Acquisition and observation of boll stalk abscission zone
Apply ethephon 200 mg · L-1, 400 mg · L-1 and 800 mg · L-1 to cotton petioles with the same growth and at full flowering stage.After 24 hours, the effects of different concentrations of ethrel on the formation of abscission layer of flower stalks were observed (Fig. 1). It can be seen that with the increase of ethrel concentration, the effect of abscission layer formation was more obvious, but no abscission layer was found in water treatment . After treated with 800 mg · L-1 for 24 hours, most of petioles had fallen off. The effect of 200 mg · L-1 treatmentfor 24 hours on abscission formation was not obvious compared with 400 mg · L-1 treatment. Therefore, The cotton petiole treated with 400mg.L-1 ethephon was selected as the experimental material.
After treatment with 400 mg · L-1 ethrel, the change of abscission zone at different time after treatment was observed (Fig. 2). There was no significant change in the abscission zone of boll stalk before 8 h after treatment, but at 12 h, the change of abscission zone could be observed obviously. There were broken marks between boll stalk and stem, and the abscission zone began to form. After 12 h, the broken marks gradually expanded, and finally petiole falled off.
The changes of abscission layer cells were observed by microscope.In the control group (Fig. 3), it can be observed that with the passage of time, there is no obvious morphological difference between the cells in the abscission zone of the boll stalk and the surrounding cells, and the cells are closely arranged, indicating that water treatment has no effect on the abscission cells. However, the treatment group (Fig. 3), around 12h-16h after treatment, the cells became loose, the cell walls of the exfoliated cells began to disintegrate, and obvious tissue fracture marks appeared. As time goes on, the fracture marks became larger and larger until petiole exfoliated. It can also be seen from the figure that the formation of abscission layer starts from the epidermal cells and extends to the vascular bundles of the boll stalk.
Scanning electron microscopy of treatment group showed that the number of Golgi apparatus and the number of stacking layers of Golgi apparatus were significantly more than those in the control group(Fig. 4).It indicated that the number and structure of Golgi apparatus changed during the formation of ionosphere induced by ethrel. The increased number of Golgi apparatus and stacking layers is related to a large number of physiological and biochemical reactions in the process of abscission formation.
Bioinformatics analysis of GhArfGAP gene family in upland cotton
The characteristic domain of GhrfGAP gene was obtained by Pfam protein database. TblastN sequence comparison was carried out in the whole genome of upland cotton by using "DNATOOLS" software. The GhArfGAP gene was compared by cluster multi sequence comparison tool in MEGA6.0 software, and repeated and redundant GhArfGAP gene was deleted, and then the residual base was tested by Pfam and SMART.The amino acid sequence contains the GhArfGAP conserved domain. Finally, 35 candidate genes of GhArfGAP are obtained. According to their location on chromosome, they are named GhGrfGAP1-GhGrfGAP35. The molecular weight and isoelectric point of protein amino acids encoded by the ExPasy proteomics server online tool were predicted in TableS2. The results showed that the length of 35 GhArfGAP proteins was 275-867 amino acid residues, and the longest and shortest was GhArfGAP27. The largest molecular weight is GhArfGAP20, 94.1KD, the smallest is GhGrfGAP17, 30.9KD. The maximum isoelectric point is GhArfGAP20, 9.38, and the smallest is GhArfGAP11, which is 4.96.
MEME online analysis tool was used to analyze the conserved motifs of GhArfGAP protein in upland cotton (Fig. 5). The results show that motif 1 exists in every GhArfGAP, which indicates that motif 1 may be necessary for GhArfGAP to play its function. Some motifs exist among some families, and others do not, such as motif 2 and motif 4, which indicates that these motifs are related to some functions of the family proteins. At the same time, we can find that the same family has the same motif type and order, but different families have differences. At present, the functions of motifs in the GhArfGAP gene family of upland cotton are not clear, but they may be necessary for these proteins to perform their functions.
The distribution of 35 GhArfGAP genes on chromosomes was generated by MapInspect software(Fig. 6).
the results show that 35 GhArfGAP genes were distributed on 16 of 26 chromosomes of upland cotton, and the gene distribution was relatively uniform in at At and Dt genomes of upland cotton. The most GhArfGAPs distributed on chromosome 9 of DT genome, 5 GhArfGAPs were located on it, while only one GhArfGAP gene was found on chromosome 7, 13 and chromosome 10 and 12 of At genome. It is believed that more than 3 genes are contained in the nucleotide units of about 200 kb, which is called gene cluster [17-18], but no gene cluster has been found on the chromosome of upland cotton.
