There is no trouble in the eighth day of the first month
Edit丨No worries about the eighth day of the first month
The plant cell wall is a thicker, tougher and slightly elastic structure surrounded by the outer membrane of the plant cell, which is usually divided into a primary cell wall and a secondary cell wall, of which the secondary wall is mainly formed by the polymerization of cellulose, hemicellulose and lignin.
Secondary cell walls are thick and hard, providing a physical barrier for plant survival, protecting plants from pathogens, promoting tolerance to abiotic stresses, and enhancing the ability of plant cells to transport water over long distances.
As an important medicinal and edible homologous characteristic crop, tartary buckwheat originated in the southwest of the mainland and belongs to the dicotyledonous plants of the genus Buckwheat of the family Tateaceae.
Tartary buckwheat seeds are not only rich in nutrients, but also rich in bioactive substances, such as flavonoids, phenolic acids, triterpenoids and alkaloids, etc., which have extremely high health care effects on the human body.
Materials and methods of the experiment
Plant materials were tested: thick-shelled tartary buckwheat variety 'Pintary buckwheat 1' and thin-shelled tartary buckwheat variety 'Guiheimi 15', plasmid pMD19-T, pB-WA(V)HS-ccdb-GLosgfp plasmid, Escherichia coli DH5α, Agrobacterium tumefaciens GV3101, etc.
At the sprout stage, 6-leaf stage and full flowering stage of 'Pintartary Buckwheat 1' and 'Guiheimi 15' plants, the roots, stems, leaves and flowers of the plants were placed in labeled 2mL centrifuge tubes, and then quickly put in liquid nitrogen and transferred to -80°C for storage.
When the plants planted in the experimental base bloomed in a large area, the buckwheat flowers that bloomed on the same day were selected, and the bottom of the selected petal sepals was marked with an oily marker, and the seeds at different development stages were taken by the same method and stored at -80 °C for later use.
The total RNA extraction kit was used to extract the total RNA of the roots, stems, leaves, flowers, and grains of 'Pintary buckwheat 1' and 'Guiheimi 15' plants at different development stages, and three biological replicates were set up in the experiment.
The total RNA of the grains of the conventional cultivar Tartary buckwheat and 'Pin tartary buckwheat No. 1' was extracted as a template, and the first-strand cDNA for amplification of the full-length CDS sequence of the gene was synthesized by reverse transcription using the kit.
The total RNA of roots, stems, leaves, flowers, and grains at different developmental stages of 'Pintary buckwheat 1' and 'Guiheimi 15' was used as templates, and reverse transcription was used for qPCR.
FtNAC16的引物FtNAC16-qf和FtNAC16-qr,并设计内参基因Actin7的引物FtActin7-f和FtActin7-r。
The first-strand cDNA obtained by reverse transcription of roots, stems, leaves, and flowers at the sprout and seedling stage and the 6-leaf stage of 'Pin Tartary Buckwheat 1' was used as a template, and qPCR analysis was performed using the kit.
Three biological replicates and three technical replicates were set up, and the flowers of 'Pin tartary buckwheat 1' were used as the control, and the calculation method of 2-ΔΔCt was used. The relative expression of the target gene was calculated, the value of 2-ΔΔCt of flowers was set to 1, and the expression folds of roots, stems and leaves were calculated.
The primer design was the same as that in 1.2.5, and the first-strand cDNA obtained by reverse transcription of the grains of the thick-shelled tartary buckwheat 'Pin tartary buckwheat 1' and the thin-shell tartary buckwheat 'Guihei rice 15' were used as templates.
The kit was used for qPCR analysis, and 3 biological replicates and 3 technical replicates were set up in the experiment, and the grain development of 'Guihei Mi 15' was used as the control for 4 days.
Analysis of experimental results
The cDNA of the grains of 'Pin Tartary Buckwheat 1' was used as a template to amplify the full-length CDS sequence of FtNAC16 to obtain a target band of expected size.
