Plant materials and growth conditions
All plants used in our experiments were grown in a greenhouse at 25 °C, with a 16 h light/8 h dark cycle.
To examine gene expression at different stages of different maturity varieties, the leaves were collected from CCRI50, CCRI74, G11 and BM at the cotyledon and 1-, 2-, 3-, 4- and 5-leaf stages. Each sample included three repetitions. The growth period of CCRI50 is about 105 days. After Shuxun Yu and others investigation found that it has an obvious character of early maturity . The growth period of CCRI74 is 100 days and it has obvious early maturity . The growth period of G11 is about 123 days, which was identified by Guoxin rural technical service association . BM plants have the characteristics of late emergence, tall and loose, it belongs to late maturing varieties . As Gu described, these varieties are often used for testing and preservation in our laboratory . All samples were quickly frozen in liquid nitrogen and stored at −80°C for subsequent experiments. In addition, two early-maturing varieties, CCRI50 and CCRI74, and two non-early-maturing varieties, G11 and BM, were used for expression pattern analysis. The cotton varieties were planted in the field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences (Anyang, Henan, China). Different tissues, including roots, stems, leaves, buds, fiber, petals and sepals were harvested from CCRI50 .
Gene cloning and sequence analysis
To amplify the CDS and promoter of GhAP1 (Gh_D03G0922), we designed primers using Oligo7. The primers used in this study are listed in Additional file 1: Table S1. The full-length CDS and promoter fragment of GhAP1 was cloned from cDNA and DNA of CCRI50 leaves, respectively. The fragments were inserted into PBI121 vector and transformed into Escherichia coli competent cells (E. coli DH5a) for sequencing. The genomic and coding sequences of GhAP1 were submitted to Gene Structure Display Server online software (GSDS2.0) (http://gsds.cbi.pku.edu.cn/) to predict gene structures. Multiple sequence alignment was conducted using DNAMAN software.
Total RNA was isolated using RNA prep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (Tiangen, China). One microgram of total RNA was prepared for cDNA synthesis in a 20 μl reaction system using a Prime Script™ RT reagent kit with gDNA Eraser (RR047A) (TaKaRa, Japan). The cDNA was diluted 5 times for qRT-PCR. Transcript levels were detected using a 7500 Real-Time PCR system (Applied Biosystems) and UltraSYBR Mixture (Low ROX) (CW2601M) (CWBIO, China). The 10 μl reaction volume contained the following components: 5 μl of 2×UtraSYBR Mixture, 0.2 μl of the PCR forward primer (10 μM), 0.2 μl of the PCR reverse primer (10 μM), 1 μl of cDNA and 3.6 μl of ddH2O. The optimal PCR amplification procedure was used as follows: a pre-denaturation step at 95℃ for 10 min; 40 cycles of 95℃ for 10 s, 60℃ for 30 s and 72℃ for 32 s, a melting curve step at 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s and 60℃ for 15 s. GhActin and AtUBQ10 were used as reference genes. The 2-ΔΔCT method was applied to calculate relative expression levels . All reactions were performed with three technical replicates.
The CDS sequence of GhAP1 without termination code was amplified from CCRI50 cDNA using PCR. GhAP1 was cloned into PBI121-GFP to construct 35-GhAP1::GFP vector and transformed into Agrobacterium tumefaciens strain LBA4404. Approximately 200 μl LBA4404 strains were added into LB liquid medium containing kanamycin, rifampicin and streptomycin, shaking to OD600=1.8-2.0 at 28℃. The culture was centrifuged for 10 min at 4000 rpm. Adjust the OD600=1.5 using the transformation medium. The bacteria were left in the dark at room temperature for 3 h before injected into the tobacco leaves. The fluorescent signal was observed by fluorescence microscope after 2 days (dark culture for 24 h and normal culture for 24 h).
Genetic transformation of Arabidopsis
The CDS sequence of GhAP1 was inserted into PBI121 vector to construct 35S::GhAP1 and transformed into the Agrobacterium tumefaciens strain LBA4404 chemically competent cells. Arabidopsis was infected by dipping flower method . The LBA4404 cells containing 35S::GhAP1 were cultured in the liquid medium and shaken to OD600=1.2-1.6 at 28 ℃. The culture was centrifuged for 5 min at 5000 rpm to remove the supernatant. The bacteria was resuspended using a transformation medium (0.217 g 1/2MS + 5 g sucrose + 20 μl silwettl-77 per 100 ml H2O) to adjust the OD600 = 0.8-1.0. The flower buds of WT plants were immersed in the suspension for 50 s and the transformed plants were placed in the dark condition for 24 h. After one week, the Arabidopsis plants were genetically transformed again. The seeds harvested form the transformed WT plants are T0 generation. The positive plants were screened using the 1/2MS medium containing kanamycin. Phenotypic observation and data statistics were carried out in T3 generation.