Relative expression analysis of GhArfGAPgene
The phylogenetic tree analysis showed that GhArfGAP13 , GhArfGAP15, GhArfGAP25 and GhArfGAP34 were clustered into one group(Fig.S1),and suggested these genes might be related to the formation of abscission layer. Therefore, qRT- PCR was used to analyze the expression of these genes. The results (Fig.7) showed that GhArfGAP13 was expressed in root, stem and leaf, and the highest expression level was found in leaves, about 2 times higher than that in roots, and the expression level in stem was about 1.5 times of that in root; GhArfGAP15 was the highest expression in stem, but almost no expressing in root; GhArfGAP25 expression was relatively high in stem and leaf, and also almost no expressing in root; the expression level of GhArfGAP34 was the highest in roots, relatively low in leaves, and almost no in stems.These data indicated that GhArfGAP13, GhArfGAP15, GhArfGAP25 and GhArfGAP34 were expressed in all tissues of upland cotton, but the expression levels in different tissues were different, which might be related to the functions of these genes in upland cotton.
Furthermore, the expression of these four genes in different time periods of ethrel induced abscission formation was detected (Fig. 8). In general, the expression levels of GhArfGAP15 and GhArfGAP25 genes increased first and then decreased, and both showed a downward trend at 12 h. The difference was that the peak of GhArfGAP15 expression appeared at 8 h, and the peak of GhArfGAP25 expression appeared at 16 h; the expression levels of GhArfGAP13 showed an alternating rise and fall, and the peak appeared at 4 h; The expression level of GhArfGAP34 increased first, then decreased, and then increased, and the peak appeared at 4 h. except that the expression level of GhArfGAP13 at 0 h was lower than that of the control group, the other expression levels were higher than that of the corresponding control group. There was no significant change in the expression of the four genes in the control group. The changed expression of GhArfGAP13, GhArfGAP15, GhArfGAP25 and GhArfGAP34 indicated that these four genes all played a certain role in the formation of abscission layer.
Structural analysis of GhArfGAP25 protein
According to the results of qRT-PCR, we cloned GhArfGAP25 and expected to study its role in the formation of abscission layer.The structure of the protein expressed by this gene was analyzed.TMHMM tool analysis showed that the protein had no transmembrane domain(Fig. 9A).Online tool signalP 4.1 server predicted that GhArfGAP25 protein does not contain signal peptide sequence,and it is a non secretory protein(Fig. 9B). Studies have shown that ArfGAP protein is involved in intracellular material transport, which indirectly proves that the prediction is reliable[19-20].Analysis of secondary structure of GhArfGAP25 protein by NPS showed(Fig. 9C) that the secondary structure of GhArfGAP25 protein is mainly composed ofα- helix and random coil, in addition to someβ-turn and extended chain. There are 158α- helices, accounting for 33.83% of the total protein; 237 irregular coils, accounting for 50.75% of the total protein; 25β-turns, accounting for 5.35% of the total protein; 47 extended chains, accounting for 10.06% of the total protein.SWISS-MODEL [21-24] was used to predict the tertiary structure of GhArfGAP25 protein. The results are shown in the (Fig. 9D). The main structure wasα-helix, irregular curl and a small amount ofβ-angle.
Analysis of expression in transgenic Arabidopsis plants
In order to further study GhArfGAP25, we transferred GhArfGAP25 into arabidopsis thaliana by agrobacterium transformation and obtained verified by GUS staining(Fig. 10).Fortunately, we got transgenic arabidopsis.
We analyzed the expression of GhArfGAP25 gene in different tissues of wild-type and transgenic arabidopsis by qRT-PCR(Fig. 11) .The results showed that the expression of GhArfGAP25 in roots, stems and leaves of transgenic Arabidopsis was higher than that of wild type. Compared with different tissue parts of cotton, the expression of GhArfGAP25 gene was the lowest in arabidopsis stem and the highest in root, while the expression of GhArfGAP25 gene was the lowest in cotton root and the highest in leaf, indicating that the expression of the same gene was different in different tissue parts of different species.
The function of GhArfGAP25 is located in Golgi apparatus and endoplasmic reticulum
To investigate the functional localization of GhArfGAP25 gene, tobacco was used for subcellular localization(Fig. 12). The results showed that the green fluorescence was the fluorescence emitted by GFP and surrounded the tobacco epidermal cells. The green fluorescence emitted by GhArfGAP25-GFP not only appeared around the tobacco leaf epidermal cell membrane, but also appeared in the cells. Therefore, it was speculated that the function of GhArfGAP25 gene was located in Golgi apparatus and endoplasmic reticulum, which was consistent with previous studies.