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Calcineurin B subunit protein is one of the calcium ion receptors in plants, which can specifically bind to CBL-interacting protein kinase, and the CBL-CIPK complex formed plays a key role in plant growth and development and response to adversity stress.
A total of 13 PsCBLs and 20 PsCIPKs were identified in the whole genome of peas, and they were all distributed in the remaining 6 chromosomes except chromosome 3.
Except for PsCBL5, which has a sequence length of 416aa, the rest of PsCBLs are short peptides composed of more than 200 amino acids, and the protein length of PsCIPKs is about 450aa.
Except for PsCIPK18, the promoter regions of other PsCIPKs and PsCBLs contained cis-acting elements related to hormones and abiotic stress, and the most cis-acting elements in response to abscisic acid signaling were the most.
The role of calcium ions in plant genes
Calcium plays an important role in plant stress signal transduction.
When plants are subjected to abiotic stresses such as drought, high temperature, high salt and low temperature, the concentration of calcium ions in cells will change, and the calcium ion signal generated will be received by calcium ion receptors, and then cause a series of physiological and biochemical reactions.
CBL protein is one of the calcium receptors in plants, and it usually contains 4 EF hand domains and one PFPF domain, which binds to calcium ions in plants.
The C-terminal PFPF domain can bind to the NAF and FISL domains on serine and threonine protein kinases, and CBL protein can only transduce calcium ion signals after interacting with serine and threonine protein kinases to form serine and threonine phosphatase complexes, and then respond to abiotic stress by regulating the expression of downstream target genes.
CIPK is a unique class of protein kinases containing serine and threonine functional domains in plants, and its N-terminus contains a conserved Pkinase domain, which mainly plays a catalytic activation role.
The C-terminal contains NAF, FISL domain and PPI domain, which are used to bind CBL protein, and the PPI domain can also be bound by phosphatase, which can participate in hormone signal transduction pathways and regulate cell life activities.
At present, CBL and CIPK proteins have been identified and analyzed in plants such as Arabidopsis, rice and rape. The results showed that each CBL protein could interact with multiple CIPK proteins, and one CIPK protein could also interact with multiple CBL proteins.
The CBL-CIPK protein complex formed by their interaction can respond to abiotic stresses such as high salt, drought, low temperature, abscisic acid, low potassium and high pH.
The results showed that when plants were subjected to high salinity and drought stress, the high concentration of sodium ions in the cell could induce the increase of intracellular calcium concentration and the decrease of potassium concentration.
The AtCBL10-AtCIPK24 and SOS2 complexes can maintain the intracellular sodium ion concentration through vacuoles after receiving the calcium ion signal and reduce the influx of sodium ions.
In addition, when plants are subjected to high salinity and drought stress, the influx of a large number of sodium ions will cause the outflow of a large number of potassium ions in the cell.
After receiving the calcium ion signal, the AtCBL1 and 9-AtCIPK23 complexes can increase the influx of potassium ions and increase the concentration of potassium ions in the cell by regulating the potassium ion channel, thereby maintaining the life activities of plant cells.
In this study, the genome-wide identification and analysis of PsCBLs and PsCIPKs in peas in response to stress stress have not been reported in peas, and their transcriptional levels under drought stress were analyzed.
This study will help to study the molecular mechanism of PsCBLs and PsCIPKs in response to drought and other stress stresses in pea, and provide the possibility for screening pea stress resistance genes for molecular marker-assisted selection breeding.
Identification of pea CBL and CIPK gene families
A total of 13 CBL genes and 20 CIPK genes were identified from the pea genome, and they were named PsCBL1~PsCBL13 and PsCIPK1~PsCIPK20 according to their chromosomal order.
They were distributed on chromosomes 1, 2, 4, 5, 6 and 7 of peas, but PsCBLs and PsCIPKs were not distributed on chromosome 3.
In PsCBLs, the protein length range is between 213aa~416aa, the molecular weight range is 24437.04Da~44549.15Da, and the isoelectric point range is 4.5~4.93.
By screening for criteria greater than 80% for both gene alignment coverage and amino acid identity, a duplicate gene pair was identified, which may have resulted from tandem copies.
In addition, collinearity analysis showed that PsCBL and PsCIPK had genes colinear with each other on different chromosomes, such as PsCBL1 and PsCBL4, PsCBL2 and PsCBL10, PsCBL3 and PsCBL6, PsCIPK3 and PsCIPK19.
These results suggest that the amplification of the CBL gene family and the CIPK gene family in peas may be due to tandem duplications of the whole genome or small-scale genomes.
The aim of this study was to study the evolutionary relationship between pea and other species of CBL and CIPK gene family members.
In this study, the maximum likelihood method in MEGA software was used to analyze the protein sequences of 10 AtCBLs in Arabidopsis, 10 OsCBLs in rice, and 13 PsCBLs identified in peas.
The results showed that AtCBLs, OsCBLs and PsCBLs could be classified into four groups, and CBL in Arabidopsis, rice and pea was distributed in the other three groups, except for AtCBL5 and PsCBL13 in GroupI.
