragrant "Haida No. 2" pineapple was used as the test material. In the blooming period, the same flowering period, well-grown varieties are registered, harvested about 150 days after flowering, collected and sent to the laboratory in time, and varieties with no disease, no insects and no similar maturity are selected, and divided into 3 groups.
Group 1 was controlled by normal growth period; Subsequently, the second group will be taken to 1000mg. L-1 ethylene liquor soak the fruit in water for 2-3 minutes and then remove it to dry; The pulp of group 3 was then fumigated with 0.5 mg.L1-methylcyclopropene for 17 h.
All three varieties were placed at room temperature (22±1) with a relative humidity of 90%. Sampling was performed according to the maturity of each treatment, namely: daily sampling of the control group; The ETH treated group was sampled at 0.5 day intervals, and blood was collected at 0, 4, 8, 10, 12, 14 and 16 days after 1-MCP treatment.
3-5 pieces of each fruit, separate the pulp of jackfruit, then cut it into small pieces of 5mmx5mmx5mm, sample according to the ratio of 1/4, stir well, freeze liquid nitrogen, and store in an ultra-low temperature refrigerator at -80 ° C.
TaKaRacDNAsynthesis technology was used to take pineapple honey as the research object, and through RacDNAsynthesis technology, the cDNA of pineapple honey was expressed in reverse.
The cDNA of jackfruit of "Haida 2" was amplified by PCR using a 50μL polymerase chain reaction system, and the amplification sequence of PCR was: 94°C for 3 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and 72°C for 10 minutes after 35 cycles.
The PCR products were amplified for gel electrophoresis detection, the rubber was recovered, pMD20-T and pMD19-T carriers were connected and E. coli DH5α was converted, and the bacterial solution was sequenced after detection.
The online analysis software ProtParamtool was used to study the basic physicochemical properties of the protein encoded by the bromeliad NAC gene.
Using SignalP4.1 server and TMHMM technology, the transcription factors of NAC genes were studied, and the transcription factors of NAC genes were determined.
Subcellular location was predicted using soft Bayesian protein; The phosphorylation and glycosylation sites of NAC genes were analyzed by NetPhos 3.1 server and DictyOGlyc1.1 server, respectively.
Multiple pairs of NAC were compared using Clustalx and GENEDOC; An evolutionary tree was established in MEGA 7.0 using the nearest neighbor approach.
This project intends to take NAC as the research object, use Pfam34.0 database and MEME technology to conduct in-depth research on the functions of NAC, and identify the functions of NAC.
Using AheGAPDH as a reference, Primer 5.0 was used to design the non-conserved region of the gene, Oligo 6.0 was used to verify it, and finally the qualified primers were submitted to Sanggong Shanghai for synthesis.
Results & Analysis
Using jackfruit pulp cDNA as the template, AheNAC1-for/-rev, AheNAC2-for/-rev and AheNAC3-for/-rev primers were used to obtain target bands consistent with the desired gene fragment length by RT-PCR amplification and electrophoresis, and positive clones were selected and sequenced.
The molecular weight of AheNAC1, 2, 3 genes is 40.16, 38.91, 38.86kD, among the amino acid composition of the three NACs, Ser is the most, its amino acid composition is 9.7%, 10.3%, 10.0%, and the lowest is cysteine.
The proteins expressed by this gene are all hydrophilic, AheNAC1 is an unstable acidic protein, while AHeNAC2 is an unstable basic protein, and AhenAC3 is an unbalanced basic protein.
Through the analysis of three different NAC sites, the results show that the NAC sites of the three NAC sites do not contain signal peptides and transport peptides, and are non-secretory proteins.
By analyzing the transmembrane region sequences of the three genes, it was found that the number of transmembrane regions of the three genes was 0, there was no transmembrane region, and it did not belong to the cell membrane protein.
Thus, we found that NAC is a transcription factor that is normally located inside cells.
Using SoftBerryProtComp9.0 software, the subcellular localization analysis of three NAC proteins was carried out, and the results showed that the overall position of the three NAC proteins on the nucleus was 8.61, 8.86, 9.10, and their overall position on the nucleus was 8.61, 8.86, 9.10, and their distribution on the plasma membrane and vacuole was relatively small.
AheNAC3 protein is also distributed in a small number of extramembrane and peroxisomes, which suggests that NAC proteins are likely to be present in the nucleus, suggesting that AheNAC1, AheNAC2, and AheNAC3 are most likely transcription factors that play a regulatory role in the nucleus.
Analysis of glycosylation modification sites showed that AheNAC1, AheNAC2 and AheNAC3 had 1, 2 and 2, respectively.
The phosphorylation analysis of the three proteins showed that the phosphorylation levels of the three proteins were 46, 38 and 53, and the phosphorylation levels of the three proteins were 46.
The secondary structure of these phosphatases was analyzed by the SOPMA method, and the results showed that AheNAC1, AheNAC2 and AheNAC3 all contained four components: α-helix, extension chain, β-corner and random curling.
These components are predominantly randomly coiled and evenly distributed across the whole chain, followed by α helix, and are concentrated at the N- and C-terminations of the protein, while the β-corner components are low and appear only at the N-terminus of the protein.
Through the modeling of the three-dimensional structure, it was also found that α-helix, extension chain, beta corner and random curl are the folding patterns of 3 proteins, the protein tertiary structure of AheNAC1 and AheNAC2 is similar, while the protein tertiary structure of AheNAC3 is very different from AheNAC1 and AheNAC2.