The sequencing results were compared, and it was found that the amplified target band sequence was completely consistent with the reference sequence of the tartary buckwheat genome, and the CDS sequence of FtNAC16 was 1086 bp in length, encoding a total of 361 amino acids.
The analysis of FtNAC16 using BioXM2.6 software showed that the molecular weight of the protein encoded by this gene was 90.54 kD and the theoretical isoelectric point was 4.849.
The data analysis showed that the secondary structure of Ft‐NAC16 protein contained 19.39% α-helix, 61.77% irregular coil, 14.40% β-fold and 4.43% β-turn.
The results showed that the similarity between FtNAC16 and the stress-induced transcription factor NAC1 could reach 100%, which was consistent with the prediction results of the secondary structure.
FtNAC16 was compared with the NAC family protein sequence, and it was found that both FtNAC16 and its homologous protein sequences had a highly conserved NAM domain of about 150 amino acids at the N-terminus, indicating that FtNAC16 was a member of the NAC family.
Ft‐NAC16 was used to construct a phylogenetic tree by regulating NAC transcription factors in secondary wall biosynthesis in other plants.
The results showed that FtNAC16 was clustered with ZmSWN1 and ZmSWN2 in maize, AtNST2 in Arabidopsis, PtrWND2A in poplar, OsSWN1 and OsSWN2 in rice, and was most closely related to AtNST2 and PtrWND2A.
The pBWA(V)HS-FtNAC16-GLosgfp fusion protein vector and pBWA(V)HS-GLosgfp were seamlessly cloned into Agrobacterium tumefaciens GV3101 competent cells by freeze-thaw method.
The obtained positive transformants were enlarged and resuspended, and the leaves of Baccotiana benthamiana were impregnated by injection, and the expression of GFP was observed under a confocal microscope.
The fluorescence signal of the empty vector in the control group was distributed on the cell membrane, cytoplasm and nucleus of tobacco epidermal cells, while the fluorescence signal of pBWA fusion protein was only distributed in the nucleus, so it was concluded that FtNAC16 was localized to the nucleus.
The results showed that FtNAC16 had the same expression pattern at the seedling stage and the 6-leaf stage, that is, its expression levels in each tissue part of the two developmental stages were stems, roots and leaves, and the expression level of FtNAC16 in the flowers of genital tartary buckwheat was also higher.
The expression levels of FtNAC16 in the grains of thick-shelled tartary buckwheat and thin-shelled tartary buckwheat 'Tartary buckwheat 15' were detected by qPCR to detect the differences in the expression levels of FtNAC16 in the grains of tartary buckwheat at different development stages.
The results showed that the expression of FtNAC16 in Tartary buckwheat showed an upward trend and reached the highest at 7 days of grain development, which was significantly different from that of Tartary buckwheat.
Subsequently, the expression of FtNAC16 decreased at 4, 10 and 13 days of grain development, and there was no significant difference in the expression of FtNAC16 between thick-shelled tartary buckwheat and thin-shelled tartary buckwheat.
The expression of one downstream regulatory gene and two structural genes related to secondary wall biosynthesis in the grains of Tartary buckwheat at different developmental stages was detected.
The results showed that the expression of FtMYB103, FtCESA4 and FtCESA8 was the highest at 7 days of grain development, and was significantly positively correlated with the expression of FtNAC16.
In addition, the expressions of FtMYB103 and FtCESA8 increased again on the 13th day of grain development, and the trend was consistent with that of FtNAC16, which suggests that FtNAC16 has the function of regulating downstream secondary wall biosynthesis related regulatory genes and structural genes.
Discussion of experimental results
The shell of tartary buckwheat is thick and tough, which makes it very difficult to remove its husk, and has become a worldwide problem restricting the development of tartary buckwheat processing industry. The regulatory genes of secondary cell wall biosynthesis in tartary buckwheat husks were excavated, and their molecular regulatory mechanisms were analyzed.
It can provide excellent genetic resources for cultivating new varieties of tartary buckwheat with high yield, high quality and strong adaptability through gene editing and other genetic manipulation and molecular marker-assisted breeding in the future.