The collinear PsCBL3 and PsCBL6 are distributed in Group III., and the collinear PsCBL1 and PsCBL4, PsCBL2 and PsCBL10 are distributed in Group IV.
At the same time, the phylogenetic phylogenetic tree was constructed using the same method using the identified protein sequences of 20 PsCIPKs, the reported protein sequences of 26 Arabidopsis AtCIPKs and 33 rice OsCIPKs.
The results showed that AtCIPKs, OsCIPKs and PsCIPKs proteins could also be classified into four groups, and CIPKs in Arabidopsis, rice and pea were distributed in all four groups, among which the CIPK proteins in Group III and Group IV were the most abundant.
However, the CIPK protein in Group II. was the least, and only collinearity PsCIPK3 and PsCIPK19 were distributed in peas.
The phylogenetic tree analysis of PsCBLs in pea showed that PsCBL3, PsCBL5 and PsCBL6 were closely related and clustered into one class, while the rest of the PsCBLs were clustered into one class.
The motif prediction analysis showed that except for PsCBL5 without motif5, the rest of the PsCBLs contained 5 conservative motifs. Conserved domain analysis showed that all 13 PsCBLs contained EF-hand_7 conserved domains.
Analysis of expression patterns of CBL and CIPK genes in pea
In order to study the constitutive expression patterns of PsCBLs and PsCIPKs in pea, TBtools software was used to visualize the expression levels of PsCBLs and PsCIPKs in different tissue parts of "Zhongpea 6".
The results showed that the expression levels of PsCBL2, 4 and 10 were higher in the six tissues of PsCBLs, and all of them were the highest in pea whiskers, while PsCBL3 was specifically highly expressed in pea buds and flowers.
In general, most of the PsCBLs and PsCIPKs genes were specifically expressed in pea whiskers, flower buds and flowers, indicating that these genes may play an important role in the growth and reproductive development of pea.
In order to study the expression patterns of PsCBL-CIPKs under drought stress, the expression patterns of PsCBL-CIPKs in seedlings and mature leaves of pea drought-tolerant germplasm and drought-sensitive germplasm were used as materials.
The results showed that the expression levels of PsCBL2, 4, 10, PsCIPK5 and 12 were higher in the two germplasm leaves at different stages and treatments.
The results of significant difference analysis showed that the expression of PsCBL2 in the leaves of drought-tolerant germplasm at the mature stage decreased significantly, while the expression of PsCIPK11 and 19 increased significantly.
However, there was no significant difference in the expression levels of these three genes in leaves at seedling stage and drought-sensitive germplasm at different stages.
Under drought stress, the expression levels of PsCBL6 and 11 in leaves at the seedling stage of drought-tolerant germplasm decreased significantly, while the expression of PsCIPK7 increased significantly, and there was no significant difference in the expression levels of these three genes in leaves at different stages of mature and drought-sensitive germplasm.
The expression levels of PsCBL8 and PsCIPK1 in leaves at the mature stage and seedling stage of drought-tolerant germplasm and drought-sensitive germplasm were significantly decreased under drought stress.
The expression levels of PsCIPK8 and 17 were significantly increased under drought stress, but there was no significant difference in the expression levels of these four genes in the leaves of drought-tolerant germplasm seedlings and drought-sensitive germplasm at maturity stage.
Under drought stress, the expression of PsCIPK10 in the mature leaves of drought-tolerant germplasm and drought-sensitive germplasm decreased significantly, but there was no significant difference in the seedling stage.
PsCBL2, 6, 11 and PsCPK7, 11 and 19 were significantly different in drought-tolerant germplasm, but not in drought-sensitive germplasm.
Among them, the expressions of PsCBL2, 6, 11 and PsCPK11 and 19 were significantly down-regulated under drought stress, and the expression of PsCPK7 was significantly up-regulated under drought stress.
In general, under drought stress, the expression trends of PsCBL2, 6, 8, 11, 12, 13 and PsCIPK1, 6, 10 and 18 in leaves at different stages were consistent and lower than those of the control.
These results indicated that drought stress inhibited the expression of these genes in leaves, and these genes may respond to drought stress by forming complexes, while the expression levels of PsCIPK2, 7, 8, 11, 17 and 20 increased under drought stress.
It also indicated that drought stress promoted the expression of these genes in leaves, and these genes may be positive regulators in response to drought stress.
The PsCBL-CIPKs genes in drought-sensitive germplasm were significantly different under drought stress than those in drought-tolerant germplasm, and most of them were significantly reduced, which may indicate that PsCBL-CIPKs genes were more likely to respond to drought stress in drought-sensitive germplasm.
In plants, CBLs interact with CIPKs to regulate plant growth and development and respond to biotic stresses, as well as abiotic stresses such as high salt, cold, drought, pH, and hormones.
A total of 13 PsCBLs and 20 PsCIPKs were identified from the whole genome of pea, and their physicochemical properties and phylogenetic tree analysis were analyzed, and their expression patterns in different tissues and under drought stress were analyzed.