The Blastn search results showed that AheNAC1 was 84.65% similar to Loquat variety EjNAC3 and 83.9% similar to Peach Tree variety PpNAC4. The similarity of AheNAC2 with jujube zinc NAC2 was 76.96%, and the similarity of lemon NAC4 was 76.75%.
Among them, AheNAC3 has the greatest similarity with MNAC21, and the sequence of AheNAC1, AheNAC2 and AheNAC3 are all conserved regions of NAM at positions 15-139 and 16-142, respectively.
Using Clustalx and GENEDOC, we found that AheNAC has a very conserved N-terminal at the N-terminus, and can be divided into five subdomains: A, B, C, D, and E, of which the C-terminal is the least conserved and is likely to be related to the transcriptional activation of AheNAC.
MEME analysis showed that AheNAC proteins all contained 4 conserved modals, all of which were located in the NAM functional domain at the N-terminus, region A was located in Motif3, region B was located in Motif1 and Motif3, region C was located in Motif1, and Motif2 and Motif4 were located in D and E of the NAC functional domain.
On this basis, we used MEGA7.0 to establish the kinship of 105 NAC proteins associated with jackfruit.
Through the adjacent relationship analysis and previous research of the three bromeliar NACs, we found that the pineapple NACs were NAM, AheNACs and NAPs.
In Arabidopsis, 3 bromeliads NAC1 were compared with homologous sequences of ANAC092, AheNAC2 with ANAC072, and AheNAC3 with ANAC025, and their similarity was greater than 90%.
In order to explore the association between these three AheNAC genes and jackfruit fruit ripening, the expressions of AheNAC1, AheNAC2 and AheNAC3 after natural ripening, ETH ripening and 1-MCP treatment were analyzed using RT-qPCR method.
In the pulp of jackfruit, the relative expression of AheNAC1 will first increase and then decrease with the ripening of fruit during the ripening process.
The day after picking, the relative expression of AheNAC1 reached its highest value and then decreased; The expression of AheNAC2 and AheNAC3 peaked on the first day and gradually decreased after that.
Under the action of ETH and 1-MCP, the expression rates of AheNAC1 and AheNAC3 genes at each stage showed a trend of first increasing and then decreasing, and there was no significant difference between the expression rates at each stage and the normal maturity rate. ETH significantly downregulates the expression of AheNAC2.
discuss
In the previous study, we have cloned the NAC gene from a variety of fruits, and Wu Xiaoqi et al. isolated one NAC protein from peaches, which has 349 amino acids and a total length of 1050 bp.
Yang Feiying et al. cloned two NAC genes from papaya, with a total length of 609bp, 805bp, 202 and 268 amino acids; We have previously obtained 3 NAC family members from bromeliads, encoding 349, 348 and 349 amino acids.
Our previous work showed that the secondary structure of the three bromeliad NAC proteins was mainly irregularly curled, which is consistent with previous reports.
The experimental results of Li Shenglong et al. showed that all NACs are hydrophilic, 23 are acidic, 50 are alkaline, and all NACs are distributed in the nucleus.
Our previous experimental results show that bromeliad NAC is hydrophilic, of which AheNAC1 is an acid, 2 and 3 are bases, and subcells are distributed in the nucleus.
The experimental results of Yang Feiying et al. showed that NAC had no transmembrane structure and no signal polypeptide; Tan Qinliang and other studies found that pineapple NAC protein has obvious transmembrane structure and no signal peptide.
In our previous work, through the analysis of 3 bromeliad NACs, we found that 3 bromeliad NACs had no transmembrane region and no signal polypeptide in the bromeliads, which was very different from the pineapple NAC reported by Tan Qinliang et al., which is likely due to the different roles of the three genes of pineapple NAC in pineapples.
The N-terminal of NAC transcription factor consists of 150-160 amino acids and is divided into five substructures related to its DNA and protein binding, while the C-terminal of NAC transcription factor is a transcriptional activation region, and its structure and length are quite different, which can simultaneously activate or inhibit the expression of target genes.
Through the analysis of this gene sequence, we found a bromeliad NAC protein with an N-terminus containing a conserved region of NAM and 5 subregions A~E; The strong diversity of the C-terminal suggests that it has multiple functions, which is consistent with previous reports.
Previous studies by Tan Qinliang and others showed that the expression of pineapple AcoNAC1 decreased in the early and late ripening stages, indicating that AcoNAC1 showed some negative regulatory function in the ripening stage of pineapples.
The previous work of Wu Xiaoqi et al. found that the expression rate of PpNAC72 gene in peaches was high, and it showed an upward trend during the ripening stage of peaches.
Our previous work showed that AheNAC1 and AheNAC2 have a high affinity with the ANAC092 of positive regulation of plant premature aging, and superexpression ANAC092 can accelerate plant premature aging, and they show a high expression rate in the process of plant premature aging, and superexpression ANAC072 can accelerate plant premature aging.
Bibliography:
[1] Wang Wenqiu, "Study on the regulation mechanism of postharvest maturation of kiwifruit fruit mediated by miR164-NAC"
[2] Qinliang Tan, Yuanbao Cai, Xiangyan Yang, et al., "Bioinformatics and Expression Analysis of Pineapple NAC Transcription Factor Gene AcoNAC1"
[3] Mao Qi, Ye Chunhai, Li Yingzhi, et al. "Jackfruit Research Progress"
[4] Wang Junning, Ma Jing, Feng Feng, et al., "Effect of ethylene on carbohydrate metabolism and enzyme activity of jackfruit fruits after harvest"