Studies have shown that NAC transcription factors play an important regulatory role in the biosynthesis of secondary walls in plant cells.
NAC is one of the plant-specific transcription factors, and its protein sequence N-terminus contains a highly conserved DNA-binding domain composed of about 150 amino acid residues.
A NAC transcription factor gene FtNAC16 was cloned from the conventional tartary buckwheat 'Pin tartary buckwheat 1' by RTPCR technology, and the encoded protein sequence was composed of 361 amino acid residues.
Its protein sequence is N-terminus, containing a conserved domain belonging to the NAC transcription factor family, and the FtNAC16 protein localizes to the nucleus.
Studies in the model plant Arabidopsis thaliana and other plants have shown that NAC transcription factors play a decisive regulatory role in cell secondary wall synthesis as a primary switch in cell secondary wall biosynthesis.
Phylogenetic tree analysis showed that FtNAC16 was clustered with other NAC family members involved in the regulation of cell secondary wall synthesis in other plants, and was most closely related to Arabidopsis thaliana AtNST2 and poplar PtrWND2A.
The expression analysis of different tissue sites showed that FtNAC16 was highly expressed in stems, roots and flowers of plant FtNAC16 mainly in the late stage of development and high degree of fibrosis.
The secondary wall synthesis of the object, which regulates the homologous gene of NAC transcription factor NST2, may have a similar function to the homologous gene of Tartary buckwheat to regulate the synthesis of the secondary wall of cells.
The secondary wall of the fruit shell cells of Tartary buckwheat is significantly thicker than that of Tartary buckwheat, which makes the shell thick and tough, and it is extremely difficult to dehull, and the study in Arabidopsis thaliana shows that the secondary wall synthesis regulates the NAC transcription factors AtNST1, AtNST2 and AtNST3.
Through direct or indirect regulation, the secondary wall synthesizes the downstream regulatory gene MYB transcription factor, and the expression of structural genes composed of cellulose and hemicellulose synthesis in the secondary wall, thereby promoting the thickening of the cell secondary wall.
It was found that FtNAC16 was highly expressed in the early developed husks of Tartary buckwheat and Tartary buckwheat, but its expression level in the grains of Tartary buckwheat was much higher than that of Tartary buckwheat.
In addition, Arabidopsis thaliana was regulated by AtNST2 in the expression of the downstream regulatory gene AtMYB103, and the cellulose synthesis structure genes AtCESA4 and AtCESA8.
The expression patterns of its homologous genes FtMYB103, FtCESA4 and FtCESA8 in Tartary buckwheat were highly similar to those of FtNAC16 in Tartary buckwheat and Tartary buckwheat at different developmental stages.
These results suggest that FtNAC16 may regulate the thickening of the secondary wall of tartary buckwheat husk by regulating the expression of swim-regulated genes and structural genes under secondary wall synthesis.
However, the function of the gene FtNAC16 in the biosynthesis of the secondary wall of tartary buckwheat and its detailed molecular regulation mechanism still need further study.
The NAC transcription factor gene FtNAC16 was cloned from 'Pin tartary buckwheat 1', and bioinformatics, subcellular localization and gene expression analysis were performed.
It was found that FtNAC16 belongs to the NAC family and is a nuclear localization transcription factor, which is most closely related to AtNST2, which regulates secondary wall thickening in Arabidopsis.
FtNAC16 was mainly expressed in roots, stems, flowers and early developed grains, and the expression level of FtNAC16 in thick-shelled tartary buckwheat in early grain development was significantly higher than that in thin-shelled tartary buckwheat.
The expression of FtNAC16 in grains was highly positively correlated with the downstream regulatory gene FtMYB103 of secondary wall formation and the structural genes FtCESA4 and FtCESA8 of cellulose synthesis at different developmental stages of Tartary buckwheat and Tartary buckwheat.
These results suggest that FtNAC16 may play an important regulatory role in the biosynthesis of the secondary wall of tartary buckwheat.