Cloning of Protein Kinase CIPK32 Gene from Poa pratensis L. and Its Response to Abiotic Stresses

doi:10.7668/hbnxb.20193891

Abstract

Abstract:

In order to further study the molecular mechanism of CIPK gene family in response to abiotic stress and explore the role of CIPK32 gene in Poa pratensis L. under abiotic stress. The CIPK32 gene was cloned from Poa pratensis L. midnight Ⅱ by RT-PCR, and its expression in different tissues and under different abiotic stress conditions was analyzed. On-line softwares such as NCBI-CDD and SWISS-MODEL were used to analyze the protein structure, homology alignment and other bioinformatics. The results showed that the sequence ORF of CIPK32 gene of Poa pratensis L. was 1 320 bp, encoding 439 amino acids. It is predicted that the relative molecular weight of CIPK32 protein was 50.28 ku, the isoelectric point was 6.82, and the protein molecular formula was C₂₂₄₉H₃₅₇₄N₆₀₈O₆₆₅S₁₆, which belonged to unstable hydrophilic protein. CIPK32 of Poa pratensis L. contained typical STKc_SnRK3 and CIPK_C domains, and had high homology with amino acids encoded by Aegilops tauschii (XP_020198391.1), Lolium rigidum(XP_047091407.1) and Hordeum vulgare(XP_044980943.1). It also contained many binding sites such as Amidation site, N-glycosylation site, N-myristoylation site and so on. The expression level of CIPK32 gene in Poa pratensis L. was tissue-specific, and the order was leaf>stem>root. Under drought stress, the gene was up-regulated first and then decreased, and the highest value was reached when 10% PEG6000 was treated for 16 h, which was 6.2 times that of 0 h; with the increase of NaCl concentration, the expression of the gene was significantly promoted, and 200 mmol/L NaCl was 6.9 times that of 0 mmol/L NaCl. The expression of CIPK32 gene could be promoted by low concentration of NaNO₃ and low concentration of KH₂PO₄(0.1 mmol/L KH₂PO₄). To sum up, the CIPK32 gene of Poa pratensis L. has tissue specificity, and the expression of CIPK32 gene can be induced by drought, salt, nitrogen and phosphorus stress. CIPK gene family plays a potential role in abiotic stress.

Key words: Poa pratensis L., Protein kinase, CIPK32, Gene cloning, Expression analysis

CHEN Yang, WANG Qi, GAO Yansong, YOU Xue, XIONG Yi, JIN Yifeng. Cloning of Protein Kinase CIPK32 Gene from Poa pratensis L. and Its Response to Abiotic Stresses[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(4): 65-73. doi: 10.7668/hbnxb.20193891.

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Figures/Tables 10

Tab.1 Primers required for gene cloning and fluorescence quantification

引物名称 Primer name	序列(5'—3') Sequences(5'—3')	长度/bp Length
CIPK32-F	CCAGAGGGGGAATAAGGCAG	1 354
CIPK32-R	CCTTCTTAGCAGCGATTTGG
Q-CIPK32-F	TCCAATGACGAGAATAACAG	123
Q-CIPK32-R	GATGTGGTCGCTGTCTTCAA
Q-UBQ-F	AGAAGAAGACCTACACCAAG	217
Q-UBQ-R	GGTTGTAAGGCGTAGGTGAG

Fig.1 The PCR amplification products of the CIPK32 gene M.DL2000.

Tab.2 Amino acid composition of Poa pratensis L.CIPK32%

氨基酸 Amino acid	比例 Proportion	氨基酸 Amino acid	比例 Proportion	氨基酸 Amino acid	比例 Proportion	氨基酸 Amino acid	比例 Proportion
丙氨酸Ala(A)	5.5	谷氨酰胺Gln(Q)	2.3	亮氨酸Leu(L)	10.3	丝氨酸Ser(S)	4.8
精氨酸 Arg(R)	5.7	谷氨酸Glu(E)	8.7	赖氨酸Lys(K)	9.6	苏氨酸Thr(T)	6.2
天冬酰胺Asn (N)	4.1	甘氨酸Gly(G)	5.7	甲硫氨酸Met(M)	2.7	色氨酸Trp(W)	0.9
天冬氨酸Asp(D)	6.8	组氨酸His(H)	2.3	苯丙氨酸Phe(F)	4.8	酪氨酸Tyr(Y)	3.0
半胱氨酸Cys(C)	0.9	异亮氨酸Ile(I)	4.6	脯氨酸Pro(P)	3.9	缬氨酸Val(V)	7.5

Fig.2 Phosphorylation site of CIPK32 protein of Poa pratensis L.

Fig.3 Conserved Domain Database of CIPK32 in Poa pratensis L.

Fig.4 Phylogenetic tree and MEME analysis of CIPK32 amino acid sequence of Poa pratensis L.

Fig.5 Multiple sequence alignment of CIPK32-encoded amino acids between Poa pratensis L.and various plants ①.Amidation site;②.N-glycosylation site;③.Leucine zipper pattern;④.N-myristoylation site; ⑤.cAMP-and cGMP-dependent protein kinase phosphorylation site;⑥.CBL interaction site; ⑦.PP2C binding site;+.Tyrosine kinase phosphorylation site;=.Protein kinases ATP-binding region signature;#.Serine/Threonine protein kinases active-site;Red line.Casein kinase Ⅱ phosphorylation site;Blue line.Protein kinase C phosphorylation site.

Fig.6 Structure of CIPK32 protein in Poa pratensis L.

Fig.7 Analysis of CIPK32 gene expression in different tissues Different lowercase letters indicate significant differences among tissue(P<0.05).

Fig.8 Effects of abiotic stresses on CIPK32 gene expression Different lowercase letters indicate significant differences during drought,salt,nitrogen and phosphorus stress(P<0.05).

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