For the VIGS assay, approximately 250 bp fragment of GhAP1 gene was amplified from the CCRI50 cDNA and integrated into the pCLCrVA vector to construct pCLCrVA-GhAP1. The pCLCrVA-GhAP1 plasmids were transformed into LBA4404. The LBA4404 strains carrying pCLCrVA-GhAP1, pCLCrVA (negative control) or pCLCrVA-PDS (positive control) were mixed with the strain harboring pCLCrVB (helper vector) (1:1 ratio, OD600 =1.5) and co-injected into two fully expanded cotyledons of TM-1 plants. In the VIGS assay, at least 20 seedlings were used per group. For qRT-PCR detection, samples from at least 6 uniform injected plants were used. The cotton plants were cultivated in a greenhouse at 22℃ with a 16 h light/8 h dark cycle. The experiment was repeated three times. The detailed VIGS procedure was performed as previously described [57, 58].
To identify the interaction proteins of GhAP1, the buds and leaves of CCRI50 from the two-leaf, three-leaf and five-leaf stages were used to construct yeast two-hybrid library. GhAP1 was cloned into pGBKT7 to construct pGBKT7-GhAP1 plasmids and transformed into Y2H yeast receptor cells. Three colonies were randomly selected for self-activation detection. The pGBKT7 empty vector was used as a negative control. The three clones were diluted with water and placed on the defect medium of yeast growth, namely, SD/-Trp, SD/-Trp/-His/-Ade and SD/-Trp/-His/-Ade/X-a-gal.
The mating method was used to screen yeast library on SD/-Trp/-His/-Ade/-Leu plates to identify the proteins that can interact with GhAP1. The Y2H yeast transformants containing the correct pGBKT7-GhAP1 was used to prepare the yeast competent cells, and the library plasmid pGBKT7-cDNA was transferred into it. Screening was conducted on the defective culture plate of SD/-Trp/-His/-Ade/-Leu plates, and the normally growing spots were coated on the culture plate of SD/-Trp/-His/-Ade/-Leu with X-a-Gal for further screening. The spots that could grow normally and turn blue were considered to be positive spots. The positive clones were amplified via PCR and sequenced. Cotton genome blast analysis was performed to obtain the coding genes of potential proteins .
Yeast one-hybrid assay
A suitable 50 bp promoter fragment sequence of GhAP1 was selected and three copies were designed to construct pHis2 recombinant vector. The recombinant plasmid was called pHis2-L. pHis2-L construct, positive control and negative control were applied to the corresponding defect culture plates (pGAD53m+pHIS2 as negative control, pGAD53m+p53HIS as positive control). Different concentrations of 3-AT were added to the defect culture plate to screen the appropriate 3-AT concentration. The CDS sequence of GhLFY was inserted into the pGADT7 vector to construct pGADT7-GhLFY and co-transformed into Y187 yeast cells with pHis2-L constructs. The Y187 yeast cells containing pGADT7-GhLFY and pHis2-L were identified on SD/-Trp/-His/-Leu + 3-AT plates. If it can grow normally, it indicated that the GhLFY gene is the upstream gene of GhAP1.
Dual-luciferase reporter assay
Transient reporter expression was performed in tobacco leaves using a dual-luciferase reporter system . The GhAP1 promoter was amplified and inserted into the pGreenII0800-LUC vector as the reporter plasmids. The ORF of GhLFY was amplified and inserted into the pGreenII62-SK vector as the effecter plasmids. The fusion construct plasmids were transformed into Agrobacterium tumefaciens strain GV3101 (pSoup-p19). The GV3101 (pSoup-p19) cells containing the recombinant plasmids were incubated in LB liquid medium containing 50 mg L−1 kanamycin, gentamycin and rifampin until the OD600 value reached 0.5–0.6. Subsequently, the culture was adjusted to an OD600 value of 0.2 with the filtration buffer (10 mM MgCl2, 10 mM MES and100 μM acetosyringone). The culture suspensions were left for 2 h at room temperature. The effecter and reporter suspensions were mixed in a 1:1 ratio and co-infiltrated into tobacco leaves. After 2 days of infiltration, LUC and REN luciferase activity was detected using a dual-luciferase® reporter assay system (Promega, USA) on a Glomax 20/20 Luminometer (Promega, USA) according to the manufacturer’s instructions. At least six independent replicates were performed.